Biomedical subjects
M J Doenhoff
Publications and source records attributed to M J Doenhoff.
Schistosomiasis vaccines: a response to a devils' advocate's view.
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Detection of praziquantel resistance in schistosomes.
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Schistosoma mansoni cercarial glycolipids are dominated by Lewis X and pseudo-Lewis Y structures.
The oligosaccharide structures of glycolipids from cercariae of the human blood fluke, Schistosoma mansoni, were analyzed in the form of their corresponding, pyridylaminated oligosaccharides by methylation analysis, partial hydrolysis, exoglycosidase treatment, on-target exoglyco-sidase cleavage and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The six, dominant chemical structures present have been determined as: GalNAc(beta1-4)Glc1-ceramide; GlcNAc(beta1-3)Gal-NAc(beta1-4)Glc1-ceramide; Gal(beta1-4)GlcNAc(beta1-3)Gal-NAc(beta1-4)Glc1-ceramide; Gal(beta1-4)[Fuc(alpha1-3)]Glc-NAc(beta1-3)Gal-NAc(beta1-4)Glc1-++ +ceram ide (Lewis X pentasaccharide structure); Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Glc-NAc(beta1-3)GalNAc(beta 1-4)Glc1-ceramide (Lewis X hexa-saccharide structure); and, Fuc(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)GalNAc(beta1-4 )Glc1-ceramide (pseudo-Lewis Y hexasaccharide structure). These structures belong to the characterized schisto-series of protostomial glycosphingolipids. The Lewis X and pseudo-Lewis Y glyco-lipids are stage-specifically expressed by the cercarial life-cycle stage, and not by the adult or egg.
Immunochemical characterisation of Schistosoma mansoni glycolipid antigens.
The aim of this study was to investigate the occurrence, distribution and immunochemical properties of antibody-defined carbohydrate epitopes in neutral glycolipid fractions of Schistosoma mansoni eggs, cercariae and adults. The amount of extractable, antigenic, neutral glycolipids was lowest in adult worms, increasing consecutively in cercariae and eggs. The immunoreactivity of the glycolipids resided in the carbohydrate moiety in that it was periodate-sensitive. Serological reactivity, and monosaccharide component analysis, anomeric configuration and methylation-linkage analyses indicated that there were two dominant epitopes, which could be partially defined immunologically. The first epitope was detected on egg, cercarial and adult glycolipids. It was strongly recognised by mouse chronic infection sera and rabbit hyperimmune sera raised against specific egg antigens, and was defined by the monoclonal antibody M2D3H (Bickle QD, Andrews BJ. Characterisation of Schistosoma mansoni monoclonal antibodies which block in-vitro killing: failure to demonstrate blockage of immunity in vivo. Parasite Immunol 1988;10:151-168). M2D3H appeared to have the same epitope specificity as monoclonal antibody 128C3/3 (Weiss J, Magnani JL, Strand M. Identification of Schistosoma mansoni glycolipids that share immunogenic carbohydrate epitopes with glycoproteins. J Immunol. 1986;136:4275-82). The internal epitope was defined structurally by the presence of fucose 3-linked to 3,4-disubstituted N-acetylglucosamine, which was itself partially substituted by a second fucose residue, to yield the determinant -4[Fucalpha1,2Fucalpha3]GlcNAcbeta1-. The second epitope was defined by the anti-LewisX monoclonal antibody 4D1 and was found primarily on cercarial glycolipids. It was chemically characterised as the LewisX epitope of Galbeta1,4[Fucalpha1,3]GlcNAcbeta1- in a terminal position. The removal of fucose greatly diminished the binding of the anti-LewisX and M2D3H monoclonal antibodies, as well as the polyclonal chronic infection sera, to glycolipids of all three life-cycle stages and thus revealed the epitopic importance of fucose.
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.
Granulomatous inflammation and the transmission of infection: schistosomiasis--and TB too?
Immune-dependent granulomatous inflammation is important in the pathogenesis of both schistosomiasis and tuberculosis. A case has previously been made for a role for schistosome egg-induced granulomas in the onward transmission of infection. Here, Mike Doenhoff suggests that a similar hypothesis applies to tuberculosis.
A Vaccine for Schistosomiasis: alternative approaches.
An independent trial of candidate antigens for a Schistosoma mansoni vaccine has been completed recently under the auspices of the World Health Organization TDR programme. It has been acknowledged that the results of the trial failed to meet expectations and, therefore, it is appropriate that the options for future work should be considered. In this article, Mike Doenhoff describes two S. mansoni molecules-a schistosome larval protease and a high molecular weight egg and worm antigen. Both are associated with protective immunity, but they have unusual immunological properties that distinguish them from the antigens tested so far. The results suggest that alternative approaches to a schistosomiasis vaccine are still worth exploring.
Vector biology and the control of parasitic diseases.
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Is Schistosomicidal Chemotherapy Sub-curative? Implications for Drug Resistance.
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Alterations in cytochrome-c oxidase expression between praziquantel-resistant and susceptible strains of Schistosoma mansoni.
The genetic differences between praziquantel-resistant (R) and susceptible (S) strains of Schistosoma mansoni (Fallon & Doenhoff, 1994) were explored using RAPD and by cloning differentially expressed mRNAs by subtractive PCR. No differences between the 2 strains were detectable by RAPD using 41 different primers indicating that no major genomic rearrangements were present. Subtractive PCR generated a number of fragments, 1 of which was shown to correspond to an over-expressed mRNA in the R strain and to encode a fragment of the subunit 1 of cytochrome-c oxidase (SCOX1). In the absence of a complete sequence for this gene, we used EST sequences to compile a consensus sequence for the 904 bp at the 3' end that enabled us to choose primers for semi-quantitative RT-PCR. This technique showed that SCOX1 was indeed over-expressed about 5 to 10-fold in the R strain whereas the genes encoding the 28 kDa glutathione S-transferase, glutathione peroxidase, NADH dehydrogenase subunit 5 and the ATP-binding cassette family protein SMDR2 were not. In contrast, cytochrome-c oxidase enzyme activity was 4-fold lower in the R strain than in the S strain.
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.
Schistosoma mansoni: maturation rate and drug susceptibility of different geographic isolates.
The fecundities and drug susceptibilities of Schistosoma mansoni isolates from Senegal, Puerto Rico, and Kenya have been examined in mice. The Senegal parasite, obtained from the field in 1993, was shown to have a longer prepatent period (eggs first recovered in the faeces on Day 46 after infection) than those of two isolates, from Puerto Rico and Kenya, that had been maintained for a long period in the laboratory (faecal eggs recovered on Days 38 and 36 after infection, respectively). A Kenyan isolate, also collected from the field in 1994, was shown to mature more slowly than the laboratory-maintained Kenyan isolate. Tissue egg counts confirmed that early in infection the fecundity of the recently collected isolates from Senegal and Kenya was significantly lower than that of the long-term laboratory-maintained Kenyan isolate. Praziquantel and oxamniquine treatment of 8-week-old infections caused a significant (P < 0.001) reduction in worm burden in all isolates tested. However, the reduction in worm burden after praziquantel treatment of infections of the Senegal isolate (50% reduction) was significantly lower than the > 90% reductions in worm burdens after praziquantel treatment of mice infected with either of the Kenyan isolates (P < 0.001). The study confirms that despite being tolerant to praziquantel, the Senegal isolate is fully susceptible to oxamniquine. The praziquantel tolerance of the Senegal parasite is not solely attributed to the state of maturation of the parasite at the time of drug administration.
Cloning, heterologous expression and antigenicity of a schistosome cercarial protease.
A gene coding for the 30 kDa Schistosoma mansoni cercarial protease was amplified using the polymerase chain reaction (PCR) from genomic DNA templates. Cloning and sequencing of several independent PCR clones revealed the presence of an intron additional to the one described in the original cloning of the gene. The 3 exons were cloned into expression vectors so that they could be expressed as separate glutathione-S-transferase (GST) translational fusions. Recombinant bacteria carrying these expression plasmids expressed the fusion proteins at high levels. Western blotting of bacterial lysates with sera raised against the native S. mansoni cercarial protease showed that all 3 exons were recognized. Thus we have produced recombinant bacteria capable of providing large amounts of an S. mansoni antigen for immunological studies and evaluation as a candidate vaccine.
Schistosoma mansoni: anomalous immunogenic properties of a 27 kDa larval serine protease associated with protective immunity.
A cationic Schistosoma mansoni cercarial antigen was shown to be a serine protease as it was capable of hydrolysing N-acetyl-DL-phenylalanine beta-naphthyl ester (NAPBNE) after precipitation by immunoelectrophoresis, and this reaction was modulated by the serine protease inhibitors phenylmethanesulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DEP). The antigen in the immunoprecipitin arcs could also be radio-isotope labelled with tritiated DFP. The peptidolytic enzyme identified in immunoelectrophoresis with polyspecific sera and radio-isotope labelled with tritiated DFP had a relative molecular size of approximately 27 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and evidence obtained after partial purification, SDS-PAGE and immunoblotting supported this size estimate for the enzyme. A rabbit antiserum raised against the peptidolytic antigen reacted against a doublet of antigens at 27/28 kDa in immunoelectrophoresis arcs and against an antigen of 60 kDa in Western immunoblots of crude cercarial homogenate. However, the latter serum precipitated the cationic antigen in immunoelectrophoresed cercarial homogenates only after pre-incubation of the homogenates with PMSF. Fractions containing the partially purified protease also degraded radio-isotope labelled human IgG. The reactivity of a range of polyspecific and monospecific rabbit antisera in Western blots with larval extracts indicated that antibody responses against the 27/28 kDa doublet may be modulated. When immunized with material which contained the 27 kDa enzyme as a major constituent, and which was secreted by S. mansoni cercariae during transformation, only 5 of 16 mice produced antibody to this antigen that was detectable in Western blots. The 5 antibody 'responder' mice were significantly (P < 0.001) protected against challenge with a percutaneous infection of S. mansoni cercariae compared with a group of a mice also immunized with CTF, but which had not produced antibodies against the 27/28 kDa doublet. The results indicate that the 27 kDa serine protease of S. mansoni larvae is a target that is sensitive to immunological attack.
A role for granulomatous inflammation in the transmission of infectious disease: schistosomiasis and tuberculosis.
The relationship between cell-mediated granulomatous inflammation and transmission of disease in schistosomiasis and tuberculosis has been explored. In 2 experiments involving Schistosoma mansoni-infected normal and T cell-deprived mice, and infected deprived mice that had been variously reconstituted with immune or normal lymphocytes or immune serum, there was a significant positive numerical correlation between mean liver granuloma diameters and faecal egg counts in individual animals. Lymphocytes from donors with recently patent infections were more active than cells from chronically infected or uninfected donors in reconstituting egg excretion rates in deprived recipients, and mesenteric lymph node (MLN) cells were more active than spleen cells. Modulation of granulomatous activity with increasing chronicity of infection in the donors, resulting in a decrease in granuloma size around freshly produced tissue-bound eggs, was paralleled by a waning of the capacity of transferred lymph node cells to reconstitute egg excretion in the recipients. Serum taken from chronically infected donor mice over the same period and transferred to infected deprived recipients became more active in enhancing egg excretion in the recipients as the cell-mediated activity declined. A recent study in Kenya has found that S. mansoni-infected patients with concurrent human immunodeficiency virus (HIV) infection excrete fewer eggs than patients exposed to the same levels of schistosome infection, but who are not HIV-infected, thus indicating that schistosome egg excretion in humans is also immune-dependent. Attention is drawn to an apparently parallel situation in human tuberculosis, another pathogen which induces a cell-mediated granulomatous immune response. Several studies have shown that patients with tuberculosis who are also HIV-seropositive tend to have fewer tubercle bacilli detectable in their saliva than those with tuberculosis, but who are HIV-negative. This discrepancy, associated with differences in lung pathology in HIV-positive patients, suggests that in tuberculosis immune cell-mediated granulomatous inflammation causes the destruction of host tissue in a manner which facilitates onward transmission of the bacterial pathogen.
Praziquantel: an urgent and exciting challenge.
The anthelmintic drug praziquantel has proved useful in the treatment of schistosomiasis. The precise mechanism by which praziquantel kills the parasites has yet to be elucidated. Here, John Kusel and colleagues review the current theories on praziquantel action and suggest future avenues for research, which becomes urgent in the light of some reports of drug resistance.
Protection of mice against Schistosoma mansoni infection by passive transfer of sera from infected rabbits.
Sera from rabbits infected with unattenuated Schistosoma mansoni cercariae conferred significant levels of protection against S. mansoni challenge (P < 0.001) after passive transfer to mice. Infected rabbit sera were only effective in conferring protection when transferred during the first week of infection, and were not effective when administered against liver-stage worms. Immunoglobulins isolated from the infected rabbit sera with Protein A-Sepharose were shown to be responsible for the transfer of protection to mice. Immunofluorescence studies demonstrated that the sera were more reactive against the surface of three hour-old mechanically transformed schistosomula than against the surfaces of lung-stage schistosomula. The sera from infected rabbits reacted polyspecifically against antigens in cercaria, schistosomula, and the worm and egg stages of the S. mansoni life-cycle. The host parasite relationship of S. mansoni in the rabbit is discussed.