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Biomedical subjects

J P Rotmans

Publications and source records attributed to J P Rotmans.

At least 19 recordsLinked to original sources
PubMed1996

Schistosoma mansoni excretory circulating cathodic antigen shares Lewis-x epitopes with a human granulocyte surface antigen and evokes host antibodies mediating complement-dependent lysis of granulocytes.

Parasitic worms of the genus Schistosoma excrete relatively large amounts of immunogenic glycoproteins (circulating cathodic antigen [CCA]) that contain polysaccharide side chains with the trisaccharide Lewis-x (L(ex)) as a repeating unit. These carbohydrates evoke high titers of specific IgM antibodies that cross-react with the repeating L(ex) units on the surface of granulocytes. Consequently this might lead, in the presence of complement, to lysis of the granulocytes. In the present study, this hypothesis was investigated using anti-CCA mouse monoclonal antibodies (MoAbs) and polyclonal antibodies purified from sera of infected humans. By flow cytometry, it was demonstrated that the mouse MoAbs directed against CCA strongly recognized the granulocytes. It could also be shown that these MoAbs, as well as anti-CCA IgM antibodies purified from infected human sera, caused lysis of granulocytes in a complement-dependent cytotoxicity assay. Sera from healthy controls or from patients with other helminth infections resulted in negligible granulocytotoxicity. These in vitro observed phenomena may explain the mild to moderate neutropenia that occurs in schistosomiasis patients.

Animals↗
PubMed1996

Localization of alpha 2-macroglobulin protein and messenger RNA in rat liver fibrosis: evidence for the synthesis of alpha 2-macroglobulin within Schistosoma mansoni egg granulomas.

alpha 2-Macroglobulin (alpha 2M) in the rat is a strong-reacting acute-phase protein with potent protease-inhibiting and cytokine-binding properties. Production of alpha 2M is ascribed mainly to liver parenchymal cells. In the present study, we investigated, by means of immunohistochemistry and in situ hybridization, whether fibrosis in the rat liver induced by Schistosoma mansoni eggs leads to local production of alpha 2M. alpha 2M protein and messenger RNA (mRNA) in the unaffected liver tissue, as well as serum values of alpha 2M, were comparable in control rats and egg-injected rats, at 1, 3, and 8 weeks after injection of the eggs. alpha 2M was homogeneously distributed across the liver lobule. In contrast, at the sites of the granulomas, a strong increase in alpha 2M was observed. alpha 2M mRNA was expressed by granuloma cells, but not by the surrounding liver parenchymal cells. Within the granulomas, alpha 2M protein was present in numerous spindle-shaped cells and was diffusely distributed in the extra-cellular matrix. Using double-staining techniques, a subpopulation of the alpha 2M-positive cells in the granulomas appeared to be desmin-positive, suggesting a myofibroblast origin. In addition, parenchymal cells directly surrounding the granulomas contained alpha 2M protein in approximately 50% of the granulomas 1 week after injection of the eggs. In situ hybridization on consecutive sections revealed that these parenchymal cells showed only background activity of alpha 2M mRNA, suggesting uptake of alpha 2M-protein by these parenchymal cells and previous activation of alpha 2M by proteases within the granuloma. The significance of the present study is that alpha 2M is produced locally at sites of inflammation and liver fibrosis, without measurable increase of serum levels of alpha 2M. Unexpectedly, alpha 2M present at the sites of the granulomas is not produced by the liver parenchymal cells, but rather by granuloma cells.

Acute-Phase Reaction↗
PubMed1996

Antibody response patterns against Schistosoma mansoni in a recently exposed community in Senegal.

Acquired immune resistance is believed to be largely responsible for age-dependent infection and reinfection patterns in schistosomiasis. In a recently established but intense focus of Schistosoma mansoni in Senegal, the humoral immune response was studied in a random population sample of 289. Antibody levels of various isotypes to schistosome worm and egg antigens were determined by ELISA and related to egg counts (eggs per gram of feces [EPG]), age, and sex. Both IgG1 and IgG4 followed age-related patterns similar to egg counts and strongly correlated with EPG, even after allowing for age. Specific IgE levels increased slowly with age. The humoral immune response patterns in this recently infected population appeared to be largely similar to those in chronically infected communities. Thus far, the observations do not support the current hypothesis that age-related resistance to Schistosoma is determined by IgE-mediated protective immunity acquired during many years of exposure.

Adolescent↗
PubMed1996

Schistosoma mansoni: in vitro and in vivo excretion of CAA and CCA by developing schistosomula and adult worms.

In this study we describe the excretion patterns of circulating anodic (CAA) and cathodic antigen (CCA) by freshly transformed and developing Schistosoma mansoni schistosomula and adult worms. In vitro, CAA and CCA were excreted by the parasites immediately after transformation. During the first days of development CAA and CCA levels were similar, but after 1 wk more CCA was excreted. Neither feeding the schistosomula with red blood cells nor addition of colchicine influenced the rates of antigen excretion. Female worms produced more antigen than males. In heavily infected mice CCA was the first antigen detectable from the third week of infection onward. A few days later, CAA showed a steep increase, becoming the predominant antigen during the course of infection. In urine samples, obtained at the time of perfusion (7 wk), CCA was the predominant antigen. In conclusion, although CAA and CCA levels in serum and urine generally correlate well with worm burden (as determined by egg output), the present study and a literature review show that the actual quantities produced by the worms and detected in the host circulation or excreta may depend on many factors, e.g., host and parasite species, clearance rates, or duration and intensity of infection.

Animals↗
PubMed1994

The immunologically reactive part of immunopurified circulating anodic antigen from Schistosoma mansoni is a threonine-linked polysaccharide consisting of --> 6)-(beta-D-GlcpA-(1 --> 3))-beta-D-GalpNAc-(1 --> repeating units.

The gut-associated excretory antigen CAA (circulating anodic antigen) from adult Schistosoma mansoni worms was isolated by immunoaffinity chromatography. Amino acid analysis following alkaline borohydride treatment indicated that CAA is a glycoprotein, O-glycosylated at Thr. The primary structure of the released O-glycan moiety was investigated by one- and two-dimensional, homo- and heteronuclear 1H and 13C NMR spectroscopy. It was found that the major carbohydrate chains have a novel polysaccharide structure, consisting of a branched disaccharide repeating unit containing 2-acetamido-2-deoxy-beta-D- galactopyranose (beta-D-Galp-NAc) and beta-D-glucopyranuronic acid (beta-D-GlcpA). [formula: see text] The major antigenic character of CAA arises from this novel polysaccharide, which was shown to be an absolutely specific diagnostic marker in schistosomiasis. The cross-reactivity of CAA with anti-CCA (circulating cathodic antigen) monoclonal antibodies is caused by the presence of a small amount of O-linked CCA-poly-Lewis x carbohydrate chains on the CAA protein chain.

Animals↗
PubMed1994

The immunologically reactive O-linked polysaccharide chains derived from circulating cathodic antigen isolated from the human blood fluke Schistosoma mansoni have Lewis x as repeating unit.

The gut-associated excretory antigen circulating cathodic antigen (CCA) was isolated by immunoaffinity chromatography from adult Schistosoma mansoni worms, which were collected from infected golden hamsters. This antigen is probably involved in protection of the schistosome gut and is increasingly used in highly sensitive and specific immunodiagnostic assays. Amino acid analysis before and after alkaline borohydride treatment of CCA and monosaccharide analysis indicated that CCA is O-glycosylated mostly via GalNAc-Thr. After reductive alkaline treatment, the O-linked carbohydrate chains were fractionated by gel-permeation chromatography, followed by normal-phase HPLC on LiChrosorb-NH2. Carbohydrate-positive fractions were investigated by one-dimensional and two-dimensional 1H-NMR spectroscopy, fast atom bombardment mass spectrometry and collision-induced-dissociation tandem mass spectrometry. The analyses showed that the low-molecular-mass O-linked oligosaccharide alditols (the minor fraction) consist of disaccharides to hexasaccharides having the Gal beta (1-3)GalNAc-OL core in common. The major carbohydrate fraction comprises a population of polysaccharides, containing Lewis x repeating units (-3)Gal beta (1-4)[Fuc alpha (1-3)]GlcNAc beta (1-). CCA-specific monoclonal antibodies and IgM antibodies in patient sera recognized the fucosylated O-linked carbohydrate antigenic structures. Since CCA evokes a strong IgM antibody response and carbohydrate structures containing repeating Lewis x units are found on circulating neutrophils, it is proposed that the antigenic poly-Lewis x polysaccharide of CCA is involved in the induction of auto-immunity against granulocytes, resulting in the mild to moderate neutropenia observed during schistosome infection.

Animals↗
PubMed1994

Quantitative determination of circulating soluble egg antigen in urine and serum of Schistosoma mansoni-infected individuals using a combined two-site enzyme-linked immunosorbent assay.

Two enzyme-linked immunosorbent assays (ELISA-5B1 using monoclonal antibody [MAb] 114-5B1-A [IgG1] and ELISA-4D12 using MAb 114-4D12-A [IgG3]) that detect circulating soluble egg antigen (CSEA) of Schistosoma mansoni were combined into one assay. This assay showed better performance than either of the two MAbs alone in detecting egg antigen, which was demonstrated with 80 urine samples from patients infected with Schistosoma mansoni from Zaire. The lower detection limit of the combined ELISA was 90 pg of the trichloroacetic acid-soluble fraction of soluble egg antigen (SEA-TCA) per milliliter. Thirty-two serum samples and 107 urine samples from uninfected Dutch individuals were negative when tested with the combined ELISA. This assay showed the same sensitivity (86.3%) with patients' urine samples as parallel testing with ELISA-5B1 and ELISA-4D12 (85%), while ELISA-5B1 and ELISA-4D12 showed sensitivities of 81.3% and 75%, respectively. The sensitivity of the combined ELISA with 51 serum samples was 84.3%, and three of five serum samples available from the seven patients with negative urine were positive for CSEA. The concentration of CSEA calculated from a four-parameters logistic curve for samples tested showed a correlation with egg output and serum circulating anodic antigen (P < 0.0001). Circulating soluble egg antigen in urine showed a significant decrease with an increase in age of the patients in relation to serum CSEA and egg output.

Age Factors↗
PubMed1994

Quantitative diagnosis of Schistosoma infections by measurement of circulating antigens in serum and urine.

Recent research on the detection of the schistosome circulating antigens, circulating anodic antigen (CAA) and circulating cathodic antigen (CCA), has greatly expanded the scope of applications. In the present paper aspects of assay development, in particular of highly sensitive assays and of field-applicable assays, are discussed. The progress in the use of CAA- and CCA-detection for diagnosis, follow-up of chemotherapy and sero-epidemiological studies is evaluated.

Animals↗
PubMed1993

Schistosoma mansoni circulating anodic antigen but not circulating cathodic antigen interacts with complement component C1q.

Adult schistosome parasites, living in the blood vessels of their mammalian hosts, protect themselves against immune damage in a variety of ways. In addition to the tegument, the intestinal epithelium of the blood-feeding worms is permanently exposed to both the innate and the acquired immune system. In this study, we investigated whether the Schistosoma gut-associated antigens CAA and CCA (circulating anodic antigen and circulating cathodic antigen, respectively), which are excreted in relatively large quantities into the host's circulation, might play a role in evading complement attack. Of several complement components tested, only purified C1q showed significant binding to CAA, a negatively charged highly glycosylated glycoprotein. CCA, also highly glycosylated, but neutral or slightly positively charged, did not bind to C1q. CAA bound only to the collagen-like stalks of C1q and not to the globular heads. No detectable interaction of CAA with precursor human C1 was found and CAA did not induce activation of C1 in whole human serum as assessed by consumption of hemolytic C4 activity. Also CAA could not induce activation of precursor C1 in vitro. These results suggest that CAA behaves like a receptor for C1q, and might be involved in protecting the vulnerable schistosome gut against complement-mediated attack.

Animals↗
PubMed1993

Schistosoma mansoni: analysis of monoclonal antibodies reactive with gut-associated antigens.

The analysis of a series of monoclonal antibodies (mAbs) developed in our laboratory against gut-associated antigens of Schistosoma mansoni is described. It was found that mAbs that recognized epitopes of antigens in the gut and on the eggshell were mainly of the IgM isotype; these epitopes are likely to be carbohydrate in composition. Of a number of mAbs that were reactive with antigens important to the human humoral immune response, 75% appeared to be reactive with the circulating cathodic antigen.

Animals↗
PubMed1993

Detection of IgM antibodies directed against the gut-associated circulating cathodic antigen in sera from Schistosoma mansoni infected patients. Development and comparison of three enzyme-linked immunoassays.

The majority of the human IgM antibodies detected with an immunofluorescence assay (IFA) on adult worms are directed against the gut-associated circulating cathodic antigen (CCA). In order to study this phenomenon further we developed and evaluated three related ELISA methods to specifically detect IgM antibodies against purified CCA. The assays employed: 1) direct coating of CCA, 2) indirect coating of CCA via a monoclonal antibody, and 3) IgM antibody-capture by rabbit anti-mu chain antibodies. Using a group of 46 positive sera, it was found that the three ELISA's and the IFA were significantly correlated. To discriminate between positive and negative sera we used a cut-off level of average reactivity + 3 standard deviations of 50 negative sera. False negative reactions were not found in any of the ELISA's, while both in the direct and indirect ELISA one false positive reaction occurred. For further studies or diagnostic use the antibody-capture ELISA is recommended.

Animals↗
PubMed1992

Schistosoma japonicum: immunoinhibitory studies on hemoglobin digestion using heterologous antiserum to bovine cathepsin D.

Antibody against purified bovine cathepsin D was raised in rabbits, and the polyclonal antiserum was tested to determine its ability to inhibit the hemoglobinolytic activity of the crude enzyme preparation (CEP) from adult Schistosoma japonicum and its effect upon in vitro cultured Schistosoma mansoni schistosomules. The 100,000 g supernatant fraction (CEP) from lyophilized adult worms was preincubated with antiserum and subsequently incubated with hemoglobin. Hemoglobinolytic activity was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic procedures. Five hours of incubation failed to diminish hemoglobin concentration in experimentals, whereas controls treated with preimmune serum displayed hemoglobin degradation. Pepstatin inhibited hemoglobin degradation. Western blot analysis of the CEP revealed a broad band of activity at approximately 45 kDa. Schistosomules incubated in vitro either in the antiserum or pepstatin and subsequently exposed to host erythrocytes showed a marked inhibition of digestive activities. Although structural changes were not evident in the gastrodermis, some perturbation of the tegument was observed. Schistosomules fed host erythrocytes and postincubated in the antiserum displayed increased tegumental perturbation and extensive alteration of the gastrodermis, including dilation of cisternae and membrane disruption. Schistosomules exposed to preimmune serum were normal in all respects.

Animals↗
PubMed1989

Sensitive determination of circulating anodic antigen in Schistosoma mansoni infected individuals by an enzyme-linked immunosorbent assay using monoclonal antibodies.

From a panel of mouse monoclonal antibodies reactive with a repeating epitope of the schistosome circulating anodic antigen, an IgG1 monoclonal antibody was selected. This monoclonal antibody was applied in a sandwich enzyme-linked immunosorbent assay as capture antibody and as alkaline phosphatase labeled conjugate. This assay allowed a sensitive quantitation of circulating anodic antigen in serum samples of infected individuals, detecting less than 1 ng antigen/ml serum. In Schistosoma mansoni infected individuals from Zaire, the level of antigen in serum correlated with fecal egg output. The lower detection level of the immunoassay corresponded to a level of about 10 eggs/gm feces.

Animals↗
PubMed1987

Interlaboratory evaluation of indirect enzyme-linked immunosorbent assay, antibody capture enzyme-linked immunosorbent assay, and immunoblotting for detection of immunoglobulin M antibodies to Toxoplasma gondii.

One hundred and fifteen serum samples from healthy laboratory personnel and 50 consecutive samples from 19 patients with anamnestic clinical signs of toxoplasmosis were assayed by four laboratories for the presence of immunoglobulin M antibodies to Toxoplasma gondii by an indirect enzyme-linked immunosorbent assay (ELISA), an antibody capture assay with peroxidase-labeled toxoplasma antigen, and an immunoblotting assay. In addition, a commercially available antibody capture ELISA was used. Highly significant correlation coefficients were obtained between the four laboratories and the commercial test. The indirect ELISA and antibody capture ELISA showed equal sensitivity in detection of immunoglobulin M antibodies to toxoplasma in early-stage serum samples. However, in this study, the antibody capture assay discriminated better between serum samples obtained at early or late stages of toxoplasma infection.

Animals↗
PubMed1984

The effect of antigen cross-linking on the sensitivity of the enzyme-linked immunosorbent assay.

To investigate the effects of antigen cross-linking on ELISA results, we assayed antibodies to several purified antigens, using microtitration trays coated with antigens in their native and cross-linked forms. Cross-linking was achieved by a carbodiimide promoted condensation reaction. For several antigens an increase in sensitivity of the ELISA was obtained after cross-linking. The results of a double antibody sandwich assay using the same cross-linked antigens, showed that the affinity for antibodies remained unchanged after limited cross-linking. Therefore, the higher sensitivity of the ELISA is most likely due to a higher affinity of the cross-linked antigens for the plastic carrier. Cross-linking of proteins in a complex antigen preparation obtained from Schistosoma mansoni led to a large increase in sensitivity.

Animals↗
PubMed1983

Cross-linking of Schistosoma mansoni antigens and their covalent binding on the surface of polystyrene microtitration trays for use in the ELISA.

The usefulness of the enzyme-linked immunosorbent assay (ELISA) for serodiagnosis is limited by the incomplete and unequal binding of individual antigens present in a complex antigen preparation. We therefore investigated 2 modifications of the commonly used antigen coating procedure in the ELISA. Schistosoma mansoni antigens cross-linked by N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (EDC), and then adsorbed on polystyrene microtitration trays, tested with sera from patients, and from immunized rabbits, showed an increase in the ELISA results, with respect to both absorbance values and titers. Measurement of specific antibodies against low molecular weight schistosomal antigens, which is not possible in the standard ELISA, was made possible by adsorption of a cross-linked antigen preparation. Covalent binding of cross-linked antigens to modified microtitration trays, gave a further increase in ELISA readings when the antibody response of S. mansoni infected mice to lower molecular weight antigens was assayed. In assays of patients' sera, a further increase in sensitivity of the ELISA was occasionally obtained by binding cross-linked antigens covalently. With some sera the use of physically adsorbed cross-linked antigens gave optimal results.

Animals↗