Host Serum Proteins Research Articles

Antigenic analysis of a variant-specific component of Trypanosoma brucei brucei: localization on the surface coat with labelled specific antibodies

The nature of antigens present on the surface coat of Trypanosoma brucei brucei (serotype AnTat–1) trypanosomes has been investigated by light and electron microscopy using indirect immunofluorescence and immunoenzymatic labelling techniques. The use of a monospecific anticomponent V serum has shown that the variant-specific antigen, component V, is the major constituent of the surface coat. Antigens of heterologous serotype, of culture form trypanosomes, of host serum proteins or host red blood cells were not demonstrated on the surface coat.

Novel approach to the treatment of hydatid disease.

HYDATID disease is a cyclozoonotic infection of man and certain other mammals which results from the cystic intermediate stage of the cestodes Echinococcus granulosus and E. multilocularis. The growth of these two species of parasites is basically similar except that the latter undergoes tumour-like metastatic dissemination. The present knowledge about immunity to this disease is incomplete; however, recent reports from this laboratory1–3 have defined certain basic aspects of the immune response to secondary echinococcosis. These studies have demonstrated the rapid in vitro lysis of protoscoleces by the complement proteins when these are activated by surface immunoglobulins acquired in vivo by the larvae within the cysts. We also have evidence (unpublished data) that host serum proteins, including IgG and IgM immunoglobulins, are found within the cyst tissues of E. multilocularis, as previously demonstrated in E. granulosus4; Rau and Tanner3 have demonstrated that heat-inactivated serum from infected hosts is cytotoxic to the protoscolices of E. multilocularis in vitro. Based on these results, we now hypothesise that anti-Echinococcus antibodies in the serum of infected animals can “recognise” antigenic sites on the parasite but are unable to reach these sites in sufficient quantities under the natural conditions of the infection; given the opportunity of contact, these cytotoxic antibodies would subject the parasites to the deleterious effects of the immune response. We now present results which show that cysts of E. granulosus are killed when the fluid within these cysts is replaced with the fresh serum of infected animals and suggest this immunotherapy as a novel treatment of hydatid disease. Similar replacement of the fluid of E. multilocularis cysts also successfully killed the treated cysts; but the cyst mass continued to grow by surface budding.

The Cell Surface of Trypanosoma musculi Bloodstream Forms. II. Lectin and Immunologic Studies*

Living Trypanosoma musculi bloodstream trypomastigotes were agglutinated specifically with concanavalin A (ConA), wheat germ agglutinin (WGA), soybean agglutinin (SBA), and fucose-binding protein (FBP). The agglutination with these lectins of living cells from which the coat was removed by trypsinization was the same as with intact trypanosomes. Glutaraldehyde or formalin fixation did not affect the results with regard to agglutination with WGA, SBA, and FBP, but lower agglutination with ConA was observed upon fixation. By using a dense iron-dextran marker many fewer ConA marker particles were localized at the fine structural level in the intact than in trypsin-treated trypanosomes. On the basis of the results obtained by agglutination and electron microscopy, it is likely that fixation cross-links intact surface-coat components associated with the ConA binding sites. It is evident from the studies in which lectins were employed that ligands containing alpha-D-mannose, N-acetylglucosamine, N-acetylgalactosamine, and alpha-L-fucose are randomly distributed in the outer surface of the pellicular and flagellar membranes of T. musculi trypomastigotes. Results obtained with alpha-amylase- and dextranase-treated trypanosomes suggested that lectin-binding sugar ligands in the cell surface were not directly associated with alpha-1,4 or repetitive alpha-1,6 glucan-bonded polysaccharide moieties. Similar conclusions can be drawn on the basis of neuraminidase treatment with regard to N-acetylated neuraminic acids. After thorough washing, intact, but not trypsin-treated trypomastigotes were agglutinated specifically with antisera against whole mouse serum and against mouse IgG. Evidently, adsorbed constituents of mouse serum are regular components of the T. musculi surface coat. After incubation in dilute whole mouse serum or in mouse IgG solutions, also the trypsinized cells were agglutinated by the 2 antisera. No such results were obtained with trypsinized cells incubated in serum-free buffers. It was concluded that mouse serum proteins were readily readsorbed on, and firmly bound to the trypsinized cells' surfaces. Specific agglutinations were obtained with trypsinized cells after incubation in dilute rat, rabbit, bovine, and human sera and in solutions of rat and rabbit IgG in reactions with the corresponding antisera. It seems, therefore, that the host serum proteins are adsorbed nonspecifically to the cell surface of trypsinized T. musculi bloodstream forms. When examined by electron microscopy, the intact trypomastigotes were covered by an ununiform, slightly granular, fibrillar extracellular coat, applied to the entire outer lamina of the pellicular and flagellar membranes. No indication of such a coat was noted in the trypsinized organisms. Flocculent surface coat-like matrix could, however, be discerned in cells which, after trypsinization, were incubated in various sera.

Immunologic and fine structure evidence of avidly bound host serum proteins in the surface coat of a bloodstream trypanosome.

Intact, washed Trypanosoma lewisi bloodstream forms, isolated from rats, were agglutinated specifically by antisera against rat whole serum, albumin, alpha2-macroglobulin, and IgG. However, trypsinized bloodstream and intact culture forms lacking surface coat were not agglutinated by these antisera. Trypsinized bloodstream forms, incubated in dilute rat or heterologous host serum proteins, were agglutinated with specific antisera. The characteristic surface coat of intact bloodstream forms was absent from trypsinized cells; however, trypsinized and serum-incubated bloodstream forms reacquired a surface coat similar to that of intact cells. Gel-diffusion and immunoelectrophoretic results showed that rat albumin, alpha2-macroglobulin, and IgG were present in the surface coat of bloodstream forms. Results of quantitative rocket immunoelectrophoresis demonstrated that the adsorbed rat serum proteins constituted by weight about 5% of the trypanosome total surface coat protein.

Cuticular reactivity of early larval stages of Ascaris suum: Binding of fractions of immune serum to the surface of infective and parasitic stage larvae as detected by the mixed antiglobulin test

Infective stage and early parasitic larvae of Ascaris suum were evaluated for surface reactivity with serum from uninfected and hyperimmune guinea-pigs. Cuticular binding of serum components was assessed by the mixed antiglobulin test. Ensheathed infective larvae bound both normal serum proteins and 7S protein from immune serum over the entire sheath surface. Parasitic larvae recovered at 16 and 25 h post infection (h.p.i.) were poorly reactive, and binding occurred only to sites on the head and tail regions. Larvae recovered at 48 and 72 h.p.i. were highly reactive over the entire cuticle. Host serum protein was not detectable on the surface of parasitic larvae when harvested from guineapigs after a primary infection until the larvae had been in the host for 72 h. However larvae recovered from hyperimmune animals had host serum protein detectable on the cuticle as early as 16 h.p.i.

IgG Levels and Host Specificity in Hydatid Cyst Fluid

IgG was found in sheep hydatid fluid at concentrations ranging between 1 and 3 Aug/ml by the specific radial immunodiffusion method. The species-specific antigenic determinants of the host immunoglobulins were found in hydatid cyst fluid from 4 different species of hosts. The albumin levels and albumin/IgG ratios of cyst fluid were also determined. It is postulated that these proteins enter the parasite by diffusion from the surrounding host tissues. Host serum protein components have been demonstrated in hydatid cyst fluid and attempts made to quantify them. Codounis and Polydorides (1936) detected albumin concentrations of 80 to 1,200 /ug/ml in human hydatid fluid (HHF). In studies with paper electrophoresis Magath (1959) reported a total protein concentration of 75 [g/ml in HHF, of which 33 /g/ml were in the albumin region and 13.5 1tg/ml in the gamma globulins. Goodchild and Kagan (1961) observed variations in the concentrations of protein in the albumin and gamma globulin regions of the cyst fluids of Echinococcus granulosus and E. multilocularis using paper and starch electrophoresis. They also found differences between cysts of E. granulosus from different host species. Chordi and Kagan (1965) identified and characterized 10 bands of parasite origin in sheep hydatid fluid (SHF) by means of immunoelectrophoresis. Nine other bands were common to host serum and of these albumin and gamma globulin were the major components. From an examination of the electrophoretic patterns reported by these authors it is clear that some parasite antigens in SHF also have electrophoretic mobilities in the albumin and gamma globulin regions. This suggested to us that measurements of their quantities of host albumin and gamma globulin in hydatid fluid by electrophoretic methods does not provide an accurate account of the levels of those components which are truly of host origin. This report concerns the results obtained using radial immunodiffusion as a specific method for determining the levels of host Received for publication 2 December 1971. albumin and immunoglobulin in sheep hydatid fluid. The species specificity of the immunoglobulins in the hydatid fluid obtained from cysts in different hosts was also investigated. MATERIALS AND METHODS Radial immunodiffusion was performed according to the method of Mancini et al. (1965) and immunoelectrophoresis by that of Scheidegger (1955). Rabbit anti-sheep IgG serum IgG was purified from the whole sheep serum by precipitation with 33% saturated ammonium sulfate followed by dialysis against 0.005 M phosphate buffer pH 8 and separation in a DEAEcellulose column, stabilized with the same buffer. The ascending portion of the first eluted peak was used to immunize rabbits. Each animal was inoculated intramuscularly with 300 uug of purified IgG in Freund's complete adjuvant, weekly for 5 weeks. In immunoelectrophoretic tests using normal sheep serum this rabbit antiserum detected a single band in the IgG region.

Purification of Lipoprotein Antigens of Echinococcus Granulosus from Sheep Hydatid Fluid

Abstract A simple two-step procedure was devised for the preparation of a fraction of sheep hydatid fluid containing two major lipoprotein antigens. The physicochemical characteristics of the antigens were examined by gel filtration, electrophoresis, immunoelectrophoresis, and chemical analysis. The degree of contamination by host serum proteins was measured with specific precipitation of 125I-labelled antigen and found to be in the region of 1.6%. One component of the fraction was further purified by gel filtration on Sephadex G-200 and its approximate molecular weight calculated to be 160,000.