The Wellcome Trust population initiative.

This paper describes what the Wellcome Trust has done and aims to do through its population initiative. The Trust is required to spend its funds to improve the physical welfare of mankind, and in this context there can be no more important issue than the rapid changes that are occurring in the human population. The Trust's first involvement was to help fund the New Delhi population summit covered by the world's scientific academics in 1993 and, following discussions with authorities in the field, initiated its funding programme in 1995. Through this programme, the Trust hopes to bring about improved understanding of the relationship between reproductive health, population growth, and sustainable development and create cadres of high quality research scientists in relevant disciplines. Uniquely, funding is available under this programme to suitably qualified applicants from any country other than the USA.

PIP: In 1993, the Wellcome Trust entered the population field by contributing funding to the 1993 New Delhi population summit that called for an integrated global policy on population and sustainable development and provided a framework for discussion at the UN's 1994 International Conference on Population and Development. This meeting stimulated the Trust to explore how it could contribute to population research by reviewing current international expenditure in population-related aid and research. As a result of this analysis, the Trust committed up to 50 million British pounds over a period of 5 years to a new funding initiative to improve understanding of the relationship between reproductive health, population growth, and sustainable development while contributing to the training of cadres of high quality researchers. This paper presents an overview of the following funding activities of the Trust's population studies panel: 1) research training fellowships in reproductive biology; 2) research training fellowships in population studies and reproductive health; 3) project and program grant support in the fields of population studies and reproductive health; 4) development of an international research center in Africa; and 5) other awards and activities such as funding of a World Health Organization consultation on preclinical and clinical requirements for non-latex male condoms and a consultation on the toxicology of intrauterine quinacrine. Future developments will involve research training, reproductive health-related research, and identification of research priorities in population and development. The Trust will fulfill its mandate of supporting research conducive to improving the physical conditions of humankind by offering funding to qualified applicants from any country except the US.

Identification of an antigen of Onchocerca volvulus of possible diagnostic use.

Adult Onchocerca volvulus worms obtained by enzyme digestion from nodules of infected Mexicans were radio-isotope labelled by the chloramine-T or Bolton-Hunter methods. No antigenic determinants were detected in extracts of worms labelled by the chloramine-T method but 3 antigens were detected in extracts of the Bolton-Hunter labelled worms. Two were present in such small amounts that it was impractical to investigate them further, but a major component of mol. wt 20 kDa was purified by gel filtration and used in a serological survey of inhabitants of villages in Southern Mexico. Using the 20 kDa antigen, which is superficially located on both sexes of O. volvulus, sera from both non-endemic and endemic regions were analysed by radio-immunoprecipitation of this antigen. In Southern Mexico, the average sensitivity of the test was 92%, and the specificity 98%. Whilst the 20 kDa antigen did not detect antibodies in the sera of Trinidadians infected with Wuncheria bancrofti or Mansonella ozzardi, this antigen detected high levels of antibodies in Indians exposed to W. bancrofti.

Surface proteins of a filarial nematode: a major soluble antigen and a host component on the cuticle of Litomosoides carinii.

The surface composition of three stages in the life cycle of Litomosoides carinii, a filarial parasite of rodents, has been studied using radio-iodination techniques. Confirmation that radiolabelled components were confined to the parasite surface was achieved using light and electron microscope autoradiography. Biochemical analysis of extracts of radiolabelled parasites by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that one major component (mol. wt. 55 000) could be solubilized with the aid of detergents. This component, which was present on male and female adult worms and on post-parasitic third stage larvae, accounted for about one-third of the total proteins available for surface iodination, and was antigenic in infected hosts. The remaining surface components could be solubilized only with urea and SDS under reducing conditions. The 55 000 mol. wt. surface antigens of male and female adult worms exhibited identical two-dimensional tryptic maps, but the similar 55 000 mol. wt. antigen of post-parasitic third stage larvae was different. There was, however, some sharing of antigenic determinants between adult and larval surface components. The principal protein present in detergent extracts of surface-radio-labelled blood microfilariae was host serum albumin.

Characterization of surface and excretory-secretory antigens of Toxocara canis infective larvae.

The surface antigens of Toxocara canis infective larvae have been identified by radio-iodination and compared with the excretory-secretory (ES) products released by the larvae in vitro. Common antigens, of molecular weight 32 000 and 120 000 are found on the larval surface, in the ES material and in culture supernatant following surface iodination of living T. canis larvae. The 120 000 antigens consist of three closely migrating bands in each of these preparations. However, one prominent ES component, of molecular weight 400 000, is not found on the larval surface. Additional molecules of 55 000 and 70 000 are present in the ES material, but while these may be discerned in surface preparations there appears to be more heterogeneity of surface molecules in this size range. Both sets of molecules are antigens to infected patients and experimental animals. A comparison of characterized human sera show that a radio-immunoprecipitation assay correlates with the established ELISA test (r = 0.89), and that all labelled molecules are antigenic to the infected host.

Some properties of the surface of nematode larvae.

The nature of complement binding to the surface to infective larvae of Trichinella spiralis and Nippostrongylus brasiliensis differs. When worms were incubated in serum from uninfected hosts, washed and incubated in fluorescent reagent the whole surface of T. spiralis fluoresced but in N. brasiliensis fluorescence was confined to the anterior end and some internal organs. The outer structure of the cuticle of the T. spiralis larvae was shown not to contain ATP-ase, thus differing from many cell membranes.

Cross-reactive surface antigens on three stages of Brugia malayi, B. pahangi and B. timori.

Surface antigens of three stages of three species of the filarial nematode genus Brugia have been analysed by radio-iodination and immunoprecipitation. These surface antigens have been shown to be characteristic for each stage by polyacrylamide gel electrophoresis. For example, infective larvae and adult worms have relatively complex patterns while microfilariae have few bands which are not found when other stages are radio-isotope labelled by the same technique. The surface antigens of Brugia malayi, B. timori and B. pahangi adult worms are all closely homologous, as are the surface antigens of infective larvae of the same three species, and of microfilariae of B. malayi and B. pahangi. Immunoprecipitation revealed that antibody raised in mice against one stage or species reacted with surface antigens from other stages and species. For example, sera raised against B. pahangi male adults reacted strongly with surface antigens from all three species. This cross-reactivity was dominant despite the apparent stage-specificity of the surface pattern seen on SDS-PAGE analysis. Moreover, in cross-immunization experiments, infective larvae were able to stimulate a secondary antibody response in mice previously primed with microfilarial surface antigens. The major microfilarial surface antigens (of mol. wt 65-70 000 Daltons) were recognized by serum antibody from microfilariae-, infective larvae- or adult-infected animals. Thus, although the dominant antigens from each stage are of different molecular weight, cross-reactions with stage-specific antisera suggest that there must be shared epitopes on Brugia surface antigens from each stage. Such shared antigenic determinants dominate the immune response, although other evidence, including the differences in molecular weight, indicates the existence of stage- and species-specific components.

In vitro induction of human helper T cell activity by Trypanosoma brucei.

Proliferation of peripheral blood T lymphocytes from unsensitized donors in response to fractions of Trypanosoma brucei was observed to be monocyte-dependent. The activated T cells displayed enhanced 'helper' activity, but no 'suppressor' activity during pokeweed mitogen (PWM)-induced immunoglobulin (IgM and IgG) synthesis, and were resistant to the inhibitory effects of theophylline on E rosette formation. Whilst in vivo studies have failed to reveal excessive T cell proliferation in patients with sleeping sickness, these results suggest a possible role for T cells in the induction of hypergammaglobulinaemia characteristic of this disease. Trypanosome fractions were not inhibitory to PWM-induced proliferation, and actually enhanced immunoglobulin synthesis. Thus it is unlikely that direct inhibition by the parasite per se is a major factor in the generation of immunosuppression by the T. brucei subgroup.

Functional abnormalities in the beige mouse eosinophil assessed using T. spiralis as a target.

These studies on eosinophils from beige mice have shown that such cells possess morphologically abnormal granules and that they also have an impaired capacity to interact in vitro with a non-phagocytosable target such as the infective larva of Trichinella spiralis. However, beige mice with these functionally abnormal eosinophils are able to control a T. spiralis infection as well as the normal heterozygote mice. A morphological study of beige eosinophils revealed the presence of structurally distinct lysosomal secretion granules. Some resembled granules in normal eosinophils while others were grossly enlarged and contained multiple crystalloids. When these peritoneal eosinophils were allowed to interact with T. spiralis in vitro in the presence of specific antibody and/or complement, they behaved differently. Cells containing only large granules adhered loosely and temporarily; they were not observed to degranulate. In contrast, cells containing a mixture of granules, or only small granules were able to adhere and degranulate. Despite the defects in the eosinophils of beige mice, the course of an infection with the nematode parasite T. spiralis in beige animals was similar to that in normal animals. Therefore, if eosinophils are crucial in the control of this infection as suggested from other studies, the defect in beige eosinophils is not sufficient to prevent an apparently normal response to the parasite.

Antigenic analysis of Brugia timori, a filarial nematode of man: initial characterization by surface radioiodination and evaluation of diagnostic potential.

The antigenic composition of Brugia timori has been investigated with surface labelling techniques and defined sets of parasite molecules have been identified on infective larvae, adult worms and microfilariae. Iodinated preparations from all three stages were assessed for immunodiagnostic potential with a small number of serum samples from human filariasis patients. In these tests, reaction with infective larval antigen was the clearest indicator of infection. Reactivity to microfilarial antigens however, correlated poorly with incidence of infection. These experiments show that levels of anti-parasite antibody appear to increase as filarial disease becomes more severe. In contrast to some reports, antibody to microfilarial surface antigens is present in sera from several patients with circulating microfilariae. The immunodiagnostic potential of these tests is indicated by the detection of a few individuals who have high levels of antibody but no outward signs of infection.

Restricted sets of parasite antigens from the surface of different stages and sexes of the nematode parasite Nippostrongylus brasiliensis.

Surface molecules of parasitic stages of the nematode Nippostrongylus brasiliensis can be readily iodinated by the chloramine T technique, and assessed for antigenic reactivity with humoral antibody from infected animals. Free-living infective larvae are less amenable to analysis by this, or similar methods, but within 18 hr of larvae entering the host, new macromolecular surface antigens can be detected. The parasites change their surface antigens twice more in the course of the maturation to the adult stage. Surface antigens are stage-specific: lung larvae (L3), intestinal larvae (L4) and gut-living adults each possess characteristic sets of cuticular molecules. Single stage infections result in antibody reactive only to the antigens from the homologous stage. The adult surface appears to bear the greatest number of antigens, one of which is found only on the male worm. The composition of these antigens does not differ grossly between adult worms from a naive or immune host, or worms established after the adaptation of a 'trickle' (multiple low dose) infection. There appears to be an interesting contrast between the rapidity and extent of changes in surface antigens in the early phases of infection, and the stability of adult antigens analysed at different points in the host immune response.

Antibody response to stage-specific Trichinella spiralis surface antigens in strong and weak responder mouse strains.

The antibody response to antigens on the cuticular surface of Trichinella spiralis was compared in two strains of mice, NIH mice, which control the parasite relatively strongly, and C3H, which reject this nematode more slowly. The evolution of the antibody response to the nematode surface was monitored by the appearance of antibodies that mediate the adherence of eosinophils to the worm and the appearance of antibodies that recognize molecules on the surface that can be labeled with 125I. Antibody responses were measured using three life cycle stages; muscle stage larvae, immature and mature intestinal stages, and newborn larvae. We found that NIH mice responded immunologically to the four molecules found in extracts of surface-labeled T. spiralis adult worms, but that C3H mice did not produce antibodies to one of these molecules until much later in the infection. NIH mice produced antibodies against both infective larvae antigens by day 4 after infection, whereas C3H recognized only one before day 30. Variations in antibody response also occurred against antigens on the surface of newborn larvae: antibody to one of these antigens was not present in C3H immune sera until day 30. Sera from the two strains likewise differed in their ability to mediate eosinophil adherence to each parasite stage, and in some of the life cycle stages, these differences corresponded to the recognition of specific antigens found on the parasites' surface. The links between recognition of defined parasitic antigens and the fate of the parasite in genetically different strains of mice are discussed.