Biomedical subjects

A E Bianco

Publications and source records attributed to A E Bianco.

At least 19 recordsLinked to original sources

PubMed1992

The relationship between Trichuris trichiura transmission intensity and the age-profiles of parasite-specific antibody isotypes in two endemic communities.

The present study compares parasite-specific antibody responses in two Caribbean communities with high and low levels of Trichuris trichiura transmission. The age-dependency of antibody levels suggest that IgG1 and IgG2 levels relate to the current intensity of infection (as assessed by density of eggs in stool (e.p.g.) and reflect the age-intensity profile at the population level. IgG4, IgE and IgA levels persist into early adulthood and the subsequent decline is gradual. In the low transmission area, lower infection levels are reflected in lower parasite-specific antibody levels (of all isotypes) in the community as a whole. Despite a significantly greater past experience of infection in the high transmission area, antibody levels are not maintained at significantly higher levels throughout adulthood. The production of IgA appears to require a threshold for triggering, and a vigorous IgA response is maintained into early adulthood only in the high transmission village where peak intensity is greatest and the age-convexity of intensity is most marked. Experimental and theoretical studies focusing on the dynamic nature of host-helminth interactions in hosts exposed to high and low infection levels, and the putative role of acquired immunity, are discussed in relation to the data presented.

Adolescent↗

PubMed1992

Host strain, H-2 genotype and immunocompetence do not affect the survival or development of Onchocerca lienalis infective larvae implanted within micropore chambers into mice or rats.

The survival, growth and development of Onchocerca lienalis 3rd-stage (L3) larvae implanted into mice within micropore chambers has been studied with a view to developing a vaccination model for studies of protective immunity in onchocerciasis. The influence of host genetics on worm recoveries and development (growth and moulting rate) was analysed in a panel of inbred mice (CBA, BALB/c, DBA/2, SJL, 129J, C57BL/10 (B10), C3H/He and NIH), together with mice of BALB and B10 backgrounds with different major histocompatibility complex (H-2) genes (BALB/c, BALB.K, BALB.B and B10, B10.D2/n, B10.BR, B10.S). Parasite recoveries and development were similar in all mouse genotypes tested. They were unaffected by procedures designed to block or modulate phagocytic cell function with carbon or carrageenan, or to suppress inflammation by treatment with hydrocortisone acetate. A comparison of chambers sealed with membranes designed to admit (5.0 microns pore size) or exclude (0.2 microns pore size) host cells demonstrated no effect on the percentage recovery of living larvae, although dead larvae were more frequently retrieved when cells were excluded. Recoveries and rates of development of larvae implanted into immunodeficient scid mice and athymic Hooded rats were similar to those recorded in immunocompetent controls. We conclude that host genetic factors and immunocompetence are not significant determinants of survival, growth or development of O. lienalis larvae implanted within micropore chambers into naive mice. Despite its limitations, the use of this system merits further investigation as an approach to the study of protective immunity against developing larvae in onchocerciasis.

Animals↗

PubMed1991

Humoral immune responses in human infection with the whipworm Trichuris trichiura.

The humoral immune response to infection with Trichuris trichirua was investigated by ELISA and immunoblotting using human sera from the Caribbean island of St Lucia. Immunoblot analysis of the degree of cross-reactivity with the related trichuroid Trichinella spiralis and with the other commonly co-existent nematodes, Ascaris lumbricoides and Toxocara canis, was carried out using selected sera. The IgM, IgA, IgE, and IgG subclass antibody levels were measured in ELISA using a detergent solubilized extract of adult T. trichiura. The IgG and IgE responses were highly Trichuris specific. Anti-T. trichiura IgM responses were totally cross-reactive with A. lumbricoides and were completely ablated by pre-incubation of sera with Ascaris antigen. The IgG response was predominantly of the IgG1 subclass with a minimal IgG3 response. Only 1 person out of 130 tested had a detectable IgG3 response. The IgG2 response appeared to be directed primarily against carbohydrate or polysaccharide antigens as pre-treatment of the ELISA plates with poly-L-lysine was necessary before a response could be detected. These data are the first demonstration of human isotypic responses to infection with T. trichiura.

Animals↗

PubMed1991

Age-dependency of infection status and serum antibody levels in human whipworm (Trichuris trichiura) infection.

This study examines the age-dependency of the relationships between human infection with whipworm (Trichuris trichiura) and parasite-specific antibody level measured by ELISA against an extract of adult worms after preincubation of the sera with Ascaris lumbricoides adult worm extract. The convex age-profile of parasite infection intensity is shown to be mirrored by an age-dependent change in age-class mean levels of IgG (all subclasses except IgG3), IgA, IgM and IgE. Mean antibody levels rise with increasing acquisition of infection in childhood and decline as the intensity of infection falls in adulthood. Immunoblot analysis of selected sera from different age-classes indicates that antigen recognition is similarly dependent on infection intensity. In individual children, antibody levels correlate positively with acquisition of infection, consistent with a simple model of antigen dosage specifying the magnitude of the humoral immune response. In adults, IgG4 correlates positively and IgA negatively with intensity of infection, suggesting involvement of these isotypes in functional roles of immune blockade or effector mechanisms, respectively.

Adolescent↗

PubMed1991

Immunity to Onchocerca volvulus microfilariae in mice and the induction of cross-protection with O. lienalis.

Inbred CBA/Ca mice were vaccinated with Onchocerca volvulus or Onchocerca lienalis microfilariae (mf) and challenged 100 days later with the homologous or heterologous parasite. There was a major reduction in mf recoveries from immunised animals compared with controls when the sensitising and challenge infections were both of O. volvulus mf. Resistance was manifest within one week of challenge, and rose from an 87% reduction in recoveries on day 7 to a 96% reduction on day 15. Similar results were obtained for homologous sensitising and challenge infections with O. lienalis mf. Protection afforded by sensitisation with the heterologous parasite was also high, although the expression of resistance appeared to be delayed. Vaccination with O. lienalis mf led to a 45% level of cross-protection against O. volvulus at 7 days after challenge, which rose to 94% by day 15. Vaccination with O. volvulus mf stimulated cross-protection against O. lienalis at levels of 49% and 80% as measured 7 and 15 days after challenge. It is concluded that the mouse model may be of value in analysing immune responses directed against O. volvulus mf and that the cross-protection between Onchocerca species is an advantage that should be exploited in characterising the antigens involved.

Animals↗

PubMed1990

Developmentally regulated expression and secretion of a polymorphic antigen by Onchocerca infective-stage larvae.

In order to analyse the developmental biology of Onchocerca spp. with a view to identifying molecules with specialised functions, we have devised a novel method for labelling proteins synthesised by larvae during growth in the vectors. Pulse labelling of Onchocerca lienalis by micro-injections of [35S]methionine into blackflies have revealed a major acidic protein of 23 kDa which is developmentally expressed almost exclusively by infective, third-stage larvae. The protein appears to be antigenically conserved between O. lienalis and Onchocerca volvulus, but exhibits size polymorphisms both among species and among individual organisms. It continues to be elaborated after terminal differentiation of the parasite in flies, but not by post-infective larvae entering the phase of development in the vertebrate host. A shift in temperature from 26 degrees C to 37 degrees C triggers secretion of the 23-kDa molecule as a discrete event 24-72 h after transmission. The labelling technique has been successfully employed with filarial species that develop in mosquitoes, and in principle should be widely applicable to the study of endoparasite gene expression within arthropods.

Animals↗

PubMed1989

Biochemical and immunochemical characterisation of a 20-kilodalton complex of surface-associated antigens from adult Onchocerca gutturosa filarial nematodes.

Surface radioiodination of adult Onchocerca parasites reveals a restricted range of proteins associated with the cuticle. We present data to show that prominent among these is a complex of low-molecular-weight proteins which can be released in soluble form by homogenisation of surface-labelled Onchocerca gutturosa in phosphate-buffered saline (PBS). One of this groups of proteins, designated gp20, has a molecular mass of 20,000, is glycosylated with two N-linked carbohydrate side chains, and has a basic pI. Other PBS-soluble, 125I-labelled proteins of similar size appear not to be glycosylated. A distinct group of molecules are released only in the presence of reducing agents, and are likely to be cuticular collagens. The low-molecular-weight components are antigenic and cross-reactive with Onchocerca volvulus infection sera. Cross-reactions are also observed in immunoprecipitation experiments using sera from Brugia-immunised animals and infected humans. Comparative two-dimensional analyses of these immunoprecipitates reveal at least two Onchocerca specific components. As an alternative to radiolabelling and PBS homogenisation, incubation of worms in medium containing the reducing agent 2-mercaptoethanol resulted in a similar set of molecules being released into the medium. Since surface antigens of O. gutturosa from bovines and O. volvulus from humans appear similar in size and are antigenically cross-reactive, the more readily available parasite is being used to study further the properties of these molecules and to provide reagents for raising antisera reactive to the equivalent O. volvulus antigens.

Animals↗

PubMed1989

Extracellular proteases of Onchocerca.

Two important events in infection by Onchocerca parasites involve cutaneous tissue migration by larval stages. L3 larvae migrate from the blackfly bite site to subcutaneous locations for adult development, and microfilariae from subcutaneous nodules to distant regions of the skin and sometimes the eye. By analogy to other tissue-invasive helminth larvae, it has been proposed that migration of Onchocerca larvae through cutaneous tissue is facilitated by secretion of proteolytic enzymes. To test this hypothesis, neutral protease activity capable of degrading a model of cutaneous extracellular matrix was assayed using live L3 larvae of O. lienalis and microfilariae of O. cervicalis and O. cervipedis. Five hundred L3 larvae degraded most of the matrix within 24 hr of incubation. Substrate gel electrophoresis and other protease assays showed a 43-kDa serine elastase was secreted by O. lienalis L3 larvae. Larvae and adults of the free-living nematode, Caenorhobditis elegans, by contrast, did not secrete neutral proteases and large numbers of motile C. elegans juveniles and adults produced no degradation of the extracellular matrix. Expression of Onchocerca neutral protease activity was stage specific. No protease activity corresponding to that seen in L3 larvae was found in adult worms. Microfilariae of O. cervicalis and O. cervipedis produced both a serine and a metalloprotease, but the level of protease activity of these microfilariae was substantially lower than that of L3 larvae, and no significant protease activity was detected in extracts of O. lienalis microfilariae. Uterine microfilariae of O. cervicalis had different protease species than skin microfilariae, suggesting that changes in protease expression parallel other morphologic and biochemical changes in the development of skin microfilariae. The serine protease of L3 larvae probably plays an important parasitic function, facilitating L3 migration from the blackfly bite site to distant regions of the body where adults will develop and form nodules. The protease activity of microfilariae, while individually considerably less than that of L3 larvae, may still contribute to the tissue destruction seen with heavy skin densities of microfilariae.

Animals↗

PubMed1989

Fate of developing larvae of Onchocerca lienalis and O. volvulus in micropore chambers implanted into laboratory hosts.

Infective larvae of Onchocerca lienalis and O. volvulus implanted subcutaneously within micropore chambers into laboratory hosts moulted to the fourth stage (L4) and underwent limited development and growth. Similar recoveries of O. lienalis L4 larvae in the range of 33-66% were obtained from chambers implanted into CBA and BALB/c strains of mice, jirds, and the natural bovine host. A relatively constant proportion of larvae survived up to 24 days post implantation and thereafter recoveries declined, although some worms were still alive after 96 days. Recoveries of O. volvulus L4 larvae from chambers given to normal or T-cell deprived mice were equivalent to one another and to those obtained with O. lienalis. Moulting of O. lienalis in chambers was observed on days 3 and 5, in close accordance with the timing of the third moult in cattle following systemic infection. Moulting of O. volvulus occurred between days 3-6. Morphological changes in developing larvae included a small but significant increase in length, a transient increase in width, and early development of the spicular primordia and genital tube. L4 larvae of O. lienalis, but not those of O. volvulus, exhibited 3 distinct caudal papillae not present on infective larvae.

Animals↗

PubMed1989

Parasite development and adaptive specialization.

The complex life-cycles of parasitic animals are a product of exploiting the process of development to generate organisms with different biological potentials within a species. Successive stages of the parasite adapt to functions such as host invasion and transmission, and to the colonization of a variety of niches, often involving different hosts, tissues or cells. Understanding the molecular basis of adaptive biology among parasites is a major challenge that lies at the heart of research in contemporary parasitology. Differences in scale at the levels of genomic complexity and cell biology exist between most parasitic protozoa and helminths, rendering a cautionary note to making generalized observations. The two principal approaches used to gain insights into adaptive specializations are to start with a biological activity and identify the molecule, or select molecules with particular properties and deduce function. Both have their part to play, with their inherent advantages and limitations, and both are illustrated with examples of studies on adaptive biology among parasitic nematodes.

Adaptation, Physiological↗

PubMed1989

A semi-automated system of intrathoracic injection for the large-scale production of Onchocerca lienalis infective larvae.

Intrathoracic injection of Onchocerca lienalis microfilariae (mf) into Simulium ornatum sl. is a highly efficient procedure for routine production of infective stage larvae (L3). This bovine parasite provides a useful analogue of Onchocerca volvulus from man, which presents far greater difficulties for L3 production outside endemic areas of onchocerciasis. Using novel systems for the infection and caging of adult blackflies in the laboratory, mean yields of 10.1-38.6 L3 per fly have been regularly obtained. Cyclical maintenance of either the parasite or vector is not a requirement for the success of this approach. An automated device has been developed to control deliveries of mf inoculations, that permits an experienced operator to perform injections at a rate of approximately 200 flies an hour. Mass recoveries of larvae by Baermann extraction appeared to be highly efficient, contrasting an earlier report on the application of this method to Onchocerca L3. Typical performance of these techniques in routine use over a period of time is illustrated with data on 18,000 inoculated blackflies, which yielded over 100,000 L3. Viability of the L3 obtained by the procedures described was confirmed by successful transmission of patent infections to laboratory-reared calves.

Animals↗

PubMed1988

Preparation and evaluation of the specificity of Parafilaria bovicola antigen for detection of specific antibodies by ELISA.

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in bovine sera against Parafilaria bovicola nematodes was developed and its sensitivity was compared with the immunodiffusion (ID) method. An exoantigen of P. bovicola which was shown to contain four major polypeptides was used in these procedures. The serological reactivity of the antigen polypeptides was defined by using the enzyme-linked immunoelectrotransfer blot technique (EITB) and whole-worm extract proteins. It identified only four serologically reactive polypeptides with sera from one experimentally infected calf and a verified field case. These two positive sera reacted mainly with four major antigens which coincided in molecular weights of the polypeptides of the exoantigenic preparation, namely, 43, 39, 28 and 25 KDa. Calves experimentally infected with P. bovicola showed a positive reaction with ELISA at 4 months after inoculation, and after this period a rapid increase in serum antibody response occurred. In these cases the ID reaction was observed for the first time at 7 months after inoculation. The specificity of an ELISA method using crude exoantigen preparation of P. bovicola was tested for the diagnosis of bovine parafilariasis. No cross-reactivity was detected when the P. bovicola exoantigen preparation was tested against sera from calves experimentally infected with Onchocerca lienalis, as well as against the sera from cattle naturally infected with Dictyocaulus viviparus or from cattle chronically infected with Ostertagia ostertagi. In addition, testing of 740 field sera from cattle in areas non-endemic and endemic for P. bovicola indicated a specificity of the antigen preparation used. Forty sera from laboratory-confirmed field cases of P. bovicola infection were tested by ELISA and immunodiffusion. All of these sera were ELISA positive, whereas only 70% of these were positive in the ID test. Seven (2.1%) of 328 sera from 21 herds from non-endemic P. bovicola areas were ELISA positive, as opposed to none in the ID test. Of the 94 sera from six herds in areas endemic for P. bovicola infection, 51 (54%) were ELISA positive whereas only 24 (26%) were positive in the ID test. When 56 slaughtered cattle, with varying degrees of meat condemnations due to parafilariasis, were tested for P. bovicola specific antibody, 91% of the serum samples were positive by ELISA. These results suggest that the exoantigen of P. bovicola can be used in a sensitive and reliable serological detection of parafilariasis by ELISA.

Animals↗

PubMed1988

Parasite-specific immune responses to Onchocerca lienalis microfilariae in normal and immunodeficient mice.

The model of Onchocerca lienalis microfilariae (mf) in CBA mice has been employed to examine the immunological mechanisms underlying the destruction of skin-dwelling mf in onchocerciasis. Comparative studies among immunologically intact (CBA/H) or deficient (CBA/N, T-cell-deprived) syngeneic animals demonstrated that levels of mf of a primary infection were reduced most rapidly in fully immunocompetent mice. Significant reductions in recoveries of a secondary infection were evident in CBA/H (80%) and CBA/N (44%) mice, but not in T-cell-deprived animals. The establishment of primary and secondary infections was apparently not influenced by complement, as judged by C3 depletion with Cobra Venom Factor. Eosinophilia was demonstrated to varying degrees in all infected animals; similar levels occurred in CBA/H and CBA/N mice which were greatly elevated after mf challenge. In contrast, the eosinophil response of T-cell-deprived mice was weak and not potentiated during secondary infection. Type I immediate hypersensitivity responses to soluble mf antigen (mf-Ag) were mounted by all groups, but significantly less strongly in T-cell-deprived mice. Type IV delayed responses were generally weak, although CBA/N mice reacted strongly in the early phase of primary infections. During the first 2 weeks of infection CBA/H and T-cell-deprived mice mounted rapid IgM responses to mf-Ag. Subsequently, levels of IgG2a, IgG2b and IgG1 increased in all mice. There was a potentiated IgG2a, IgG2b and IgG1 response in all groups following challenge, with levels of IgG1 highest in CBA/H mice. IgE responses were also detected by passive cutaneous anaphylaxis during primary and secondary infections. Peak levels of parasite-specific antibodies coincided with the timing of mf clearance.

Animals↗

PubMed1988

Patterns of antigen expression in asexual blood stages and gametocytes of Plasmodium falciparum.

The stage specificity and localization of 12 Plasmodium falciparum antigens were determined by immunofluorescence using acetone-fixed parasites reacted with monospecific antibodies against cloned antigens. Antibodies were prepared by immunization of rabbits with recombinant proteins or by affinity purification of human plasma against cloned antigen adsorbents. Most of the antigens occurred predominantly in mature asexual parasites, two were abundant in ring stages and three were absent in rings. Four of the 12 antigens were detected in asexual stages but not in gametocytes. Grouping of antigens by localization within blood stages was difficult because of the complexity of fluorescence patterns observed. With some antibodies, fluorescence was apparently distributed evenly over the parasites, but in other cases label was concentrated within discrete compartments or organelles. Extraparasitic intraerythrocytic fluorescence was also observed.

Animals↗

PubMed1988

Moulting and exsheathment of the infective larvae of Onchocerca lienalis (Filarioidea) in vitro.

Fourth-stage larvae (L4) of the cattle filarial parasite Onchocerca lienalis were produced from third stage forms (L3) and maintained in vitro using a variety of culture media and feeder cell layers. Up to 50% of the L3 larvae moulted to the fourth stage in the presence of two jird (Meriones unguiculatus) cell lines, a monkey kidney cell line and also with Vero cells. Moulting took place 2-5 days after of the initiation of the cultures: These L4 parasites could be maintained in a healthy condition for up to 88 days, although no significant increases in size of these larvae were observed during the culture period. The medium giving the most promising results, both in terms of moulting and survival, was 199 supplemented with heat inactivated foetal bovine serum (20%) and glucose (2 mg/ml). The feeder layer cells could be replaced by fresh jird red blood cells. Moulting of parasites occurred in medium alone but the viability was reduced under these conditions.

Animals↗

PubMed1987

A cDNA clone expressing a rhoptry protein of Plasmodium falciparum.

Antibodies from immune humans were used to select a cDNA clone expressing an asexual blood stage antigen of Plasmodium falciparum. The expressed fused polypeptide was used as an affinity reagent to purify human antibodies specific for the corresponding parasite antigen. Western blotting and immunoelectronmicroscopy demonstrated that the antigen was a 105 kDa protein located in the rhoptries of merozoites. The cDNA encodes the carboxy terminus of the rhoptry antigen, a sequence rich both in charged and hydroxy amino acids.

Amino Acid Sequence↗

PubMed1987

Putative glycophorin-binding protein is secreted from schizonts of Plasmodium falciparum.

A cDNA clone expressing an antigen of Plasmodium falciparum, selected by screening an expression library cloned in Escherichia coli, encodes a portion of the protein identified as a glycophorin-binding protein [Kochan et al. (1986) Cell 44, 689-696]. Human antibodies affinity-purified on extracts from this clone were used to characterize the antigen by immunoblotting. This protein was present in all isolates tested, restricted to mature trophozoites and schizonts. It was abundant in culture supernatants at the time of merozoite release but present in minor amounts if at all in merozoites. The pattern of antigen distribution over schizont-infected cells observed by immunoelectron microscopy differed from that of the precursor of the major merozoite surface antigens in that most of the antigen appeared to be located over the erythrocyte cytoplasm without any obvious association with organelles. It thus appears unlikely that this antigen is present on the merozoite surface prior to schizont rupture.

Amino Acid Sequence↗

PubMed1987

Resistance to Onchocerca lienalis microfilariae in mice conferred by egg antigens of homologous and heterologous Onchocerca species.

Embryonic stages of various Onchocerca species have been used to stimulate resistance in CBA mice to challenge injections with the microfilariae of Onchocerca lienalis. Comparable levels of resistance to challenge (29-37% reductions) were conferred by living, freeze-killed, or sonicated organisms administered with Freunds' Complete Adjuvant (FCA). Antigens extracted in saline, or with the detergent sodium deoxycholate, were also protective. Adjuvants enhanced the protective effect, particularly FCA (78% reduction), Freunds' Incomplete Adjuvant (74% reduction), aluminum hydroxide (70% reduction) and Bordetella pertussis (70% reduction). Detergent extracts prepared from intact embryos with n-octyl glucoside also stimulated significant levels of protection against microfilarial challenge when given with FCA (37-45% reductions). Levels of resistance induced by immunizations with intact organisms were greatest following subcutaneous (s.c.) injection over the neck or by intramuscular inoculation. Soluble extracts were also particially effective given by s.c. inguinal or intraperitoneal injection. A time-interval of greater than 3 weeks between the completion of immunization and challenge was required for the expression of immunity. Cross-protection against challenge with O. lienalis microfilariae was also afforded to mice by immunization with intact embryos or detergent extracts of Onchocerca gutturosa (45 and 34% reductions), Onchocerca gibsoni (66 and 47% reductions) or Onchocerca volvulus (58 and 41% reductions). It is concluded that the embryonic stages of both human and animal parasites provide a source of cross-protective antigens of value in studies on resistance to Onchocerca microfilariae in experimental hosts.

Animals↗