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Biomedical subjects

P Hagan

Publications and source records attributed to P Hagan.

At least 55 records · Page 3Linked to original sources
PubMed1996

Intracerebral NMDA injection stimulates production of interleukin-1 beta in perinatal rat brain.

Susceptibility to NMDA neurotoxicity peaks in the early postnatal period in rats. Although indirect evidence suggests that interleukin-1 beta is a mediator of NMDA neurotoxicity in perinatal rats, direct confirmation of NMDA-induced interleukin-1 beta production in the brain has not been reported previously. The primary goal of this study was to determine if intracerebral injection of a neurotoxic dose of NMDA stimulates interleukin-1 beta production acutely. We used a rat-specific interleukin-1 beta ELISA to quantify brain tissue homogenate interleukin-1 beta content, and an immunocytochemical assay with a monoclonal antirat interleukin-1 beta antibody to visualize its distribution. NMDA (10 nmol) was injected stereotaxically into 7-day-old rats, using coordinates that targeted the striatum and overlying dorsal hippocampus. Interleukin-1 beta concentrations were measured in samples from the injected and contralateral cerebral hemispheres 0-12 h later; In addition, the impact of treatment with the noncompetitive NMDA antagonist MK-801 on interleukin-1 beta production was assessed. We found marked increases in tissue content of interleukin-1 beta in the lesioned hemisphere; values peaked at 6 h post injection. Treatment with MK-801 (1 mg/kg) blocked NMDA-induced increases in interleukin-1 beta. Preliminary immunocytochemical analysis demonstrated high concentrations of interleukin-1 beta-immunoreactive cells in the lesioned hippocampus, and concurrent increases in interleukin-1 beta immunoreactivity diffusely in the ependyma at 6 h after NMDA administration. Our data provide the first direct evidence that NMDA-induced excitotoxic injury stimulates interleukin-1 beta production in vivo.

Animals↗
PubMed1996

Immunity and morbidity in infection due to Schistosoma haematobium.

It has been suggested that immunologic factors, rather than transmission factors, may play a particularly important role in the regulation of schistosome populations. Support for this view comes from several studies of the epidemiology of Schistosoma haematobium infection, although evidence is accumulating from both field and laboratory studies that immunity in schistosomiasis need not be a concomitant immunity. Correlations between immunologic parameters such as peripheral blood eosinophil counts and specific anti-worm immunoglobulin E (IgE) antibody levels, and apparent resistance to reinfection following chemotherapy have been reported and used as evidence in favor of the existence of an acquired immunity. Specific anti-worm IgG4 subclass blocking antibodies have also been described. The relationship between infection intensity and morbidity may not be as simple as has hitherto been assumed, especially since the methods used to generate data on the prevalence and intensity of infection have serious limitations that may only be overcome with improved or alternative sampling techniques.

Animals↗
PubMed1995

Corticotrophin-releasing hormone (CRH)-binding protein interference with CRH antibody binding: implications for direct CRH immunoassay.

Direct immunoassay of plasma corticotrophin-releasing hormone (CRH) is potentially subject to interference from high levels of CRH-binding protein (CRH-BP) that exist in the human circulation. In this study, we tested the effect of CRH-free, native CRH-BP (6.4 nmol/l) purified from human plasma, CRH-BP diluent alone, normal human plasma (containing 5.8 nmol endogenous CRH-BP/l) and normal sheep plasma (containing no CRH-BP) on the binding of 125I-labelled CRH tracer to five N-terminal and four C-terminal CRH antibodies. All anti-(1-20)CRH N-terminal antibody dilution curves displayed marked inhibition of binding in the presence of purified CRH-BP and human plasma in comparison with the curves with the control diluent or sheep plasma. Almost no inhibition of binding was obtained with any of the C-terminal antibodies (all directed against epitopes within the last six amino acids of CRH) and the four dilution curves were nearly superimposable. Liquid-phase CRH IRMAs were then developed with different combinations of two of each of the N- and C-terminal antibodies, using radiolabelled IgG prepared from purified C-terminal antisera as tracer and raw N-terminal antisera as the link antibodies to the separating system. The addition of dilutions of purified CRH-BP over the range 1.25-20 nmol/l to the IRMA standard curve in assay buffer resulted in a dose-dependent reduction in the signal; with 5 nmol CRH-BP/l, a level commonly found in human plasma, the reduction in binding was 67% and 81% in two different IRMAs at a CRH concentration of 631 pmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Partial characterization and kinetics of expression of Sm15, a Schistosoma mansoni tegumental antigen.

Differential antibody screening of an adult Schistosoma mansoni cDNA expression library constructed in lambda gt11 identified a partial cDNA clone, A70. This cDNA encodes a fusion protein recognized by antibodies raised against highly irradiated schistosomula and adult worm tegumental membranes but not by anti-egg antibodies. Anti-tegumental membrane antisera affinity-purified on the A70 cDNA fusion protein were used for Western blotting analysis and indirect immunofluorescence, resulting in the identification of a 15-kDa protein (Sm15) in the tegument of adult worms. This is one of the principal tegumental antigens recognized by antibodies from mice protectively vaccinated with adult worm tegumental membranes. Sm15 is much smaller than the protein encoded by its gene, suggesting that it results from a highly processed precursor. It was found that Sm15 behaves as an integral membrane protein upon partitioning in Triton X-114 and that it is present in worms of 2 weeks or older but not in schistosomula or miracidia. The affinity-purified antibodies also revealed the presence of a 23-kDa antigen in whole-worm homogenates that is apparently coexpressed with Sm15. The 23-kDa antigen was not found associated with membranes and is probably a soluble protein. A further series of Western blots were undertaken using antibodies affinity-purified from serum raised against schistosomula. In this case, the 23- and 15-kDa products were not recognized, but rather soluble proteins ranging from 45- to 150-kDa were detected in almost all larval stages investigated. The results suggest that the precursor is differentially processed during maturation.

Animals↗
PubMed1994

Non-covalent antibody-mediated drug delivery.

High background activity produces imaging problems when scanning with antibodies. The following work is directed towards reducing this background. The murine monoclonal antibody (MAb) CHA-255 selectively binds 111In-nitrobenzyl EDTA, a molecule referred to as a 'hapten'. Balb/c mice studies indicate that if the antibody is administered prior to the hapten, it predictably modifies the biodistribution and pharmacokinetics of the hapten. The pharmacokinetics for the hapten were proportional to antibody dose and inversely proportional to the time interval between injection of the antibody and the hapten. A hybrid MAb was produced by the enzymatic digestion of CHA-255 and ZCE-025, an anticarcinoembryonic antigen (CEA) MAb, followed by joining of the two via a thioether linkage. The result was a F(ab')2 with affinity for both CEA and the hapten. The pharmacokinetics of the hapten were again dependent upon the kinetics and distribution of the hybrid antibody. Data in tumour models are also presented for 111In-nitrobenzyl EDTA and 111In-thioureabenzyl EDTA (TUBE), a newer hapten. The data indicate that the antibody-hapten system is capable of targeting tumour quickly while normal tissue rapidly becomes depleted of radioactivity. We conclude that the hapten-antibody technique shows some advantages over directly labelled MAb as a targeting system.

Animals↗
PubMed1994

The epidemiology, immunology and morbidity of Schistosoma haematobium infections in diverse communities in Zimbabwe. I. The study design.

Recent studies on the immunology and epidemiology of Schistosoma haematobium infection have led to several new hypotheses regarding the relationships between the transmission of infection and the development of resistance to infection and also the associations between particular antibody and cellular reactivities with resistance or susceptibility to reinfection after chemotherapy. The study will extend the information on the development of protective immune responses during Schistosoma haematobium infection by examining these responses in groups of children and adults from areas in which differences in the peak prevalence of infection have been reported. Our working hypothesis is that, in areas of intense transmission, resistance to infection develops sooner than in areas where transmission is not so intense. We will investigate whether or not this is the case by monitoring levels of reinfection after chemotherapy and by trying to relate observed resistance and susceptibility to reinfection to serological and cellular reactivities. Exposure to infection, cercariometry and malacological studies will also be completed in both areas. The possibility that resistance to infection is associated with T lymphocyte responses classed as Th-2 type will be investigated by assessing reactivities to defined antigens of S. haematobium. This study will generate data which will be used to develop and test mathematical models of the epidemiological determinants which are important in human schistosomiasis.

Adolescent↗
PubMed1994

Schistosomiasis research and the European Community.

Schistosomiasis research within the framework of the Commission of the European Communities 'Science and Technology for Development' (CEC/STD) Programme is targeted at three specific problems: diagnosis of infection and disease; the dynamics of transmission, immunity, and morbidity; and the need for improved tools and strategies for control. Several important advances have been made over the past decade. Improved methods of diagnosis by detection of circulating antigens are in an advanced stage of development and have already undergone field trials in several epidemiological settings. Treatment and reinfection studies combined with immunological observations have allowed the elucidation of possible mechanisms leading to acquired resistance, and have shown that repeated chemotherapy with praziquantel can substantially reduce morbidity. Other projects have studied the epidemiological and ecological determinants of transmission, infection and disease in various endemic situations and also in newly established, epidemic foci where remarkable observations on chemotherapeutic responses were made. Important advances have been made towards the development of a vaccine. The glutathione-S-transferases of the major species of schistosomes have been cloned, sequenced and expressed, and their biological function studied. In a variety of vaccine formulations and animal systems GST has been able to confer protection against infection and to reduce worm fecundity. GST and a series of other crude and defined antigens have been evaluated with varying results in Schistosoma japonicum and S. bovis in cattle. Much work has yet been done, however. Recommendations as to possible future directions for research are provided.

Clinical Trials as Topic↗
PubMed1993

Development of a two-site immunoradiometric assay for rat/human corticotrophin-releasing factor. Application to the measurement of interleukin-1 beta-stimulated production of hypothalamic CRF in vitro.

The hypothalamic hormone corticotrophin-releasing factor (CRF) is a highly conserved, 41-residue peptide, the N terminal region of which rarely induces antibody production, which has hindered the development of two-site immunometric assays. A synthetic N terminal peptide, CRF1-20-Cys-Tyr-NH2, was conjugated to bovine serum albumin through the cysteine thiol group, and used to prepare N terminal directed CRF-specific antibodies. The same peptide, conjugated through the cysteine thiol group to activated thiol-Sepharose, was used to affinity purify N terminal CRF-specific antibodies, and these were used in conjunction with a radioiodinated C terminal directed monoclonal anti-CRF antibody for the development of a specific, sensitive two-site immunoradiometric assay for CRF. To test the utility of the assay, hypothalami were stimulated in vitro with interleukin-1 beta, a putative regulator of CRF secretion, and CRF was measured in hypothalamic homogenates and conditioned media. Interleukin-1 beta dose-dependently stimulated synthesis and secretion of CRF, demonstrating the applicability of the immunoradiometric assay, and confirming previous reports that interleukin-1 beta can directly stimulate CRF secretion from the rat hypothalamus in vitro.

Amino Acid Sequence↗
PubMed1993

Structure of the gene encoding a putative Schistosoma mansoni tegumental antigen precursor.

In order to obtain the complete gene encoding the putative precursor of a 15-kDa Schistosoma mansoni tegumental antigen (Sm15), two cDNAs (A70 and A184) and two fragments of independent genomic clones were subcloned and sequenced. The collated sequence contains 4700 nucleotides and represents the full length open reading frame of the gene, encoding a protein of 1032 amino acids with a calculated molecular mass of 116,900. Thus, the gene encodes a much longer protein than that identified in the tegumental membranes, suggesting that it encodes a precursor that is subsequently highly processed. A 964-bp region composed of 5 closely related repeats was found to be present within the translated frame. The predicted protein is highly acidic and there is no indication of hydrophobic domains that may represent transmembrane regions or indicate attachment of a GPI anchor. The coding region has no homologies in the currently available data bases. In the 5' non-transcribed area a copy of the SM alpha repeat family is present. The coding region is preceded by putative CCAAT and TATA boxes that may be involved in the control of expression.

Amino Acid Sequence↗
PubMed1993

Is tumour necrosis factor alpha the molecular basis of concomitant immunity in schistosomiasis?

Prior to the development of high levels of resistance to infection with schistosomes, some mechanism appears to limit the number of productive worms in individuals, since children do not become superinfected, despite continued exposure to infection. One way in which infection levels might be limited, is through the generation of a concomitant immunity. Concomitant immunity results in the destruction of newly invading schistosomula whilst established adult worms continue to survive. Recent studies have provided evidence that TNF alpha enhances worm fecundity and is essential for granuloma formation. TNF alpha may therefore be important in worm reproduction and transmission, since the granuloma may serve to assist the passage of the eggs out of the tissues. With the additional evidence that the cytotoxic activity of lymphokine-activated macrophages against schistosomula may be, at least in part, due to the action of TNF alpha, we propose that TNF alpha may also be responsible for the phenomenon of concomitant immunity.

Animals↗
PubMed1993

Endotoxin-stimulated production of rat hypothalamic interleukin-1 beta in vivo and in vitro, measured by specific immunoradiometric assay.

Regulation of a number of aspects of the acute-phase response, including induction of fever and activation of the hypothalamo-pituitary-adrenal axis, occurs within the hypothalamus. The acute-phase response appears to be co-ordinated by the inflammatory cytokine interleukin-1 (IL-1). A number of studies using hybridization techniques to measure IL-1 gene expression and immunocytochemistry to localize immunoactive IL-1 have established the concept that the central nervous system, and in particular the hypothalamus, is a site of IL-1 production, and that levels increase in response to inflammatory stimuli. In this report we present data on the levels of IL-1 beta produced in the rat hypothalamus using quantitative immunoassay techniques. Bacterial endotoxin, administered to rats in vivo, evoked increases in hypothalamic IL-1 beta levels which were significant within 1 h, and reached maximum levels at 5-10 h. The response to endotoxin was dose-related, and levels reached in hypothalamic extracts corresponded to intrahypothalamic levels of the order of 20 ng/ml. During short-term in-vitro culture of rat hypothalami, endotoxin stimulated a dose-related increase in both the synthesis and the secretion of IL-1 beta, which reached similar levels to those seen after in-vivo stimulation. Hypothalami obtained from animals stimulated with endotoxin in vivo did not, however, show any evidence of persistent stimulation of IL-1 beta production when subsequently cultured in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Immunosuppressive activity of corticotrophin-releasing factor. Inhibition of interleukin-1 and interleukin-6 production by human mononuclear cells.

Corticotrophin-releasing factor (CRF) from the hypothalamus stimulates corticotrophin (ACTH) secretion. Concentrations of CRF in the peripheral circulation are normally low, and increase during pregnancy due to CRF secretion by the placenta [Cunnah, Jessop, Besser & Rees (1987) J. Endocrinol. 113, 123-131], although CRF in maternal blood does not appear to stimulate the hypothalamo-pituitary-adrenal (HPA) axis [Potter, Behan, Fischer, Linton, Lowry & Vale (1991) Nature (London) 349, 423-426]. We have examined the possibility that the placental. CRF might contribute to the suppression of the maternal immune system, which is necessary to prevent rejection of the foetus, by studying endotoxin-evoked cytokine production by monocytes as a model for activation of the immune response. CRF inhibited endotoxin-evoked cytokine production from human mononuclear cells (MNCs), the fraction of peripheral blood containing monocytes. The effects of CRF were reversed by a specific CRF receptor antagonist, and were additive with glucocorticoid inhibition of cytokine secretion. Anti-interleukin-1 (IL-1) antisera inhibited endotoxin-evoked IL-6 production; however, the CRF effect was not additive, suggesting that CRF inhibition of IL-6 production may be secondary to CRF inhibition of IL-1. These results suggest a role for CRF as an immunosuppressant during pregnancy.

Cells, Cultured↗
PubMed1992

Alterations in an indium-111 Fab' under conditions of utilization.

This study was conducted to investigate alterations that occur in an indium/111 Fab' of a monoclonal antibody following its in vivo administration. Patients were infused with 111In-Fab' of the monoclonal antibody ZCE-025. Serum and urine specimens were collected from these patients. Starting materials, serum, urine and controls samples were studied by electrophoresis. Animal distribution studies were performed in normal Balb/c mice and, in some cases, nude mice bearing a carcinoembryonic antigen (CEA)/producing human colon tumour since the antibody targets CEA. The studies indicated that the molecule circulated almost totally intact for at least 4 h and to a considerable extent for 24 h, with some evidence for in vivo fragmentation by 24 h. Evidence was also obtained suggesting the formation of a high molecular weight species in some patients. Shortly after infusion, some of the 111In in the urine appeared as the intact Fab', but within hours the majority migrated electro-phoretically as low molecular weight species. We conclude that while the majority of the 111In-Fab' of this particular antibody remains intact and immunoreactive following its administration, the molecule is structurally changed to some degree shortly after its infusion into humans. Since each monoclonal antibody is unique, the degree and rapidity of degradation of its Fab' in vivo could vary markedly from the above and possibly adversely effect its utility as a radiopharmaceutical.

Animals↗
PubMed1992

Reinfection, exposure and immunity in human schistosomiasis.

In this review Paul Hagan will focus on the results of the reinfection studies that have been completed on Schistosoma mansoni in Kenya and Brazil and on S. haematobium in The Gambia. The review will also draw on the data from other immunological, molecular and epidemiological studies that have provided information that may be relevant to an understanding of immunity in human schistosomiasis. No attempt will be made to describe the latest advances that have been reported from studies of the animal models of schistosomiasis; these are adequately dealt with elsewhere.

Journal Article↗