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A Suhrbier

Publications and source records attributed to A Suhrbier.

At least 55 records · Page 3Linked to original sources
PubMed1992

Rapid visual assay of cytotoxic T-cell specificity utilizing synthetic peptide induced T-cell-T-cell killing.

Synthetic peptides are widely used to define the specificity of CD8+ cytotoxic T-lymphocyte (CTL) clones. When many peptides need to be tested by the standard chromium release assay large numbers of a CTL clone are required. Specific synthetic peptide epitopes induce CTL clones to kill each other. This phenomenon can be directly visualized using an inverted microscope and forms the basis for a convenient assay, which can be performed with as few as 100 CTL per peptide and does not require radiolabelled targets.

Cells, Cultured↗
PubMed1991

BLT esterase activity as an alternative to chromium release in cytotoxic T cell assays.

Granules released by cytotoxic T cells (CTL), during recognition and killing of target cells, contain granule enzyme A. This serine protease has an esterase activity, which is easily measured using the substrate benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT). BLT activity, routinely used as an assay for granule release, provides an alternative to the standard chromium release assay as a measure of CTL-mediated killing. The two methods were highly comparable when either exogenous synthetic peptide or endogenously produced epitopes were used as targets and human CTL clones acted as effectors. The advantages of the BLT assay are that it uses inexpensive non-radioactive reagents, the assay can be run over any period between 4 and 30 h and can be performed with as few as 10(4) CTLs if synthetic peptide epitopes are used.

Amino Acid Sequence↗
PubMed1991

Role of single amino acids in the recognition of a T cell epitope.

T cell epitopes can be defined by the use of synthetic peptides, which when added to APC efficiently mimic naturally processed Ag. Free peptide is thought to bind to cell-surface MHC glycoproteins and the TCR then recognizes the resulting complex. The specificity of a tetanus toxin-specific human Th cell clone was investigated using a complete replacement set of peptides in which every amino acid within the minimal T cell epitope was replaced by each of the 19 alternative genetically coded amino acids. Within the minimal epitope, found to be YSYFPSVI (tetanus toxin 593-600), a small number of substitutions could be made without significant loss of activity, defined as substitutions giving peptides whose activity fell within +/- 3 SD of the mean parent response. Y593 could be substituted with F, W, M, L, V, and I; S594 with G and T; Y595, F596, and P597 with no other amino acids; S598 with A; V599 with S, and I600 with L. Rank ordering of the substitutions allowed a precise description to be made of MHC and/or TCR interaction with each amino acid side chain within the epitope. Simplified theoretic calculations based on this study indicate that class II T cell recognition has a specificity greater than 1 in 10(8). Competition experiments indicate that Y595, F596, P597, and I600 are critical for binding of this epitope to its restricting element, HLA DR4Dw14.

Amino Acid Sequence↗
PubMed1991

Inhibition of HLA B8-restricted recognition by unrelated peptides: evidence for allosteric inhibition.

A panel of synthetic peptides representing human lymphocyte antigen (HLA) B8, other class I and class II restricted T cell epitopes and two B cell epitopes, were all able to compete with recognition of a HLA B8 restricted epitope by a cytotoxic T cell clone. Competition was obtained when the competitor peptides were added either before or after the target epitope. The target epitope also had a slow off rate, implicating allosteric inhibition. The presence of non-specific, allosteric binding sites may interfere with experiments attempting to define immunologically relevant MHC binding specificities.

Allosteric Site↗
PubMed1991

Immunity to the liver stage of malaria.

The search for subunit vaccines against malaria has concentrated on asexual and sexual blood stage and sporozoite antigens. In recent years the search for the basis of the protection against sporozoite challenge obtained in mice immunized with irradiated sporozoites has focused attention on the liver or exoerythrocytic (EE) stage of the malaria life cycle. Here, Andreas Suhrbier looks at the various immune responses that appear to be active against this stage, which was once thought to be immunologically insignificant. The liver stage of malaria has thus emerged as a legitimate target for vaccine development.

Journal Article↗
PubMed1991

The development of exo-erythrocytic schizonts of Plasmodium berghei in vitro from gamma-irradiated and non-irradiated sporozoites: a study using confocal laser scanning microscopy.

Confocal scanning laser microscopy has been used to study the distribution of antigens expressed by the liver stages of Plasmodium berghei in cultured hepatoma cells. The 3-dimensional images obtained of intact parasites clearly show complex patterns of antigen expression not apparent when using conventional IFAT or immunoelectron microscopy. A liver-stage specific antigen (Pbl 1) was shown to be confined to the parasitophorous vacuole; the vacuole has extensive diverticulae extending into the host cell. Small parasites were detected for the first time in 'mature' cultures. These did not represent a distinct population, but the 'tail' of a broad continuum of parasite sizes. Irradiated sporozoites produce a transient population of slow-growing parasites which express a very limited range of antigens de novo in the invaded hepatoma cell. A comparison of the reactivity of normal EE parasites with anti-circumsporozoite antibody and with anti-Pbl 1 suggests that the former reagent may reliably be used to identify sporozoites invading host cells, but should not be used to determine the number of parasites that successfully undergo intrahepatic development. Anti-Pbl-1 indicates on 33% of invaded sporozoites identified by anti-CSP subsequently differentiate.

Animals↗
PubMed1990

An ultrastructural study of the exoerythrocytic schizonts of Plasmodium cynomolgi and P. knowlesi in Macaca mulatta.

Exoerythrocytic schizonts of Plasmodium cynomolgi and P. knowlesi were examined by electron microscopy in biopsy samples of primate livers. With maturity the parasitophorous vacuole membrane becomes highly sculptured by the addition of a discontinuous dense thickening, the distribution of which can be a distinguishing character between these two species. The parasitophorous vacuole membrane follows the contours of the parasite faithfully with a minimal surrounding vacuole. The marked destruction of the cytoplasm of the host hepatocyte by most of the parasites studied however gave the distinct, but erroneous, appearance of a large parasitophorous vacuole at the light microscope level. The mature parasite often exhibited a highly invaginated surface contour with the result that the cytoplasm of the host cell and parasite became intimately interdigitated, this interweaving is unlikely to be recognized in light microscopic studies.

Animals↗
PubMed1990

An antigen specific to the liver stage of rodent malaria recognized by a monoclonal antibody.

Vaccines currently being evaluated against malaria are based on proteins derived from the blood, sporozoite and sexual stages. Antigens from the liver stage, which is now recognized as the major target of protective sporozoite induced immunity, have received comparatively little attention. This paper describes the generation of a monoclonal antibody (MoAb), which recognizes an antigen specific to the liver stage of the rodent malaria Plasmodium berghei. The antigen is expressed throughout liver stage development and appears to be localized to the parasitophorous vacuole membrane. The MoAb did not affect the growth of liver stages cultured in vitro nor could protection be demonstrated in vivo following passive transfer of the antibody.

Animals↗
PubMed1990

Survival and antigenic profile of irradiated malarial sporozoites in infected liver cells.

Exoerythrocytic (EE) stages of Plasmodium berghei derived from irradiated sporozoites were cultured in vitro in HepG2 cells. They synthesized several antigens, predominantly but not exclusively those expressed by normal early erythrocytic schizonts. After invasion, over half the intracellular sporozoites, both normal and irradiated, appeared to die. After 24 h, in marked contrast to the normal parasites, EE parasites derived from irradiated sporozoites continued to break open, shedding their antigens into the cytoplasm of the infected host cells. Increasing radiation dosage, which has previously been shown to reduce the ability of irradiated sporozoites to protect animals, correlated with reduced de novo antigen synthesis by EE parasites derived from irradiated sporozoites.

Animals↗
PubMed1990

The development and routine application of high-density exoerythrocytic-stage cultures of Plasmodium berghei.

Methods are reviewed for the culture of the exoerythrocytic stages of Plasmodium berghei wherein development reproducibly reflects growth observed in vivo in laboratory rodents. The combination of these methods with the culture of both asexual and sexual blood stages has allowed the completion of the entire vertebrate phase of malaria development in vitro. The development of new methods for high-density exoerythrocytic-stage culture combined with robust statistical analysis of parasite growth by morphological (light microscopy), or DNA probe methods now allows the critical and precise evaluation of chemotherapeutic or immunological treatments. These methods are illustrated by data obtained on pyrimethamine, primaquine and a hydroxynaphthoquinone. Some of the new avenues of research made feasible by the high-density cultures, e.g., direct immunization to produce monoclonal antibodies and biochemical studies are discussed.

Animals↗
PubMed1990

A liver-stage specific antigen of P. berghei identified by a monoclonal antibody.

Stage-specific immunity (to the sporozoite, the asexual blood-stages and the sexual stages of malaria) has been well documented and antigens from each stage are being tested for their potential as vaccine candidates. Recently it has become clear that the liver stage can also be the target of protective immune responses; however, only the circumsporozoite protein has been identified as a protective liver antigen. It is critical for vaccine evaluation and development to identify other liver antigens and assess their potential role in immunity. In this paper we describe a monoclonal antibody, which recognizes a liver-specific antigen of Plasmodium berghei (referred to as Pbl1). Passive immunization studies using this antibody suggest that it may influence the course of sporozoite-induced infections.

Animals↗
PubMed1989

Contrasts in antigen expression in the erythrocytic and exoerythrocytic stages of rodent malaria.

The time and site of expression of five antigens, recognized by monoclonal antibodies raised against blood-stage parasites, were studied in the exoerythrocytic stage of Plasmodium berghei using indirect immunofluorescent antibody staining. Two monoclonal antibodies (W 3.5, I 2.6), which stain the cytoplasm of infected erythrocytes, did not stain the cytoplasm of the infected liver cell but stained the parasite itself suggesting a difference in the antigenic architecture of the erythrocytic and exoerythrocytic parasites. Another antibody (17.6.1) revealed a further difference in the antigenic composition of the blood and liver-stage parasites being expressed almost exclusively in the former. Two others (C139 and 17.3.9) showed broadly similar patterns of expression in these two stages of the malarial life-cycle.

Animals↗
PubMed1989

Expression of the precursor of the major merozoite surface antigens during the hepatic stage of malaria.

The precursor of major merozoite surface antigens (PMMSA) and its proteolytic products are candidates for an asexual blood stage vaccine. Previous authors have shown that PMMSA epitopes are expressed in the liver or exoerythrocytic (EE) stage of malaria. Using Plasmodium berghei, we show that the molecular weight of the liver stage PMMSA is similar to that of the blood stage and that both EE and blood stage proteins are similarly processed. In the EE stage, it was synthesized toward the end of schizogony and appeared first to localize to the rough endoplasmic reticulum and then, as the cytomeres began to form, to the parasite plasmalemma. The EE and blood stage merozoites expressed similar amounts of this antigen as determined by indirect immunofluorescence.

Animals↗
PubMed1988

Immunoelectron microscopic localization of circumsporozoite antigen in the differentiating exoerythrocytic trophozoite of Plasmodium berghei.

The distribution of the circumsporozoite (CS) antigens in the 24 hour exoerythrocytic trophozoite of P. berghei was studied using Lowicryl immunogold electron microscopy. These antigens were present on the plasmalemma of the parasite, in disrupted areas of the host cell cytoplasm adjacent to the trophozoite and around inclusions of the host cell cytoplasm. There was evidence of a redistribution of the CS antigens away from the pellicular region of the sporozoite.

Animals↗
PubMed1988

The fate of the circumsporozoite antigens during the exoerythrocytic stage of Plasmodium berghei.

There has been considerable interest in the circumsporozoite proteins due to their potential use in anti-malarial vaccines. Previous authors have shown that these proteins persist from the invading sporozoite throughout the growing exoerythrocytic or liver stage. We show that the different distributions of these proteins seen during the development of the exoerythrocytic parasite of Plasmodium berghei closely follow morphological changes, which can be recognized under the light microscope. At the end of the exoerythrocytic cycle, the majority of the remaining circumsporozoite proteins were associated with the spongy stroma in which the emerging exoerythrocytic merozoites lay. Cell-mediated immunity originally directed against sporozoites might recognize the stroma as a second target resulting in the indirect destruction of the exoerythrocytic merozoites.

Animals↗
PubMed1987

Recognition, calcium and the control of desmosome formation.

Since desmosome formation requires the participation of two adjacent cells, a crucial initiating event must be recognition between desmosomal adhesion molecules. Studies of mutual desmosome formation between different cell types suggest that the recognition mechanisms are highly conserved between different tissues and different species of animals. A further requirement for desmosome formation is an adequate extracellular concentration of Ca2+ (greater than 0.1 mM). Keratinocytes, MDCK cells and MDBK cells all show Ca2+-induced desmosome formation. The desmosomes of these cells also show variable stability to reduction in [Ca2+] and Ca2+ chelation. Desmosome formation at low [Ca2+] is triggered by tunicamycin in keratinocytes, suggesting that the carbohydrate moieties of desmosomal glycoproteins may be involved in the Ca2+ control mechanism. The desmosomal glycoproteins appear to bind Ca2+, while the desmosomal adhesion molecules known as desmocollins, like other Ca2+-dependent adhesion molecules, yield a soluble fragment on trypsinization in the presence of Ca2+. For desmocollins the soluble fragment has a relative molecular mass of 42,000.

Animals↗