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A C Rice-Ficht

Publications and source records attributed to A C Rice-Ficht.

36 records · Page 2Linked to original sources
PubMed1994

Interleukin-10 is expressed by bovine type 1 helper, type 2 helper, and unrestricted parasite-specific T-cell clones and inhibits proliferation of all three subsets in an accessory-cell-dependent manner.

Murine interleukin-10 (IL-10) is produced by type 2 helper (Th2) cells and selectively inhibits cytokine synthesis by type 1 helper (Th1) cells, whereas human IL-10 is produced by and inhibits proliferation and cytokine synthesis by both Th1 and Th2 subsets. This study reports that bovine IL-10 mRNA is expressed by Th0, Th1, and Th2 clones of bovine T cells specific for either Babesia bovis or Fasciola hepatica but not by two CD8+ T-cell clones. The antigen-induced proliferative responses of all three subsets of CD4+ cells were inhibited by human IL-10, and low levels (10 U/ml) of exogenous human IL-2 restored the suppressed response. However, proliferation of one Th1 clone was never inhibited but was enhanced by IL-10. Human IL-10 also inhibited the expression of gamma interferon and IL-4 mRNA in Th0 clones. In the absence of accessory cells (AC), the responses of Th clones to concanavalin A or IL-2 were not inhibited by IL-10, whereas antigen-specific responses of Th1 and Th2 cells were reduced when IL-10-pretreated macrophages were used as AC. Together, our results with bovine T cells support the concept that IL-10 primarily affects AC function and does not directly inhibit CD4+ T cells and demonstrate that the immunoregulatory effects of IL-10 are not selectively directed at Th1 populations, as they are in mice.

Animals↗
PubMed1994

CD4+ T-cell clones obtained from cattle chronically infected with Fasciola hepatica and specific for adult worm antigen express both unrestricted and Th2 cytokine profiles.

The well-established importance of helper T (Th)-cell subsets in immunity and immunoregulation of many experimental helminth infections prompted a detailed study of the cellular immune response against Fasciola hepatica in the natural bovine host. T-cell lines established from two cattle infected with F. hepatica were characterized for the expression of T-cell surface markers and proliferative responses against F. hepatica adult worm antigen. Parasite-specific T-cell lines contained a mixture of CD4+, CD8+, and gamma/delta T-cell-receptor-bearing T cells. However, cell lines containing either fewer than 10% CD8+ T cells or depleted of gamma/delta T cells proliferated vigorously against F. hepatica antigen, indicating that these T-cell subsets are not required for proliferative responses in vitro. Seventeen F. hepatica-specific CD4+ Th-cell clones were examined for cytokine expression following concanavalin A stimulation. Biological assays to measure interleukin-2 (IL-2) or IL-4, gamma interferon (IFN-gamma), and tumor necrosis factor and Northern (RNA) blot analysis to verify the expression of IL-2, IL-4, and IFN-gamma revealed that the Th-cell clones expressed a spectrum of cytokine profiles. Several Th-cell clones were identified as Th2 cells by the strong expression of IL-4 but little or no IL-2 or IFN-gamma mRNA. The majority of Th-cell clones were classified as Th0 cells by the expression of either all three cytokines or combinations of IL-2 and IL-4 or IL-4 and IFN-gamma. No Th1-cell clones were obtained. All of the Th-cell clones expressed a typical memory cell surface phenotype, characterized as CD45Rlow, and all expressed the lymph node homing receptor (L selectin). These results are the first to describe cytokine responses of F. hepatica-specific T cells obtained from infected cattle and extend our previous analysis of Th0 and Th1 cells from cattle immune to Babesia bovis (W. C. Brown, V. M. Woods, D. A. E. Dobbelaere, and K. S. Logan, Infect. Immun. 61:3273-3281, 1993) to include F. hepatica-specific Th2 cells.

Animals↗
PubMed1993

Identification of two Th1 cell epitopes on the Babesia bovis-encoded 77-kilodalton merozoite protein (Bb-1) by use of truncated recombinant fusion proteins.

Previous studies have demonstrated the serologic and T-cell immunogenicity for cattle of a recombinant form of the apical complex-associated 77-kDa merozite protein of Babesia bovis, designated Bb-1. The present study characterizes the immunogenic epitopes of the Bb-1 protein. A series of recombinant truncated fusion proteins spanning the majority of the Bb-1 protein were expressed in Escherichia coli, and their reactivities with bovine peripheral blood mononuclear cells and T-cell clones derived from B. bovis-immune cattle and with rabbit antibodies were determined. Lymphocytes from two immune cattle were preferentially stimulated by the N-terminal half of the Bb-1 protein (amino acids 23 to 266, termed Bb-1A), localizing the T-cell epitopes to the Bb-1A portion of the molecule. CD4+ T-cell clones derived by stimulation with the intact Bb-1 fusion protein were used to identify two T-cell epitopes in the Bb-1A protein, consisting of amino acids SVVLLSAFSGN VWANEAEVSQVVK and FSDVDKTKSTEKT (residues 23 to 46 and 82 to 94). In contrast, rabbit antiserum raised against the intact fusion protein reacted only with the C-terminal half of the protein (amino acids 267 to 499, termed Bb-1B), which contained 28 tandem repeats of the tetrapeptide PAEK or PAET. Biological assays and Northern (RNA) blot analyses for cytokines revealed that following activation with concanavalin A, T-cell clones reactive against the two Bb-1A epitopes produced interleukin-2, gamma interferon, and tumor necrosis factors beta and alpha, but not interleukin-4, suggesting that the Bb-1 antigen preferentially stimulates the Th1 subset of CD4+ T cells in cattle. The studies described here report for the first time the characterization, by cytokine production, of the Th1 subset of bovine T cells and show that, as in mice, protozoal antigens can induce Th1 cells in ruminants. This first demonstration of B. bovis-encoded Th1 cell epitopes provides a rationale for incorporation of all or part of the Bb-1 protein into a recombinant vaccine.

Amino Acid Sequence↗
PubMed1993

Babesia bovis-specific CD4+ T cell clones from immune cattle express either the Th0 or Th1 profile of cytokines.

The central role of T cells in the immune response against hemoprotozoan parasites, both as helper cells for T-dependent antibody production, and as effector cells acting directly or indirectly on intracellular parasites through the elaboration of cytokines, has prompted us to investigate the bovine cellular immune response against B. bovis antigens. T cell clones generated from four B. bovis-immune cattle by in vitro stimulation with soluble or membrane associated merozoite antigen were characterized for reactivity against various forms of antigen and different geographical isolates of B. bovis and B. bigemina. The clones were categorized into seven different groups based on differential patterns of reactivity. This panel of T cell clones and additional clones specific for either the 77 kDa merozoite apical complex associated protein (Bb-1) or the 42 kDa major merozoite protein (MSA-1) were analyzed for cytokines. Biological assays to measure IL-2/IL-4, IFN-gamma and TNF-alpha/TNF-beta and Northern blot analysis to detect mRNA encoding bovine IL2, IL-4, IFN-gamma, TNF-beta and TNF-alpha revealed the differential production of cytokines by clones with different antigen specificities. Two Bb-1-specific T cell clones produced the Th1 pattern of cytokines: IL-2, IFN-gamma, TNF-beta and TNF-alpha, but not IL-4. Clones specific for the 42 kDa protein produced undetectable levels of all cytokines, but expressed an unrestricted or Th0 pattern of cytokine mRNA: IL-2, IL-4, IFN-gamma and TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Eggshell precursor proteins of Fasciola hepatica, I. Structure and expression of vitelline protein B.

Antibody raised against the major eggshell protein of Fasciola hepatica (vitelline protein B, vpB) is employed to isolate cDNAs from an expression library and to localize the protein in whole worms. Two cDNAs corresponding to the protein are homologous through the N-terminal and C-terminal coding regions and widely divergent internally. No repeated regions are apparent and no significant sequence homology is seen with chorion proteins of other genera although the amino acid composition closely reflects that of other chorion proteins. Microheterogeneity observed in the vpB is due to the presence of multiple coding sequences, transcripts and a gradient of post-translational modification. Relative transcription of the vpB mRNA throughout the female reproductive tract is demonstrated.

Amino Acid Sequence↗
PubMed1992

Eggshell precursor proteins of Fasciola hepatica, II. Microheterogeneity in vitelline protein B.

At least 3 structural protein precursors of the eggshell are synthesized and stockpiled in the extensive vitelline cells of the liver fluke Fasciola hepatica L. One of these, vitelline protein B, consists of a closely related family of proteins that owes its apparent electrophoretic heterogeneity to variations in the Tyr to DOPA conversion as well as to subtle variations in the primary sequence. The efficiency of the Tyr to DOPA conversion ranges from a maximum of about 90% to a minimum of 55% in the protein. Trypsin digestion in borate buffer at pH 8 was used to produce DOPA-peptides for sequencing. Notably, trypsin does not cleave Arg/Lys-DOPA sequences at borate concentrations greater than 0.15 M. Peptides with DOPA-containing sequences most frequently have flanking amino acids such as Lys, Ser, or Asp on the N-terminal side and Gly or Asp on the C-terminal side. All protein variants fall within a narrow molecular weight range (30-33 kDa), a pI range of 6.9 to 8.3, and the collective majority would appear to share a common N-terminal sequence up to residue 28. The results suggest some combination of the following: variations in post-translational hydroxylation, alternative post-transcriptional splicing and/or the existence of multiple gene copies of eggshell precursors. The latter have been shown to occur in the blood fluke Schistosoma mansoni [15].

Amino Acid Sequence↗
PubMed1992

Bovine helper T cell clones recognize five distinct epitopes on Babesia bovis merozoite antigens.

Helper T cell clones from two Babesia bovis-immune cattle were characterized for use in identification of potentially protective immunogens of B. bovis merozoites. Proliferation assays with 11 CD4+ clones revealed a differential pattern of response to soluble cytosolic antigen, membrane-enriched antigen, detergent extracts of the membrane-enriched antigen, soluble culture supernatant exoantigen, and different geographical isolates of B. bovis as well as Babesia bigemina parasites. When the data were combined, the clones could be grouped according to five different patterns of response. One group recognized only the membrane-enriched fraction of New World and Australian parasites. Four remaining groups recognized antigens found in the cytosolic as well as the membrane-enriched fraction, and clones representative of each group were used to identify cytosolic antigens fractionated by anion-exchange chromatography with the use of fast-performance liquid chromatography. One clone (C97.3C3), which responded to all B. bovis isolates and to B. bigemina, recognized a single peak of activity that eluted with 0.25 M NaCl and contained protein bands of 70 and 75 kDa. The remaining clones were stimulated by a second antigenic peak that eluted between 0.35 and 0.45 M NaCl and contained protein bands of 42, 47, 56, and 84 kDa. The majority of the clones produced interferon, whereas tumor necrosis factor alpha/tumor necrosis factor beta production was less frequent. These studies provide the basis for using helper T cell clones to identify potentially protective immunogens of B. bovis and delineate a minimum of five helper T cell epitopes recognized by two immune cattle.

Animals↗
PubMed1992

Induction of proliferative responses of T cells from Babesia bovis-immune cattle with a recombinant 77-kilodalton merozoite protein (Bb-1).

A major portion of a Babesia bovis-specific gene encoding a 77-kDa merozoite protein (Bb-1) produced during natural infection in cattle and in microaerophilous culture was subcloned into the pGEX1N expression vector. Recombinant Bb-1 protein fused to glutathione S-transferase (Bb-1-GST) was used to examine cellular immune responses in B. bovis-immune cattle. Sera from rabbits immunized with Bb-1-GST reacted with fusion protein and with the native antigen present in crude B. bovis but not with B. bigemina merozoites. Bb-1-GST but not GST induced strong proliferation of T lymphocytes from these immune cattle, and Bb-1-reactive T-cell lines which consisted of a mixed population of either CD4+ and CD8+ cells or CD4+, CD8+, and "null" (gamma delta T) cells were established by in vitro stimulation of peripheral blood mononuclear cells with the recombinant fusion protein. Three CD4+ CD8- and three CD4- CD8+ Bb-1-specific T-cell clones were identified after limiting-dilution cloning of the cell lines. The studies described here demonstrate that the 77-kDa protein of B. bovis contains T-cell epitopes capable of eliciting proliferation of two types of T cells in immune cattle, an important consideration for the design of a recombinant subunit vaccine.

Animals↗
PubMed1990

Isolation and characterization of a gene associated with a virulent strain of Babesia microti.

Babesia microti genomic DNA was purified from parasitized murine erythrocytes, digested with mung bean nuclease and used to construct an expression library in lambda gt11. Polyspecific antisera from mice infected with virulent B. microti organisms (ATCC30221) were used to screen the genomic library for genes encoding major immunogens. High titer antisera selected a recombinant phage, Bm13, containing 3.3 kb of B. microti DNA. Hybridization analysis confirmed the parasite origin of the clone; affinity-purified antibody revealed a native molecular weight of 54,000 for the B. microti protein encoded by the recombinant. Only genomic DNA isolated from the virulent strain of B. microti contained sequences which hybridized to Bm13. Genomic DNA prepared from the Peabody attenuated strain of B. microti or from Babesia bovis DNA did not contain any complementary sequences. These data suggest a possible role for the gene in the virulence of the organism.

Animals↗
PubMed1989

A histidine-rich protein from the vitellaria of the liver fluke Fasciola hepatica.

The vitellaria are an extensive network of glandular cells and ducts distributed throughout the peripheral tissues of the liver fluke Fasciola hepatica. Eggshell precursor proteins are produced and stockpiled in the vitelline cells of mature flukes. Vitelline protein C has an extraordinary composition: the amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA) and histidine each comprise about 20% of the residues, while glycine represents 41-42% in all variants of what appears to be a microheterogeneous protein family. Protein C has an apparent molecular weight of 16,000-17,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although the protein appears homogeneous following polyacrylamide gel electrophoresis in Tris-glycine with SDS and a acetic acid-urea, electrophoresis in borate, however, suggests that the vitelline protein consists of four or more closely related proteins weighing from 16,000 to 18,500. Isoelectric focusing of the protein family in the presence of 8 M urea resolved only two species having pI values of 6.89 and 6.99. A single N-terminus having the sequence H-H-W-D-G-DOPA-G-DOPA-G was detected. The primary structure of vitelline protein C is characterized by a repeated motif consisting of (G-X)n, where X is Ser, DOPA, or His. Most of the His occurs as G-H repeats in a pepsin-resistant fragment of the protein. Previously, a 31-kDa protein, representing up to 6% of the total protein in the fluke, was reported [Waite, J. H., & Rice-Ficht, A (1987) Biochemistry 26, 7819-7825] to contain significant levels of DOPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Babesia bovis: gene isolation and characterization using a mung bean nuclease-derived expression library.

Genomic DNA prepared from erythrocyte cultures of Babesia bovis merozoites was digested with mung bean nuclease and used to construct a lambda gt11 expression library of B. bovis recombinants. Immunoscreening with two polyclonal antibody probes detected multiple recombinants from which two, designated Bb-1 and Bb-3, were chosen for further analysis. Monospecific immunoglobulins isolated from the screening sera using nitrocellulose-bound fusion proteins were employed to determine the native molecular weight and the intracellular location of the babesial proteins encoded by the recombinants. Clone Bb-1 encodes an antigen of 77,000 Da located at the apical end of the intraerythrocytic parasite. A protein of 75,000 Da encoded by clone Bb-3 is associated with the infected red blood cell cytoplasm and/or membrane but not with the merozoite.

Animals↗
PubMed1987

Presclerotized eggshell protein from the liver fluke Fasciola hepatica.

Trematode parasites protect their eggs with a tough tanned eggshell. Eggshell precursor proteins are synthesized and stockpiled within the extensive vitellaria of the animal. A major eggshell precursor protein with an apparent molecular weight of 31,000 and pI of 7.4 was isolated from the vitellaria of Fasciola hepatica. This protein, which represents 6-7% of the total protein in mature Fasciola, is unique in containing rather high levels of the amino acid 3,4-dihydroxyphenylalanine (DOPA), i.e., 110 residues per 1000. Other prominent amino acids are glycine, aspartic acid, and lysine. A prominent DOPA-containing tryptic peptide derived from eggshell precursor protein has the sequence Gly-Gly-Gly-DOPA-Gly-Gly-DOPA-Gly-Lys. DOPA residues disappear during the maturation of the eggshell and by treatment in vitro with mushroom polyphenol oxidase. This disappearance may be related to the formation of cross-links in the eggshell protein.

Amino Acid Sequence↗
PubMed1984

Characterization of the genes specifying two metacyclic variable antigen types in Trypanosoma brucei rhodesiense.

Bloodstream trypanosomes evade the immune system of their mammalian host by sequentially expressing a large number of different variable surface glycoproteins (VSGs). In contrast, metacyclic trypanosomes, the final developmental stage in the tsetse fly, express a much more restricted set of VSGs. These metacyclic VSGs are the first to be exposed to the immune system of the mammalian host after infection and may offer the potential for the eventual development of a vaccine. We have identified cDNAs for two VSGs in cDNA libraries prepared from amplified metacyclic populations of Trypanosoma brucei rhodesiense and show that they correspond to two different metacyclic serotypes. Determination of the cDNA sequences shows that metacyclic VSG mRNAs are similar to VSG mRNAs expressed during the bloodstream stage. Southern blots demonstrate that the metacyclic VSG genes are located near chromosomal telomeres. No evidence of gene rearrangement associated with expression of these VSGs was found.

Animals↗
PubMed1984

DNA rearrangements of the variable surface antigen genes of the trypanosomes.

The trypanosome genome contains several hundred (and perhaps several thousand) genes for the trypanosome variable surface glycoproteins (VSGs). In an individual trypanosome only one of these genes is expressed at a given instant; the others are transcriptionally silent. This differential gene expression is responsible for the sequential antigenic variation displayed by trypanosomes. It is mediated by two types of genomic rearrangements of these VSG genes. The best understood rearrangement type is the formation of a transcriptionally-active expression-linked extra copy (ELC) of a transcriptionally-silent basic copy (BC) gene. This duplication and translocation event places the ELC near a chromosomal end (a telomere) where it is apparently located downstream from a strong promotor. Some VSG genes are not expressed via this ELC mechanism. These genes, which seem to already be near telomeres, are activated by a different non-duplication associated ( NDA ) type of mechanism. We have used recombinant DNA techniques to clone and determine the sequences of genes expressed by both the ELC and NDA mechanisms. Comparison of these sequences reveals that sequences flanking the VSG coding regions are similar. This indicates that there is a sequence correlation between the two mechanisms of expression. We have also shown that when bloodstream trypanosomes expressing a specific VSG via the ELC mechanism are established in culture, the resultant procyclic trypanosomes rapidly stop synthesizing the VSG mRNA (and the VSG) but retain the ELC of the VSG gene. This demonstrates that transcription of an ELC can cease without the loss of that ELC and may indicate the presence of other factors regulating VSG gene transcription.

Animals↗
PubMed1983

Comparison of the expression-linked extra copy (ELC) and basic copy (BC) genes of a trypanosome surface antigen.

A recombinant clone of an expression-linked extra copy (ELC) gene of a trypanosome-variable surface glycoprotein was sequenced. In addition the sequences of the corresponding cDNA and portions of the two basic copy genes were determined. Comparison of these sequences reveals that the 5' boundary of the ELC-transposed segment (2.2 kb) occurs within a repetitive sequence about 700 bp upstream from the start codon of the coding sequence. This sequence does not contain internal symmetries and is not homologous with the repetitive sequence at the 3' boundary. The first 35 nucleotides of the cDNA are different than the corresponding ELC sequence and presumably were transcribed from another genomic location. A restriction fragment containing predominantly sequences outside of the 5' boundary hybridizes to a Pst I fragment whose length is variable in different trypanosome clones. This hybridization pattern is similar to that observed using probes for surface glycoprotein genes that are expressed via the nonduplication-associated (NDA) mechanism rather than the ELC mechanism. This indicates that there is a sequence correlation between these two DNA rearrangement mechanism.

Amino Acid Sequence↗