Biomedical subjects

T A Potter

Publications and source records attributed to T A Potter.

At least 37 records · Page 2Linked to original sources

PubMed1996

Peptide epitopes from noncytosolic Listeria monocytogenes can be presented by major histocompatibility complex class I molecules.

Listeria monocytogenes is an intracellular pathogen which escapes the phagosome and resides in the cytosol of the host cell. Using Listeria innocua and a mutant strain of L. monocytogenes (listeriolysin O negative), which do not enter the cytosol of the host cell, we demonstrate class I presentation of an epitope of p60, a protein secreted by L. monocytogenes, to a class I-restricted CD8+ cytotoxic T lymphocyte clone.

Animals↗

PubMed1996

Polymerase chain reaction for the detection of Toxoplasma gondii in aqueous humor of cats.

OBJECTIVES: To develop Toxoplasma gondii B1 gene polymerase chain reaction (PCR) for use with aqueous humor of cats, and to report PCR and antibody detection results in naturally exposed cats with and without uveitis. SAMPLE POPULATION: Serum and aqueous humor samples from client-owned, healthy cats (n = 23) and client-owned cats with uveitis (n = 43). PROCEDURE: T gondii-specific IgM and IgG were measured in serum and aqueous humor from all cats. The Goldman-Witmer coefficient for ocular antibody production was calculated for cats positive for T gondii-specific IgM or IgG in aqueous humor. Aqueous humor from all cats was assessed by the B1 gene PCR. RESULTS: T gondii was detected in aqueous humor by PCR from 2 of 23 (8.7%) healthy cats and 8 of 43 (18.6%) cats with uveitis. T gondii-specific IgM in either serum or aqueous humor was detected in 5 of 8 (62.5%) cats with uveitis and T gondii in aqueous humor. All cats with uveitis and T gondii in aqueous humor had anterior segment disease. In 5 of 8 (62.5%) cats with uveitis and T gondii in aqueous humor, ocular production of T gondii antibodies was not detected. T gondii was not detected in aqueous humor from 14 of 17 (82.4%) cats with ocular production of T gondii-specific antibody. CONCLUSIONS: The presence of T gondii in aqueous humor may correlate to clinical disease in some, but not all, cats. CLINICAL RELEVANCE: T gondii-specific aqueous humor antibody tests and PCR should be used together to aid in the diagnosis of ocular toxoplasmosis in cats.

Animals↗

PubMed1995

CD8-dependent CTL require co-engagement of CD8 and the TCR for phosphatidylinositol hydrolysis, but CD8-independent CTL do not and can kill in the absence of phosphatidylinositol hydrolysis.

Most instances of MHC class I recognition and target cell killing by CD8+ CTL require the involvement of CD8. The role of CD8 in these events may be both for adhesion of the CTL with the APC, as well as for signal transduction through the TCR. The precise mechanism by which CD8 mediates signal transduction remains enigmatic. Similarly, it is unclear whether only the CD8 molecules which bind to the same class I molecule as the TCR contribute to signaling in the T cell responding to antigen. We have investigated the requirement for co-engagement of CD8 and the TCR in the induction of the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) during the interaction of CTL and APC transfected with either wild-type or mutant (CD8 non-binding) class I molecules. Our results show that for conventional CD8-dependent killing co-engagement of both CD8 and the TCR is required to initiate PIP2 hydrolysis. This requirement for co-engagement, however, can be overcome by a high density of ligand, such as that provided by high concentrations of exogenous peptide. In such situations, the binding of CD8 to non-antigenic class I molecules can elicit PIP2 hydrolysis. Therefore, during interactions between CTL and APC, which generally occur at low concentrations of antigenic peptide, triggering of PIP2 hydrolysis requires TCR and CD8 co-engagement, and the binding of CD8 to non-antigenic class I molecules does not contribute significantly to signaling within the T cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Listeria monocytogenes infects human endothelial cells by two distinct mechanisms.

Infection of endothelial cells by bacteria may be an important component of the bacteria's ability to escape host defenses and cause disease. Listeria monocytogenes cause sepsis and central nervous system infection in domesticated animals and immunocompromised humans, suggesting that this bacterium interacts with endothelial cells in a significant fashion. The experiments presented here tested the hypothesis that L. monocytogenes can invade and replicate within human endothelial cells. We found that L. monocytogenes grows readily in umbilical vein endothelial cells and that its intracellular life cycle involves phagosomal escape, F-actin-based motility, and cell-to-cell spread. We found that L. monocytogenes invaded endothelial cells by cell-to-cell spread from adherent mononuclear phagocytes which were previously infected by this bacterium. Interestingly, L. monocytogenes mutants lacking the invasion protein, internalin, bound less well to endothelial cells than did wild-type bacteria in the absence, but not the presence, of serum, and their invasion of endothelial cells was diminished under both conditions. Thus, endothelial cell infection by L. monocytogenes can occur by two distinct mechanisms: direct bacterial invasion of the endothelial cells in an internalin-mediated fashion or cell-to-cell spread from adherent, infected mononuclear phagocytes. These data support the idea that endothelial cell infection by L. monocytogenes is an important event in the pathogenesis of listeriosis.

Actins↗

PubMed1994

Analysis of coreceptor versus accessory molecule function of CD8 as a correlate of exogenous peptide concentration.

Substitution in the alpha 3 domain of class I molecules can ablate the recognition of target cells by CD8 dependent cytotoxic T lymphocytes. This effect has been attributed to a destruction of the CD8 alpha binding site on the class I molecule, a hypothesis which is consistent with results obtained in conjugate binding assays. To assess the relative contribution to CTL activation of CD8 functioning as either a coreceptor or an accessory molecule, we have compared the ability of H-2Kb ovalbumin reactive CTL to lyse M12.C3 or T2 cells transfected with an H-2Kb gene encoding a wild type or mutant (CD8 nonbinding) alpha 3 domain. To establish that the substitution in the alpha 3 domain does not alter the ability of the H-2Kb molecule to bind the antigenic peptide, we have compared the binding of the ovalbumin derived H-2Kb restricted peptide (SIINFEKL) to T2 cells expressing either the CD8 binding or the CD8 nonbinding form of H-2Kb. This peptide conjugated with FITC bound equally well to T2 cells expressing either form of H-2Kb. Upon binding of this peptide, both forms of the H-2Kb molecule underwent the same conformational change as revealed by increases in the expression of particular serological epitopes. Furthermore, inhibition of the binding of the SIINFEKL peptide to both the wild type and mutant H-2Kb was observed following pretreatment of the cells with similar amounts of other H-2Kb restricted peptides derived from Sendai and Vesicular Stomatitis viruses. When the transfected M12 cells were tested for their ability to serve as targets for an anti-H-2Kb ovalbumin CTL clone, cells expressing the mutant H-2Kb molecule required the addition of 100-fold more exogenous peptide than did cells expressing the wild type molecule in order to obtain significant lysis. These data strengthen the previous hypothesis that CD8 functions much more efficiently as a coreceptor than as an accessory molecule for T cell effector function.

Amino Acid Sequence↗

PubMed1992

The B cell antigen receptor complex: association of Ig-alpha and Ig-beta with distinct cytoplasmic effectors.

The B cell antigen receptor complex is a hetero-oligomeric structure composed of antigen binding, membrane immunoglobulin, and transducer-transporter substructures. The transducer-transporter substructure is composed of disulfide-linked dimers of immunoglobulin (Ig)-alpha and Ig-beta/gamma subunits that are products of the mb-1(alpha) and B29 (beta/gamma) genes. Although the receptor complex associates with Src family kinases that are activated after receptor ligation, the site of interaction of these and other cytoplasmic effector molecules with receptor subunits is unknown. The cytoplasmic tails of Ig-alpha and Ig-beta chains were found to associate with distinct sets of effector molecules. The Ig-alpha chain cytoplasmic domain bound to the Src family kinases Lyn and Fyn, phosphatidylinositol-3 kinase (PI-3 kinase), and an unidentified 38-kilodalton phosphoprotein; the cytoplasmic tail of Ig-beta bound PI-3 kinase and unidentified 40- and 42-kilodalton phosphoproteins. Binding activity was found to occur within a 26-amino acid sequence of Ig-alpha and Ig-beta that contains a motif [(Asp or Glu)-(any amino acid)7-(Asp or Glu)-Tyr-(any amino acid)3-Leu-(any amino acid)7-Tyr-(any amino acid)2-(Leu or Ile)] previously implicated in signal transduction via other receptors including the Fc epsilon receptor I and the T cell antigen receptor. These findings indicate that the subunits act independently to activate distinct second messenger pathways.

Amino Acid Sequence↗

PubMed1991

Co-engagement of CD8 with the T cell receptor is required for negative selection.

Although it is established that the CD8 and CD4 co-receptors are involved in T-lymphocyte recognition and activation in the periphery, it is less clear whether these molecules participate in thymic selection events. Analysis of thymic selection in mice transgenic for T cell-receptor genes or for major histocompatibility complex (MHC) genes, or mice injected with antibodies against CD8, CD4 or MHC molecules, is consistent with the participation of CD8 and CD4 in thymic selection. But antibody-mediated crosslinking of surface receptors in thymic organ cultures has indicated that CD8 is not involved in thymic deletion. We show here that mice transgenic for a mutant MHC class I molecule that cannot interact with CD8 do not delete CD8-dependent T cells reactive with the wild-type molecule. This finding unequivocally establishes that for negative selection in the thymus, CD8 must interact with the same MHC class I molecule as the T cell receptor.

Animals↗

PubMed1990

The role of beta 2-microglobulin in peptide binding by class I molecules.

Efficient transport of class I major histocompatibility complex molecules to the cell surface requires association of the class I heavy chain with endogenous peptide and the class I light chain, beta 2-microglobulin (beta 2M). A mutant cell line deficient in beta 2M transports low amounts of nonpeptide-associated heavy chains to the cell surface that can associate with exogenously provided beta 2M and synthetic peptide antigens. Normal beta 2M-sufficient cells grown in serum-free media devoid of beta 2M also require an exogenous source of beta 2M to efficiently bind synthetic peptide. Thus, class I molecules on normal cells do not spontaneously bind or exchange peptides.

Abelson murine leukemia virus↗

PubMed1990

Recognition by CD8 on cytotoxic T lymphocytes is ablated by several substitutions in the class I alpha 3 domain: CD8 and the T-cell receptor recognize the same class I molecule.

The CD8 molecule on class I-reactive cytotoxic T lymphocytes (CTLs) is believed to function as a coreceptor along with the alpha beta T-cell receptor. Whereas the alpha beta T-cell receptor recognizes polymorphic residues in the alpha 1/alpha 2 domains of the class I molecule, the CD8 molecule is believed to recognize monomorphic class I residues. Our previous experiments suggested that residue 227 in the alpha 3 domain of major histocompatibility complex class I molecules contributes to the determinant recognized by CD8. By using a panel of site-directed mutants of H-2Dd, this observation has been extended herein. Our findings indicate that for recognition by CD8-dependent CTLs, residue 227 must be either glutamic acid or aspartic acid and cannot be either basic or uncharged. However, the recognition by CD8-independent CTLs is unaffected by any of the substitutions at position 227 of H-2Dd. Similarly, alterations of other charged residues at positions 222, 223, and 229 have an analogous effect to substitution at residue 227, whereas substitutions at residues 192 and 232 do not affect the reactivity of CD8-dependent or CD8-independent CTLs. In addition, mutant H-2Dd molecules that are not recognized by CD8-dependent CTLs are unable to stimulate a primary CTL response, yet they can stimulate a secondary CD8-independent H-2Dd-specific CTL response. These findings suggest that CD8 recognition is obligatory for the priming of class I-dependent CTL responses. Since endogenous class I molecules were expressed by all of the transfected cell lines, these findings provide direct genetic evidence that CD8 and the alpha beta T-cell receptor must interact with the same class I molecule.

Amino Acids↗

PubMed1989

Substitution at residue 227 of H-2 class I molecules abrogates recognition by CD8-dependent, but not CD8-independent, cytotoxic T lymphocytes.

The CD8 (Lyt 2) molecule is a phenotypic marker for T lymphocytes that recognize and react with major histocompatibility complex (MHC) class I molecules. Antibody blocking experiments and gene transfection studies indicate that CD8 binds to a determinant on MHC class I molecules on the target cells, facilitating interaction between effector T lymphocytes and the target cell. The CD8 molecule may also be involved in transmembrane signalling during T-cell activation. The existence of CD8- cytotoxic T lymphocytes (CTL) and class I-reactive CTL that are not inhibited by antibody to CD8 suggests that at least some CTL do not require the CD8 molecule to interact with and lyse target cells. We have recently demonstrated that cells transfected with an H-2Dd gene that carries a mutation at residue 227 are not killed by primary CTL8. Here we show that although this mutation abrogates recognition by primary CTL, it does not affect recognition by CD8-independent CTL, suggesting that residue 227 of class I molecules might contribute to a determinant that is the ligand of the CD8 molecule.

Animals↗

PubMed1989

Effect of proviral insertion on transcription of the murine B2mb gene.

We describe three B2mb mutants caused by integration of retroviral proviruses into the B2m gene. All three insertions occurred within 500 base pairs of each other in the 5' end of the first intron of the B2mb gene. Mutant cell line i1 contained a nonfunctional Abelson murine leukemia virus-initiated transcript that spliced into B2mb exon II; mutant cell lines i7 and i18 contained transcripts complementary to B2mb exon 1. Both i1 and i7 also contained small amounts of wild-type B2mb transcript. The implications of these studies for the development of retroviral insertional mutagenesis as a strategy for cloning previously uncloned genes are discussed.

Abelson murine leukemia virus↗

PubMed1988

The Lyt-2 molecule recognizes residues in the class I alpha 3 domain in allogeneic cytotoxic T cell responses.

The involvement of the different domains of the MHC class I molecule in CTL recognition was investigated. mAbs specific for the alpha 1/alpha 2 domains of H-2Ld interfered with both the primary and secondary generation and effector function of in vitro Ld-specific CTL. mAbs specific for the alpha 3 domain of H-2Ld interfered with the generation and function of primary in vitro Ld-specific CTL; however, there was no effect on the in vitro generation of secondary CTL and only partial inhibition of their function. In vivo treatment with graft-specific antibodies to both the alpha 3 domain and the alpha 1/alpha 2 domains together resulted in a dramatic enhancement of Ld- or Dd-disparate skin grafts, whereas the individual mAbs showed minimal effects. This suggested that the class I alpha 3 domain is recognized by alloreactive CTL. Several approaches were undertaken to examine whether recognition of the alpha 3 domain determinants is mediated by the Lyt-2 molecule. When mAbs specific for the alpha 3 domain of either H-2Ld or H-2Dd were used in vivo and in vitro, the resulting CTL population was not inhibited by antibody to the alpha 3 domain and was only partially inhibited by antibody to Lyt-2. We therefore observed a correlation between the effects of antibody to the class I alpha 3 domain of the target molecule and antibody to the Lyt-2 molecule on the CTL. To further test the relationship between CTL recognition of the alpha 3 domain and the involvement of Lyt-2, we used a cell expressing a mutation in the alpha 3 domain of the Dd molecule. The mutation resulted in a single amino acid substitution of glu to lys at residue 227 of the alpha 3 domain. Consistent with an earlier report, cells expressing the mutant Dd lys molecule were not lysed by CTL from a primary stimulation against the wild-type Dd glu molecule. However, this same cell line was killed by the Lyt-2-independent secondary Dd-specific CTL generated in the presence of antibody to the alpha 3 domain in vivo and in vitro. Furthermore, cells expressing the mutant Dd lys molecule failed to stimulate a primary response. In conclusion, several independent lines of evidence indicate that residues in the alpha 3 domain of the class I molecule are involved in recognition by the Lyt-2 molecule, and that Lyt-2-mediated recognition can be specifically blocked using mAb to determinants in the alpha 3 domain.

Animals↗

PubMed1987

A single amino acid substitution in the alpha 3 domain of an H-2 class I molecule abrogates reactivity with CTL.

We previously described a somatic cell expressing a variant H-2Dd molecule that did not serve as a target for alloreactive anti-Dd CTL. The mutant cell line had been isolated by its failure to express a serological epitope present on the H-2Dd alpha 3 domain. In the present study the alpha 3 domain of the Dd molecule of this somatic cell variant was sequenced and a single nucleotide change resulting in a glutamic acid to lysine substitution at residue 227 was identified. This change was reproduced in the cloned H-2Dd gene by oligonucleotide-directed mutagenesis. Cells transfected with this mutant gene were not killed by anti-H-2Dd CTL. Because previous studies using hybrid H-2 class I molecules had established that the alpha 3 domain does not express allele-specific determinants recognized by CTL, our results raise the possibility that residues in the alpha 3 domain of H-2 class I molecules are critical for CTL recognition and constitute a conserved (or monomorphic) determinant recognized by CTL.

Amino Acids↗

PubMed1987

Expression of hemopoietic histocompatibility antigens on H-2-loss variants of F1 hybrid lymphoma cells: evidence consistent with trans gene regulation.

H-2 heterozygous marrow stem cells, lymphoid progenitor cells, and leukemia/lymphoma cells do not express hemopoietic or hybrid histocompatibility (Hh) antigens, which are important transplantation antigens recognized during the rejection of normal or neoplastic hemopoietic cells. The Hh-1b determinant of the H-2b haplotype maps to the D region of H-2. We have tested the hypothesis that gene(s) at or near H-2D of the H-2d haplotype down-regulate the expression of Hh-1b in the trans configuration. We used Abelson leukemia virus-transformed pre-B lymphoma cells (ACCb) of BALB/c X BALB.B (H-2d X H-2b) origin, as well as variant lines of ACCb, which were selected for resistance to monoclonal anti-H-2 antibodies plus complement. B6D2F1 (H-2b X H-2d), C3B6F1 (H-2k X H-2b), or B6 (H-2b) mice were infused with inocula of 5 X 10(6) B6 bone marrow cells (BMC). Proliferation of donor-derived marrow cells was judged in terms of DNA synthesis by measuring the splenic incorporation of 5-iodo(125I)-2'-deoxyuridine (IUdR) 5 days after cell transfer. B6 BMC grew much better in B6 than in F1 hybrid host mice, an expression of "hybrid resistance". As observed previously, the injection of EL-4 (H-2b, Hh-1b) tumor cells prior to infusion of B6 (H-2b, Hh-1b) BMC enhanced the growth of B6 BMC in F1 hybrid mice. Therefore, this in vivo "cold target cell competition" type of assay can be used to detect the expression of Hh-1b antigens. Unlike EL-4 (H-2b) cells, hybrid resistance was not affected by prior infusion of (H-2b X H-2d) heterozygous ACCb cells. In contrast, three ACCb variant cell lines, H-2d-, Ld-Dd-, and Dd-, enhanced the growth of B6 BMC in F1 hosts. The ACCb H-2b- cell line did not affect hybrid resistance to B6 BMC. The loss of gene expression on the H-2d chromosome at or very near the H-2Dd locus is correlated with the appearance Hh-1b, as determined by the in vivo cold target competition assay. These results support the hypothesis that heterozygous cells possess trans-acting, dominant, down-regulatory genes mapping near H-2D that control the Hh-1 phenotype of lymphoid tumor cells.

Animals↗

PubMed1987

Spontaneous deletion at the B2m locus: evidence for site-specific genetic rearrangement.

We have isolated 20 independent spontaneous mutants in the B2mb allele from a B2ma/b heterozygous murine cell line by immunoselection in vitro with a monoclonal antibody directed against the product of the B2mb allele. One class of mutants has undergone a deletion in the 5' end of the B2mb gene. The deletions appear to be identical in all of the independent clones, and extend an unknown distance upstream of the B2m gene from a region in the first intron. Southern blot analysis with the use of oligonucleotides to the wild type gene sequence mapped the breakpoint to within 39 base pairs. The high frequency of independent spontaneous mutants showing indistinguishable deletions suggests that the first intron of the B2m gene contains sequences that are highly susceptible to site-specific recombinations.

Alleles↗

PubMed1987

Mitotic recombination between homologous chromosomes generates H-2 somatic cell variants in vitro.

A spontaneously arising variant clone that does not express the H-2Dd and H-2Ld molecules was isolated by immunoselection from an (H-2b X H-2d)F1 cell line. This variant clone expresses H-2Kb, H-2Db, and H-2Kd molecules. Southern blot analysis demonstrated that the variant was heterozygous at the H-2K, I, and S loci but had lost the H-2Dd and H-2Ld genes. Karyotype analysis showed that neither of the chromosome 17s in the variant had undergone detectable deletions. Quantitative Southern blot analysis demonstrated that the variant had two copies of the H-2Db gene, whereas the parental cell line had one copy of H-2Db. The loss of the H-2Ld and H-2Dd genes, accompanied by the attainment of homozygosity at H-2Db, is consistent with a recombination between the two chromosome 17 homologues. We conclude that although mitotic recombination between homologues has been difficult to demonstrate, it may not be infrequent and may account for the development of mutant genotypes in somatic cells in vitro. Such a mechanism occurring in vivo could result in the emergence of cells that are homozygous for deleterious alleles even though the individual may be constitutionally heterozygous.

Animals↗

PubMed1985

Retroviral insertional mutagenesis of a target allele in a heterozygous murine cell line.

A clonal murine cell line that is heterozygous at the beta 2-microglobulin locus (B2m) was obtained by Abelson murine leukemia virus (Ab-MuLV) transformation of liver cells from (C57BL/6 X BALB/c) F1 fetuses. To obtain proviral insertional mutants, we superinfected a subclone of these cells, which does not express the env surface protein of the Moloney leukemia virus (Mo-MuLV, the helper virus that was used to transmit the defective Ab-MuLV genome during transformation), with Mo-MuLV. Mutant clones that fail to express the C57BL/6 allele of B2m (B2mb) were then immunoselected by using a monoclonal antibody that specifically recognizes the B2mb gene product and not that of the B2ma allele. Of 22 independent clones obtained, one contains a proviral insertion that is near or in the first exon of the B2mb gene. Surprisingly, the insertion is of the Ab-MuLV genome and not of replication-competent Mo-MuLV. This indicates that superinfection with Mo-MuLV "rescued" the defective Ab-MuLV genome, which then inserted into the B2mb gene. We conclude that when an allele-specific selection procedure exists, proviral insertion is a potential method for obtaining mutations in heterozygous mammalian cells. This approach may thereby provide a method for molecular cloning of such selectable genes, using a retroviral hybridization probe.

Abelson murine leukemia virus↗

PubMed1985

Molecular analysis of an EL4 cell line that expresses H-2Db but not H-2Kb or beta 2-microglobulin.

EL4/Mar is a variant cell line that expresses H-2Db but neither H-2Kb nor beta 2-microglobulin (beta 2m). Southern and RNA blot analysis and immunoprecipitation of metabolically labeled proteins established that the B2m gene(s), beta 2m mRNA, and beta 2m protein are normal in this cell line. Somatic cell hybridization showed that the defect in this cell line was in the synthesis of H-2Kb, and RNA blot analysis with an H-2Kb specific oligonucleotide established that the H-2Kb gene(s) in this cell line was not transcribed into a stable mRNA species. The apparent absence of beta 2m on the surface of this cell line suggests that there may be some feature of the H-2Db molecule that allows it to be expressed in the absence of detectable beta 2m.

Animals↗