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A Townsend

Publications and source records attributed to A Townsend.

At least 37 records · Page 2Linked to original sources
PubMed1995

Processing of major histocompatibility class I-restricted antigens in the endoplasmic reticulum.

We have introduced long precursor peptides directly into the endoplasmic reticulum (ER) of a mutant cell line (T2-Db) that lacks the ability to transport peptides from the cytosol to the ER in a transporter associated with antigen processing (TAP) dependent way. This was done by expressing various influenza A-derived peptides containing the naturally processed epitope ASNENMDAM (366-374) preceded by the influenza hemagglutinin ER translocation sequence. Peptides derived from these minigenes that became associated with Db were isolated and identified by combined reversed phase liquid chromatography and detection by cytotoxic T lymphocytes. Our results establish that NH2-terminal extensions of at least 40 residues can be trimmed from peptides entering the ER, but that proteolysis of larger proteins may be limited.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Expression of TAP1 by human trophoblast.

Successful placentation in the human is dependent on the trophoblast evading recognition and destruction by the maternal immune system. However, invasive cytotrophoblast express HLA-G which may be able to present peptide to T cells. Transporter proteins are essential for peptide presentation and major histocompatibility complex (MHC) class I assembly. We have determined their expression by trophoblast in relation to HLA-G, using immunohistochemistry. Anti-transporter protein antibody (TAP1) labeling closely paralleled that of MHC class I, but the intensity of its expression was much greater on the HLA-G+ extravillous cytotrophoblast than any other fetal or maternal tissue in the first trimester and at term. This suggests that the extravillous cytotrophoblast are very actively assembling MHC class I antigens with peptides. However, expression of MHC class I by the cytotrophoblast was not correspondingly elevated. This pattern could result from HLA-G being shed from the surface of the trophoblast, a process which may play a central role in protecting the fetus from maternal immune attack.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Genes encoded in the major histocompatibility complex affecting the generation of peptides for TAP transport.

The B cell line 721.174 has lost the ability to present intracellular antigens to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL). This phenotype results from a homozygous deletion in the MHC that includes the peptide transporter genes TAP1 and TAP2, and the proteasome subunits LMP2 and LMP7. Recent work has shown that such cells transfected with TAP genes load their class I molecules with endogenous peptides, and present several viral epitopes to class I-restricted CTL. These data implied that the LMP2 and LMP7 genes were not required for the presentation of most epitopes through class I molecules. By contrast, while confirming the previous reports, we have identified several epitopes that appear to require genes in the MHC in addition to the TAP for their presentation. Further analysis localizes the defect to proteolysis in the cytosol. In one case, presentation could be partially restored by re-expression of full-length LMP7. Control experiments with LMP7, from which the putative pro-region had been removed, failed to restore presentation, and this lack of effect correlated with failure of the shortened LMP7 to incorporate into the proteasome. These results suggest a role for LMP7 in the generation of a viral epitope, but leave open the possibility that additional genes within the .174 deletion are required for full restoration of antigen presentation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Elderly outpatients' understanding of a physician-initiated advance directive discussion.

OBJECTIVE: To examine elderly outpatients' understanding of advance directives (ADs), cardiopulmonary resuscitation (CPR), and artificial nutrition and hydration (ANH) with and without the benefit of a physician-initiated discussion. DESIGN: Randomized controlled trial. SETTING: University-affiliated, community-based, urban family practice residency training program. PATIENTS: One hundred patients aged 65 and older, consecutively sampled and randomly assigned to one of two discussion groups. INTERVENTIONS: Physicians' discussions based on a prepared script consisting of AD issues or health promotion issues. MAIN OUTCOME MEASURES: Test of comprehension of AD, CPR, and ANH information, using open-ended and yes-or-no questions. RESULTS: Patients in the AD and health promotion discussion groups showed good basic understanding. Younger and better-educated patients had a better working knowledge of AD-related information. Understanding of ADs was higher when the physician spent more time talking about AD-related issues after the discussion was completed. CONCLUSIONS: Many elderly outpatients have a good basic understanding of ADs, CPR, and ANH, even without explicit explanations from physicians. However, younger, better-educated patients and those who had longer unstructured discussions had greater AD-related knowledge. These factors need to be considered when framing discussions with patients about ADs and life-sustaining treatments.

Advance Directives↗
PubMed1994

A peptide encoded by human gene MAGE-3 and presented by HLA-A2 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE-3.

The human MAGE-3 gene is expressed in many tumors of several histological types but it is silent in normal tissues, with the exception of testis. Antigens encoded by MAGE-3 may, therefore, be useful targets for specific anti-tumor immunization of cancer patients. We reported previously that MAGE-3 codes for an antigenic peptide recognized on a melanoma cell line by autologous cytolytic T lymphocytes (CTL) restricted by HLA-A1. Here we report that the MAGE-3 gene also codes for another antigenic peptide that is recognized by CTL restricted by HLA-A2. MAGE-3 peptides bearing consensus anchor residues for HLA-A2 were synthesized and tested for binding. T lymphocytes from normal individuals were stimulated with autologous irradiated lymphoblasts pulsed with each of three peptides that showed strong binding to HLA-A2. Peptide FLWGPRALV was able to induce CTL. We obtained CTL clones that recognized not only HLA-A2 cells pulsed with this peptide but also HLA-A2 tumor cell lines expressing the MAGE-3 gene. The proportion of melanoma tumors expressing this antigen should be approximately 32% in Caucasian populations, since 49% of individuals carry the HLA-A2 allele and 65% of melanomas express MAGE-3.

Amino Acid Sequence↗
PubMed1994

Loss of major histocompatibility complex-encoded transporter associated with antigen presentation (TAP) in colorectal cancer.

Presentation of endogenous antigenic peptides to cytotoxic T lymphocytes (CTLs) is mediated by the major histocompatibility complex (MHC) class I molecules. These antigenic peptides derived from the cytoplasmic protein pool are transported by the recently described MHC-encoded transporters (TAP1 and TAP2) into a pre-Golgi region where they take part in the assembly of MHC class I molecules. Using an affinity-purified polyclonal antibody (AK1.7) for TAP1, we analyzed 81 colorectal carcinomas, 32 adenomas, and the respective nonneoplastic mucosa. Loss of the transporter molecule (TAP1) was observed in 14% (11 of 81) of the carcinomas, either complete (7 of 11) or focal (4 of 11), whereas adenomas and normal mucosa were always positive. This study adds further information to the understanding of the mechanisms related to the defective presentation of the MHC class I molecules by tumor cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

A method to quantify binding of unlabeled peptides to class I MHC molecules and detect their allele specificity.

A general method has been developed for measuring the stabilization of class I MHC molecules in extracts of the mutant cell lines .174/T2 and RMA-S. 35S-Met-labeled class I molecules which have been stabilized by peptides in vitro are immunoprecipitated with conformation dependent monoclonal antibodies and electrophoresed on polyacrylamide gels. The heavy and light chains are excised from the dried gel and quantified on a flat bed scintillation counter. The stabilizing effect of peptides on class I molecules in vitro correlates well with peptide binding measured by direct methods and can be therefore used to assess peptide binding affinity. We show that a peptide from HIV-1 gag (which has a high affinity for Db) is a CTL epitope restricted through Db, and also use the assay to analyse the effects of amino acid substitution on peptide affinity. In addition, the effect of a given peptide on a class I molecule within a mixture of human class I molecules can be distinguished by immunoprecipitation with the monomorphic antibody W6/32 and separation by 1-D isoelectric focussing. The technique therefore requires neither labeled peptide ligands nor allele-specific antibodies. It can be used to identify the peptide ligand of any human class I molecule, and gives a measure of peptide binding affinity. The technique should be of value in identifying epitopes recognized by CTL since we have found that these tend to bind with the highest affinities.

Alleles↗
PubMed1993

The transporters associated with antigen presentation.

Two new members of the ABC superfamily of transporter genes have recently been identified within the Major Histocompatibility Complex in man, rat and mouse. Although the exact function of these genes is not known, they have been shown to be necessary for the presentation of peptides derived from the degradation of cytoplasmic protein antigens to the cellular immune system. For this reason they have been named TAP1 and TAP2 (for Transporter associated with Antigen Presentation). Each gene encodes one membrane spanning domain and one region homologous to the ATP binding domains that characterise the superfamily. The two proteins encoded by the TAP genes form a complex that is localised to the membranes of the endoplasmic reticulum and cis-golgi. Their most likely function is to transport short peptides, that lack signal sequences, from the cytoplasm to the endoplasmic reticulum, although the evidence for this is still indirect.

Adenosine Triphosphate↗
PubMed1993

Physicians' predictions of elderly outpatients' preferences for life-sustaining treatment.

BACKGROUND: Research has shown that physicians are poor predictors of patients' life-sustaining treatment preferences. Our study examined the association between three aspects of physician experience and their ability to accurately predict patients' preferences for two different life-sustaining treatments in the event of two serious medical conditions. METHODS: Seventeen physicians predicted the treatment preferences of 57 patients and then interviewed patients regarding their actual treatment preferences. Physicians' professional experience, length of their relationship with the patient, and experience with direct feedback were measured to determine the association of these factors with the accuracy of the physicians' predictions. RESULTS: Physicians became more accurate predictors as they interviewed more patients and received direct feedback regarding the accuracy of their predictions (P < .001). Residents were more accurate than faculty in predicting patients' preferences (P < .05). CONCLUSIONS: Increased experience with life-sustaining treatment discussions improved the physicians' abilities to accurately predict patient preferences. Although possibly resulting from small sample size, neither greater professional experience nor longer relationship with a patient improved the accuracy of physicians' predictions. Future research should examine whether discussing end-of-life issues with patients more often makes physicians more sensitive predictors of patients' life-sustaining treatment preferences.

Aged↗
PubMed1992

Location of MHC-encoded transporters in the endoplasmic reticulum and cis-Golgi.

Immune recognition of intracellular proteins is mediated by major histocompatibility complex (MHC) class I molecules that present short peptides to cytotoxic T cells. Evidence suggests that peptides arise by cleavage of proteins in the cytoplasm and are transported by a signal-independent mechanism into a pre-Golgi region of the cell, where they take part in the assembly of class I heavy chains with beta 2-microglobulin (reviewed in refs 5-7). Analysis of cells that have defects in class I molecule assembly and antigen presentation has shown that this phenotype can result from mutations in either of the two ABC transporter genes located in the class II region of the MHC. This suggested that the protein complex encoded by these two genes transports peptides from the cytosol into the endoplasmic reticulum. Here we report additional evidence by showing that the transporter complex is located in the endoplasmic reticulum membrane and is probably oriented with its ATP-binding domains in the cytosol.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Assembly and function of the two ABC transporter proteins encoded in the human major histocompatibility complex.

Presentation of cytoplasmic antigens to class I-restricted cytotoxic T cells implied the existence of a specialized peptide transporter. For most class I heavy chains, association with peptides of the appropriate length is required for stable assembly with beta 2-microglobulin. Mutant cells RMA-S and .174/T2 neither assemble stable class I molecules nor present intracellular antigens, and we have suggested that they have lost a function required for the transport of short peptides from the cytosol to the endoplasmic reticulum. The genetic defect in .174 has been localized to a large deletion in the class II region of the major histocompatibility complex, within which two genes (RING4 and RING11) have been identified that code for 'ABC' (ATP-binding cassette) transporters. We report here that the protein products of these two genes assemble to form a complex. Defects in either protein result in the formation of unstable class I molecules and loss of presentation of intracellular antigens. The molecular defect in a new mutant, BM36.1, is shown to be in the ATP-binding domain of the RING11/PSF2 protein. This is in contrast to the mutant .134, which lacks the RING4/PSF1 protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Presentation of viral antigen by MHC class I molecules is dependent on a putative peptide transporter heterodimer.

Major histocompatibility complex (MHC) class I molecules present peptides derived from the endogenous protein pool to cytotoxic T lymphocytes, which can thus recognize intracellular antigen. This pathway may depend on a transporter (PSF1) to mediate entry of the cytosolic peptides into a pre-Golgi compartment where they bind to class I heavy chains and promote their stable assembly with beta 2-microglobulin. There is, however, only indirect support for this function of PSF1. Here we show that PSF1 is necessary for the efficient assembly of class I molecules and enables them to present a peptide epitope derived from endogenously synthesized viral antigen. Immunochemical and genetic data demonstrate that the PSF1 polypeptide is associated with a complementary transporter chain, which is polymorphic and is encoded by the PSF2 gene, which is closely linked to PSF1.

Antigens, Viral↗
PubMed1992

A defect in the presentation of intracellular viral antigens is restored by interferon-gamma in cell lines with impaired major histocompatibility complex class I assembly.

Surface expression of the majority of class I major histocompatibility complex (MHC) heavy chains is known to require assembly with beta 2 microglobulin (beta 2m). To define other factors involved in class I MHC assembly, we have studied two tumor cell lines that are deficient in cell surface class I (H-2) expression. The BC2 fibrosarcoma and the CMT lung carcinoma express only intracellular unassociated heavy chains despite the presence of beta 2m. As described previously, when these cell lines are treated with interferon-gamma (IFN-gamma), they are capable of assembling and transporting class I molecules to the cell surface. In this study, we have shown that in the absence of IFN-gamma these mutant cells are unable to present intracellular viral antigens, although they can be lysed by specific cytotoxic T lymphocyte (CTL) after pre-incubation with the corresponding synthetic peptide. Flow cytometric analysis demonstrated that extracellular peptide was capable of increasing twofold the surface expression of beta 2m-heavy chain complexes. Furthermore, immunoprecipitation experiments confirmed that peptide stabilizes chain association in the BC2 cell lysates. However, infecting these mutants with vectors expressing either pre-processed antigen or rapidly degraded antigen, failed to overcome their defect in the presentation of endogenous peptide to specific CTL or to mediate surface expression of class I MHC. Preincubation with IFN-gamma completely reversed the endogenous peptide presentation defect, even in mutant cells transfected with a vector encoding a cDNA for the H-2 molecule restricting CTL recognition. This last result suggests that IFN-gamma corrects the defect by a mechanism separate from simple enhancement of the number of class I molecules produced by the cell. Because there is growing evidence that endogenous peptides can participate in class I MHC assembly, the defect in these mutants could be ascribed to the lack of access to class I molecules by the endogenous peptide. This would prevent stable association of the heavy and light chains and their subsequent transport. Our data suggests that IFN-gamma reestablishes class I MHC surface expression by restoring access of endogenously synthesized peptide to class I molecules.

Amino Acid Sequence↗
PubMed1992

Structural requirements for the peptide-induced conformational change of free major histocompatibility complex class I heavy chains.

In an attempt to define the structural features of peptides which are important for inducing the folding of free class I heavy chains in the absence of beta 2-microglobulin, and to determine whether they are the same as those required to form stable major histocompatibility complex (MHC): peptide adducts, we have used a panel of peptides related to the Db-binding nonamer ASNENMDAM (influenza nucleoprotein residues 366-374) with altered primary structures, and a number of other peptides which have the Db-binding "motif". In this way, we have shown that in addition to the "anchor" residues which define this motif, the alpha amino and carboxyl groups at the N and C termini also play a major role in both inducing the conformational change in free heavy chain (HC) and formation of a stable Db:peptide complex. We also show that the importance of the key residues is affected by the primary sequence "context" in which they appear. In addition, we have extended our original finding that naturally processed epitopes induce a conformational change in free HC to the H2Kb HC, and show that the effect does not require the presence of the class I alpha 3 domain.

Amino Acid Sequence↗
PubMed1992

Association of the human invariant chain with H-2 Db class I molecules.

We describe two proteins of 24 kDa and 33 kDa (p24 and p33) which associate with H-2 Kb and H-2 Db molecules, respectively, in human cells transfected with H-2 Kb and H-2 Db genes. This association is particularly clear in the mutant cell line T2, in which association of endogenous peptide with newly synthesized class I molecules may not occur (V. Cerundolo et al., Nature 1990. 345: 449). We show that p33 is the 33-kDa form of the human invariant chain which is resident in the endoplasmic reticulum of T2 cells (P. Cresswell, Cold Spring Harbor Symp. Quant. Biol. 1989. LIV:309). The stability of the invariant chain H-2 Db complex is critically dependent upon occupation of the class I binding site by peptide ligand. In the absence of peptide, the complex is stable at 4 degrees C whereas following exposure to peptide, the invariant chain dissociates rapidly from H-2 Db molecules (half-life of 30 min at 4 degrees C). Although the interaction between the human invariant chain and murine H-2 Db is unlikely to have any functional significance, the peptide-induced dissociation of the invariant chain is consistent with a conformational change in H-2 Db on peptide binding.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1992

Short peptides assist the folding of free class I heavy chains in solution.

Previous experiments have shown that short peptides coresponding to naturally processed epitopes of viral antigens can induce a conformational change in the class I heavy chain (HC) to which they bind in the fully assembled molecule. Here, we present evidence that the mechanism for this conformational change may involve binding of peptide to a partially unfolded form of free HC, followed by its subsequent folding. These results may be important for understanding the way in which class I molecules are assembled in vivo, and how certain epitopes are selected for presentation to T cells.

Amino Acid Sequence↗