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Biomedical subjects

J McCluskey

Publications and source records attributed to J McCluskey.

At least 109 records · Page 6Linked to original sources
PubMed1991

T cell receptor and immunoglobulin gene rearrangement analysis as a laboratory aid in the diagnosis of human malignant lymphoproliferative diseases.

The identification of clonal rearrangements of the immunoglobulin heavy chain and T cell receptor beta chain gene loci by Southern blot analysis has led to advances in the diagnosis and classification of lymphoproliferative disorders. This paper reviews our experience with this technique over a three and a half year period. Specimens from 99 patients with suspected haematological malignancy were tested for leucocyte immunophenotype and immunoglobulin or T cell receptor gene rearrangement. Genotyping provided evidence of clonality in malignancies from 28 patients and demonstrated malignant cell lineage in eight patients not formally deduced from immunophenotyping alone. Our findings suggest that this technique can be employed in conjunction with immunophenotyping to aid in the determination of malignant cell lineage derivation and identification of malignant cell clonality, as well as potentially estimating the extent of disease, detecting relapse, and monitoring disease progression.

Gene Rearrangement↗
PubMed1990

Expression of human immunodeficiency virus 1 (HIV-1) envelope gene products transcribed from a heterologous promoter. Kinetics of HIV-1 envelope processing in transfected cells.

The expression of human immunodeficiency virus 1 (HIV-1) envelope glycoprotein products was studied in cells transfected with env gene constructs transcribed from an SV40 promoter. Gene constructs possessing the complete tat, rev (tat+ rev+) and env genes were transiently expressed in COS-1 cells as precursor SU-TM (gp160), SU.TM (gp120 x 41), and nucleolar rev protein. In addition, envelope glycoprotein was detected on the surface of those transfected COS-1 cells expressing abundant levels of env protein. Transfected constructs possessing a mutated tat translational initiation codon (tat-rev+) were expressed in COS-1 cells with at least a 10-fold increase in the level of envelope glycoprotein expression compared to the analogous constructs with an intact tat AUG codon (tat+ rev+). Mutation of the rev initiation codon (tat+ rev-) and (tat-rev-) resulted in no detectable expression of env products but expression of these proteins could be rescued by co-transfection of a cDNA encoding the rev gene. Subgenomic tat/rev transcripts were detected following transfection of all of the gene constructs indicating splicing of the env mRNAs transcribed from a heterologous promoter. Unspliced env transcripts were only detected in the cytoplasm of cells transfected with (rev+) constructs or with the (tat- rev-) construct in the presence of the rev cDNA supplied in trans. In contrast, unspliced transcripts were detected in the nuclear and total cellular RNA of all transfected cells. Expression of rev protein was localized to the nucleolus of transfected COS-1 cells. These results indicate that the export of unspliced env mRNA to the cytoplasm is facilitated by the expression of rev. Following env synthesis, the conversion of SU-TM (gp160) to SU.TM (gp120 x 41) was not quantitative. After a 20-h pulse-chase, only 40% of the SU-TM (gp160) present at the start of the chase period was subsequently accountable as mature SU (gp120), and approximately 30% of the detectable SU (gp120) was found in the culture medium of transfected COS-1 cells. The findings indicate that the surface expression of SU.TM (gp120 x 41) derived from heterologous gene transcripts is modulated (i) the co-expression of rev, (ii) the efficiency of proteolytic processing of SU-TM (gp160), and (iii) the degree of SU (gp120) shedding and/or secretion from the cell.

Animals↗
PubMed1990

The extracellular domains of MHC class II molecules determine their processing requirements for antigen presentation.

We have evaluated the relative contributions of the extracellular and cytoplasmic domains of MHC class II molecules in determining the Ag-processing requirements for class II-restricted Ag presentation to T cells. Hybrid genes were constructed to encode a heterodimeric I-Ak molecule in which the extracellular portion of the molecule resembled wild type I-Ak but where the connecting stalk, transmembrane and cytoplasmic domains of both the alpha- and beta-chain were derived from the class I molecule H-2Dd. Mutant I-Ak molecules were expressed as heterodimeric membrane glycoproteins reactive with mAb specific for wild type I-Ak. Fibroblast and B lymphoma cells expressing either wild type or mutant I-Ak molecules were able to process and present hen egg lysozyme (HEL) and conalbumin to Ag-specific, I-Ak-restricted, T cell hybridomas or clones. The mutant-expressing cells presented native and peptide Ag less efficiently than the wild type-expressing cells, suggesting that the disparity in presentation efficiency was not due to a difference in Ag processing. CD4 interaction was intact on the mutant I-Ak molecules. Presentation of native Ag by mutant and wild type-I-Ak-expressing cells was abolished by preincubation with chloroquine, or after paraformaldehyde fixation. After transfection of a cDNA encoding the gene for HEL, neither mutant nor wild type-I-Ak-expressing cells presented endogenously synthesized HEL to a specific T hybrid. Newly synthesized mutant I-Ak molecules were associated with invariant chain. These data demonstrate the ability of hybrid class II molecules to associate intracellularly with invariant chain and degraded foreign Ag in a conventional class II-restricted processing pathway indicating that the extracellular domains of class II molecules play a dominant role in controlling these Ag-processing requirements.

Animals↗
PubMed1990

Structure function analysis of in vitro mutated CD4 and major histocompatibility complex class II gene products.

The Class II major histocompatibility molecules are implicated in the initiation of antigen-driven autoimmune responses by CD4-positive T cells. In order to study the structure and function of CD4 and MHC Class II molecules, strategies were developed with the intent of generating secreted forms of these molecules by in vitro mutagenesis of the respective genes. A full length cDNA encoding an expressible human CD4 molecule was mutagenized to introduce a premature stop codon corresponding to residue 367 located 8 amino acids amino terminal to the start of the predicted trans-membrane region. Following DNA-mediated gene transfer of the mutant gene, secreted CD4 was detected in the supernatant of transiently transfected COS-1 cells. Surface expression of the membrane-bound form of CD4 was detected under the same conditions. In an attempt to create a secreted form of the mouse Class II molecule I-Ak the exons encoding the connecting stalk, transmembrane and cytoplasmic domains of both the alpha and beta chains were replaced by the corresponding exons from the gene encoding a secreted Class I-like molecule, Q10b. Transfer of these genes into mouse L cells failed to generate detectable secreted I-A molecules. In view of the secretion of CD4 reported in other mutagenesis studies, it is concluded that very subtle differences in the structure of the COOH-terminus can influence the folding, solubility or transport of the CD4 molecule. In addition, the assembly of heterodimeric Class II molecules may require membrane anchorage of the separate chains or some other contribution from the COOH-terminal domains of the alpha and beta chains.

Animals↗
PubMed1989

Autoantibodies to neurons and to the cytoskeleton in small cell carcinoma with paraneoplastic sensory neuropathy.

Autoantibodies to neurons and to the cytoskeleton were demonstrated in the serum of a patient with small cell carcinoma of the lung (SCCL) and paraneoplastic sensory neuropathy. The serum reacted by immunofluorescence with the nuclei of neurons, and with cytochalasin B-sensitive "stress fibres" of cultured cells. The serum also reacted by immunofluorescence with the nuclei of some cultured cell lines. Immunoblotting experiments with brain tissue, with SCCL, HeLa and other cultured cells showed that the serum reacted with 1-4 bands of 35-39 kDa apparent m.w. Two dimensional immunoblotting showed that these molecules had a neutral pI. Antibodies, affinity-purified by elution from the 35-39 kDa bands, gave staining of the nuclei of neurons and of cultured cells and immunoblotted the same 35-39 kDa antigens. These observations show that the anti-neuronal autoantibody reacts not only with neurons and with SCCL but also with some cultured cell lines. Molecular mimicry has been invoked as the basis for the reactivity of this autoantibody with exposed epitopes on SCCL and on neurons.

Animals↗
PubMed1988

T cell activation by purified, soluble, class I MHC molecules. Requirement for polyvalency.

To examine the nature of the interaction of the TCR with the MHC class I Ag, we have studied the stimulation requirements of an H-2Dd-reactive T cell hybridoma, using a homogeneous, purified preparation of a molecularly engineered soluble counterpart of the class I Ag, H-2Dd/Q10b. We demonstrate that this monovalent, soluble MHC Ag is incapable of stimulating the release of IL-2 from this T cell hybridoma. However, the same preparation of the purified protein can elicit a dose-dependent response when made multivalent either by covalent coupling to soluble, high m.w. dextran or to agarose beads, or by adsorption to polystyrene tissue culture plates.

Animals↗
PubMed1988

Cell surface expression of the amino-terminal domain of A kappa alpha. Recognition of an isolated MHC antigenic structure by allospecific T cells but not alloantibodies.

To evaluate the relationship between structure and function of the alpha 1 and beta 1 domains of class II MHC molecules, a strategy has been developed whereby isolated alpha 1 or beta 1 domains can be expressed on the surface of transfected mouse L cells as a part of a hybrid class II/class I molecule. The construction and expression of a chimeric class II/class I gene encoding the first two exons of A kappa alpha linked to the C2, TM and cytoplasmic exons of H-2Dd (A kappa alpha 1/DdC2) are described. High levels of A kappa alpha 1/DdC2 protein were detected on transfected L cells with a DdC2-specific mAb. An anti-Ia xenoantiserum specifically bound to A kappa alpha 1/DdC2-transfected L cells, although 12 different mAb reactive against I-A kappa alpha and an anti-Ia kappa alloantiserum did not bind these cells. However, alloreactive Ia kappa-specific CTL lines were able specifically to lyse cells expressing the hybrid A kappa alpha 1/DdC2 molecule. This indicates that the isolated A kappa alpha 1 domain preserves some of the structure of the native molecule and demonstrates the recognition by T cells of domain-specific A alpha allodeterminants.

Animals↗
PubMed1988

Signals controlling alternative splicing of major histocompatibility complex H-2 class I pre-mRNA.

The use of alternative splice acceptor sites during the removal of intron 7 in pre-mRNA splicing produces two forms of H-2Kb protein: the predominant form, derived from a transcript that has spliced at the upstream splice acceptor site for exon 8 (long exon 8), and a Kb molecule derived from a transcript that has spliced at the downstream acceptor site for exon 8 (short exon 8). We have identified a potential lariat branch point adenosine for the upstream acceptor splice site. This adenosine is found 28 bp from the splice junction and is contained in the sequence AGTGATGG. D-region genes, which use only the downstream splice site, have the sequence AGTGGTGG. We have used in vitro mutagenesis to change this A of the H-2Kb gene to G and have made the reciprocal change in H-2Dd. Elimination of this adenosine in H-2Kb alters the pattern of pre-mRNA splicing and results in a predominance of the Kb molecules with short exon 8 encoded sequences. However, the addition of an adenosine in H-2Dd is not sufficient to direct splicing to the upstream site.

Animals↗
PubMed1986

Analysis of hybrid H-2D and L antigens with reciprocally mismatched aminoterminal domains: functional T cell recognition requires preservation of fine structural determinants.

Studies of immune recognition of hybrid class I antigens expressed on transfected cells have revealed an apparent general requirement that the N(alpha 1) and C1(alpha 2) domains be derived from the same gene in order to preserve recognition by virus-specific H-2-restricted and allospecific T cells. One exception has been the hybrid DL antigen in which the N domain of H-2Ld has been replaced by that of H-2Dd. Cells bearing this molecule serve as targets for some virus and allospecific CTL. Because cells expressing the reciprocal hybrid LD (N domain of H-2Dd replaced by that of H-2Ld) antigen have not been available, it has not been possible to evaluate whether this exception stemmed from the relatedness of H-2Ld and H-2Dd or whether the DL antigen fortuitously preserved some function of the parent molecule as a rare exception. To assess this question, and to evaluate the contribution of the N and C1 domains of H-2Ld and H-2Dd to serologic and T cell recognition, we have constructed the reciprocal chimeric gene pLD (the N exon of H-2Ld substituted for that of H-2Dd), introduced this into mouse L cells by DNA-mediated gene transfer, and analyzed the expressed product biochemically, serologically, and functionally. Transformant L cells expressing either LD or DL antigens were both reactive with a number of anti-H-2Ld or anti-H-2Dd N/C1-specific monoclonal antibodies, indicating the preservation in the hybrid molecules of determinants controlled by discrete domains. Mab binding was generally greater with cells expressing hybrid DL antigen than with those transformants expressing LD molecules. Moreover, the amount of beta 2M associated with DL antigens was more than that associated with LD. Cells expressing hybrid DL antigens were recognized as targets by bulk and cloned allospecific anti-H-2Dd and anti-H-2Ld CTL, whereas cells expressing LD molecules were not recognized by any of the T cells tested. VSV-specific H-2Ld-restricted CTL failed to lyse VSV-infected targets expressing either DL or LD. These results indicate that T cell reactivity of cells expressing the DL hybrid antigen is an exception to the observed general requirement for class I antigens to possess matched N and C1 domains for functional T cell recognition by T cells restricted to parental antigens.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1986

Serologic and T cell recognition of truncated transplantation antigens encoded by in vitro deleted class I major histocompatibility genes.

Gene constructions encoding a leader sequence and the C2, transmembrane, and intracytoplasmic exons of both H-2Ld (pLd-C2) and H-2Dd (pDd-C2) were expressed as cell surface proteins when stably introduced into mouse L cells. Distinct proteins of 17.5 and 16 kd precipitated from pDd-C2 transformants reacted with a C2 domain-specific monoclonal antibody, whereas the pLd-C2 transformants expressed a single 19 kd glycoprotein. A xenoantiserum specific for the exon VI/VIII fusion peptide of H-2Dd precipitated only the 16 kd product, suggesting that this protein was derived from an alternately spliced transcript lacking exon VII of the native gene. CTL specifically recognized the pLd-C2 and pDd-C2 gene products, indicating the existence of CTL reactive against polymorphic determinants in the C2 domain of class I antigens.

Animals↗
PubMed1986

Alternative processing of H-2Dd pre-mRNAs results in membrane expression of differentially phosphorylated protein products.

Two distinct mRNA species encoding the mouse major histocompatibility antigen H-2Dd have been identified in BALB/c spleen cells as well as in cultured cell lines expressing this cell surface glycoprotein. The alternate transcripts of H-2Dd arise from either removal or inclusion of exon VII (encoding I2) during pre-mRNA processing. The relative levels of each kind of H-2Dd transcript varied considerably between different cell types, and in all cells examined both forms of alloantigen were expressed on the cell membrane. Antigen derived from both types of transcript reacted with H-2Dd-specific monoclonal antibodies, whereas only protein lacking the 13 amino acids of I2 reacted with a specific antiserum raised against a predicted exon VI/VIII fusion peptide. Those H-2Dd proteins translated from full length, but not smaller, transcripts were phosphorylated in resting and phorbol myristate acetate-stimulated BALB/c spleen cells, suggesting that the major site of in vivo phosphorylation is within the highly conserved sequence encoded by exon VII. Thus alternative splicing of pre-mRNA transcripts is a mechanism which leads to membrane expression of two forms of H-2Dd, one of which lacks a major site of phosphorylation.

Animals↗
PubMed1986

Genetic engineering of an H-2Dd/Q10b chimeric histocompatibility antigen: purification of soluble protein from transformant cell supernatants.

We have constructed a recombinant class I gene in which 5' sequences of H-2Dd are linked to the 3' half of a Qa subregion gene, Q10b. This hybrid gene would be expected to direct the synthesis of a protein containing the N and C1 domains of H-2Dd covalently linked to the C2 domain of the secreted, nonpolymorphic, Q10b antigen. Following DNA-mediated gene transfer into mouse L cells, transformants were analyzed by radiolabeling and immunoprecipitation. These cells secreted a molecule reactive with anti-H-2Dd monoclonal antibodies that identify epitopes on the N and C1 domains as well as with an anti-Q10 carboxyl-terminal peptide antiserum. The H-2Dd-derived antigen is associated with beta 2-microglobulin and is readily purified in milligram amounts from culture supernatants by immunoaffinity chromatography.

Animals↗
PubMed1986

Alternative protein products with different carboxyl termini from a single class I gene, H-2Kb.

In addition to mRNA encoding the canonical form of the murine class I antigen H-2Kb (348 amino acid residues), mRNA that would encode a shortened form of H-2Kb (missing 9 amino acids from the C-terminus) has been identified in C57BL/6 spleen cells by RNase-protection studies. The alternative transcripts of H-2Kb arise through the use of different AG acceptor splice sites for exon VIII. The existence of a shortened H-2Kb protein was demonstrated by sequential immunoprecipitation. Lysates of spleen cells that had been labeled with [35S]methionine and [3H]histidine were precleared with rabbit anti-peptide serum reactive with the C-terminus of the canonical H-2Kb. The shortened form of H-2Kb was immunoprecipitated from this lysate with H-2Kb alloantiserum. Both forms of H-2Kb were isolated by NaDodSO4/PAGE. Tryptic peptide mapping confirmed that these molecules differed only at their C-terminus. The shortened form of H-2Kb is also found in a B-cell line (R8) but not in three cloned T-cell lines or in a T-cell lymphoma (EL4), suggesting that regulatory events are involved in producing the two forms of H-2Kb. Putative lariat branch points involved in these alternative splicing events for the 3' coding region of H-2 class I pre-mRNAs are proposed.

Animals↗
PubMed1986

An immunogenetic study of juvenile chronic arthritis.

Ninety-seven patients from Western Australia and Victoria suffering from juvenile chronic arthritis (JCA) for longer than 1 year were studied with particular reference to immunogenetic markers in the sub-groups of the disease. In the pauci-articular group, the antigens DR5, DR8 and BW35 were increased in frequency. The phenotypes within the sub-groups appeared distinctive despite the absence of any increase in the frequency of single antigens. Complement allotyping did not prove to be different from controls or within the sub-groups for JCA. This study supports the concept that patients with JCA have specific genetic characteristics and that sub-groups of the disease are genetically distinct.

Arthritis, Juvenile↗