Biomedical subjects
Publications and source records attributed to M M Davis.
Of 10 distinct cloned DNA copies of mRNAs expressed in T lymphocytes but not in B lymphocytes and associated with membrane-bound polysomes, one hybridizes to a region of the genome that has rearranged in a T-cell lymphoma and several T-cell hybridomas. These characteristics suggest that it encodes one chain of the elusive antigen receptor on the surface of T lymphocytes.
Comparison of the sequence of a cloned T cell-specific cDNA with those of cross-reacting cloned cDNAs isolated from a thymocyte library indicates the presence of variable, constant and joining regions remarkably similar in size and sequence to those encoding immunoglobulin proteins. Together with the evidence for somatic gene rearrangements reported in the accompanying paper, this strongly suggests that the TM86 cDNA clone encodes one chain of the T-cell receptor for antigen.
A putative T-cell receptor gene was isolated from the DNA of the helper hybridoma, 2B4 . Analysis of the sequence components of this rearranged gene indicates the presence of separate variable (V), diversity (D) and joining (J) region elements analogous to those of heavy-chain immunoglobulins. These findings further support the belief that the products of this locus are involved in antigen recognition by T cells. There is no evidence of somatic mutation between the putative germ-line and the expressed variable-region gene.
The genomic structure of the joining (J) and constant (C) regions of the locus encoding the beta-chain of the murine T-cell receptor has been analysed. The gene segments are arranged tandemly (J-C/J-C) within a 15-kilobase region. The two constant-region genes are almost identical, differing by only four amino acids, all in carboxy-terminal portions. Each C region comprises four exons encoding an external globular domain, a small hinge-like region, a transmembrane region and a cytoplasmic tail plus 3'-untranslated region. The two clusters of J regions each contain 7 distinct elements, 12 of which may be functional.
Recently, complementary DNA clones encoding one chain of the T-cell receptor for antigen have been isolated from both murine and human cell lines. Sequence analysis of these clones indicates that they encode elements analogous to the variable (V), constant (C) and joining (J) regions of immunoglobins and that this corresponds to the beta-chain subunit of the T-cell receptor complex. These genes are rearranged in the genomes of specific T-cell lines and hybrids but not in other cell types. Analysis of the components of one such rearranged gene, 2B4, isolated from the pigeon cytochrome c-specific, H-2E-restricted T helper (TH) hybridoma, and its unrearranged (liver) counterpart, indicate that an 8-nucleotide sequence 3' to the rearranged variable region is not derived from either the germ-line V- or J-region gene segments. As this sequence lies at a similar position to the diversity (D) region in immunoglobulin heavy-chain genes, we postulated the existence of an array of germ-line D-region elements that would contribute significantly to the number of different beta-chain molecules which could be created. Here we describe the localization and sequence of one such D-region element, found approximately 650 nucleotides 5' to the first JT cluster. This element seems to be involved in both functional (V-D-J) and non-functional (D-J) rearrangements. Our observations, combined with previous results, indicate that variable-region formation (V-D-J joining) in the T-cell receptor beta-chain gene follows the 12/23-base pair (bp) rule of rearrangement established for the recombination of immunoglobulin gene segments, but that the organization of the heptamer and nonamer element found surrounding the D region is significantly different.
By subtractive cDNA hybridizations, we have isolated a new species of T-cell receptor cDNA clone whose predicted amino acid sequence has homology to variable, constant, joining and diversity segments of immunoglobulins and T-cell receptors. The corresponding genomic sequence is also rearranged in several T-cell DNAs. The four potential N-linked glycosylation sites, frequency of expression and predicted molecular weight (27,800) of this molecule make it a likely candidate for the alpha-chain of the T-cell receptor. Expression data also indicate that this gene may be activated at a later stage of T-cell differentiation than the beta-chain.
Analysis of three new T-cell receptor beta-chain variable regions together with those in the literature indicates that they have both remarkable similarities and differences with those of immunoglobulin. Less than 10 V regions appear to predominate in the thymus. V beta sequences are much more heterogeneous at the amino acid level than are immunoglobulin V regions and they appear to diverge between species much more quickly, apparently the result of additional hypervariable regions. Three of these putative new hypervariable regions lie outside of the classical immunoglobulin binding site, an indication that important interactions may be occurring in these regions with polymorphic MHC determinants.
The striking number of human and murine immunodeficiency disorders which map to the X chromosome suggests that genes localized on this chromosome must have important roles in lymphocyte development. At least seven distinct disorders in the human and two in the mouse disrupt lymphocyte maturation, particularly that of B cells, at characteristic stages. As functional genes mapping to the X chromosome in one mammal are found on the X chromosome in all other mammals, the same genes regulating lymphocyte development are expected to be found on the X chromosome in mouse and man. Investigations into the possible mechanisms of these X-linked disorders have been hampered by the lack of molecular probes for the genes or gene products affected; because of this, and the possibility of correlating one or more of the several hundred B- or T-cell-specific genes with a specific mutation, we surveyed 15 different B- and T-cell-specific cDNA clones for localization to the X chromosome. We report here the characterization of one of these murine cDNA clones, which hybridizes with a large, X-linked gene family, designated XLR (X-linked, lymphocyte-regulated). We show that the XLR gene family is closely linked to the X-linked immunodeficiency described in the CBA/N mouse strain (xid), by restriction fragment length polymorphism (RFLP) analysis of DNA from mice congeneic for xid. This finding, together with data on the expression of the XLR locus in B cells, indicates that this gene family either includes the locus defined by the xid mutation or is adjacent to it in a gene complex which may be important in lymphocyte differentiation.
One of the most extensively studied X-linked immunodeficiency disorder is the xid mutation of the mouse strain CBA/N. This mutation may involve a maturational defect as xid animals are unable to raise antibodies to soluble polysaccharide antigens, a function normally attributed to late-stage B cells. Moreover, studies using monoclonal antibodies have defined a B-cell surface antigen (BLA-2 or 14G8) that is expressed on most or all immature B lymphocytes, but not on a subpopulation of mature splenic B lymphocytes; this late-stage, 14G8 antigen-negative splenic B-cell subpopulation is apparently absent from mice bearing the xid defect. In the accompanying paper we describe the isolation of a cDNA clone recognizing a family of genes on the X chromosome, at least some of whose members are closely linked to the xid trait. We report here that this gene family, XLR, is transcribed in certain B- and T-cell lineage tumours, but not in macrophage tumours, or liver or kidney cells. We show that it is transcribed principally in late-stage, 14G8-negative B-cell tumours and plasmacytomas, but not in immature B-cell or pre-B-cell tumours. We are able to detect transcription in all of 12 plasmacytomas (secretory B-cell tumours) derived from mice with normal X chromosomes, but not in three plasmacytomas carrying the xid mutation. These data, combined with the restriction fragment length polymorphism analysis linking the XLR gene family to the xid mutation, suggests that the xid defect occurs within a member of this gene family.
The antigen receptor on T lymphocytes has recently been characterized as a heterodimeric, transmembrane glycoprotein consisting of disulphide-linked alpha (acidic) and beta (basic) subunits of relative molecular mass (Mr) 40,000-45,000 each. The genes encoding these proteins have been cloned and shown to resemble immunoglobulin genes in both overall structure and the requirement for DNA rearrangement before expression. In humans, three additional proteins, termed the T3 complex, are found associated with the clonotypic receptor, and a role for T3 in receptor expression has been proposed. Despite these recent advances in characterizing the antigen receptor complex, there is as yet little understanding of T-cell maturation, particularly the stage of T-cell ontogeny at which the genes encoding the antigen receptor and its associated structures are expressed and assembled. In the adult, stem cells destined to differentiate into T cells arise in the bone marrow and migrate to the thymus, where T-cell precursors proliferate, develop a preference for recognizing antigens in the context of self MHC molecules and are released to the periphery. Recently, cells that have the properties of immature murine thymocytes have been isolated and described. We have now analysed these cells with a series of molecular probes and we describe three distinct patterns of T-cell antigen receptor gene rearrangements in developing thymocytes.
A new T-cell receptor gene lies just 5' to the J alpha C alpha coding regions. Its placement in this location suggests a novel mechanism for the regulation of expression of one T-cell receptor polypeptide to another during ontogeny. Rearrangement of this locus occurs very early in thymic differentiation and its RNA expression parallels that of the gamma-chain in thymic subpopulations, making this a possible candidate for the recently described delta-chain of the T-cell receptor.
Thymus-derived lymphocytes (T cells) use clonally distributed antigen receptors to recognize peptide fragments associated with products of the major histocompatibility complex (MHC) (refs 1-4). On most murine and human T cells the T cell receptor (TCR) is composed of disulphide-linked alpha and beta chains (TCR alpha/beta), each of which contains constant and variable domains, and which are associated with the invariant chains of the CD3 complex. It has been demonstrated, however, that a distinct CD3-associated TCR is expressed on a small subset of T cells or immature thymocytes which fail to express either CD4 or CD8 (refs 7-14), the molecules associated with class II or class I MHC antigen recognition. Instead of TCR alpha/beta, these cells express heterodimers of gamma and delta chains (TRC gamma/delta). The genes encoding alpha, beta, and gamma have been isolated and characterized. A new murine T cell receptor (Cx) gene which undergoes rearrangement and expression early during T cell ontogeny has recently been identified 5' of the murine J alpha C alpha gene locus. Here we isolate and sequence the homologous transcript from PEER, a human cell line that expresses a TCR gamma/delta, and show that it encodes a protein with characteristic V, D, J, and C segments. Using probes derived from this transcript, we have shown that both PEER and MOLT-13, another TCR gamma/delta-expressing cell line, rearrange this locus and express two sizes of transcripts differing in the 3' untranslated region. Using a synthetic peptide derived from the deduced C region sequence, we have prepared antisera that precipitates the delta chain of the TCR from both PEER and MOLT-13, thus demonstrating that Cx and its human homologue code for the delta chain of the TCR.
The T-cell receptor delta-chain variable region can be assembled from as many as four distinct gene segments, V, D1, D2 and J, more than any other antigen-receptor gene. In fetal thymocytes V----D joinings are as common as D----J or VDJ rearrangements and one V gene segment predominates. Analysis of rearrangements at TCR gamma and delta loci during fetal ontogeny suggests abrupt changes and possible coordinate control in the rearrangement and expression of these loci.
PURPOSE: We sought to determine the accuracy of helical CT in the detection of pulmonary metastases. METHOD: Four anesthetized dogs with metastatic osteosarcoma underwent helical CT with a collimation of 5 mm, a pitch of 2, and a reconstruction interval of 5 mm. All macroscopically evident metastases were recorded at autopsy. CT images were independently reviewed by 10 radiologists and compared with pathologic results. Alternate slices in the dog with the most metastases were microscopically examined in their entirety. RESULTS: Pathologic examination of the lungs revealed 132 macroscopically evident pulmonary metastases, of which 74 (56%) were detected by at least one reader. Forty-four of the 99 (44%) metastases of < or = 5 mm in diameter were detected by at least one reader compared with 30 of 33 (91%) metastases of > 5 mm in diameter (p < 0.0001). The 10 readers reported a total of 107 false positives. Complete microscopy of alternate slices in the dog with the most metastases (n = 68) revealed an additional 38 micrometastases of < or = 3 mm in diameter. None of the 32 micrometastases of < or = 1 mm were detected by CT. CONCLUSION: Helical CT has some limitations in the detection of pulmonary metastases.
Obesity, now recognized as an independent risk factor for cardiovascular disease, is closely associated with hypertension. Complex mechanisms link increasing body weight with increasing blood pressure. Treatment of the obese patient with hypertension requires consideration of physiologic changes related to obesity hypertension. Lifestyle modification, including weight reduction and increased physical activity, can directly influence blood pressure levels and improve blood pressure control in obese, hypertensive patients. Clinical trials are needed to determine the most effective antihypertensive drugs for the obese, hypertensive patient. Antiobesity drugs offer viable adjunctive pharmacotherapy for obesity hypertension, but additional long-term studies are needed to support their safety and efficacy.
OBJECTIVE: To characterize the obstacles faced by physicians regarding administration of a 7-valent pneumococcal conjugate vaccine (Prevnar) to all children younger than 2 years and to high-risk children from 2--5 years of age during the months immediately following national recommendations. DESIGN: Semistructured telephone interviews. PARTICIPANTS: Convenience sample (n = 24) of pediatricians and family physicians. RESULTS: Eighteen physicians were recommending pneumococcal vaccine and 6 were not. Those who were recommending pneumococcal vaccine had encountered resistance from parents and variations in private and public insurance coverage. Physicians who were not recommending pneumococcal vaccine expressed concern about the cost of the vaccine and general caution in adopting new vaccine recommendations. Respondents offered several suggestions for improving the vaccine recommendation process. CONCLUSIONS: This analysis of physicians' early decision making regarding pneumococcal vaccine reflects obstacles to vaccine implementation that may arise with the introduction of other childhood vaccines.
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