Search PubMed⌕ Search

Biomedical subjects

L F Thompson

Publications and source records attributed to L F Thompson.

At least 37 records · Page 2Linked to original sources
PubMed1997

T cell signalling through CD73.

CD73 (ecto-5'-nucleotidase), a glycosyl phosphatidylinositol (GPI) anchored purine salvage enzyme expressed on the surface of human T and B lymphocytes, catalyzes the conversion of purine and pyrimidine ribo- and deoxyribonucleoside monophosphates to the corresponding nucleosides. The cellular distribution, cDNA sequence, and structure of CD73 are reviewed. CD73 serves as a costimulatory molecule in activating T cells. A Jurkat.T cell line transfected with the CD73 cDNA revealed that neither enzymatic activity nor the GPI anchor is necessary for T cell activation in vitro via CD73, while expression of p56kk, CD45 and the T cell receptor are required. Models for the transmission of signals via CD73 and other GPI-anchored proteins are discussed. CD73 generated adenosine functions in cell signalling in many physiologic systems, including intestinal epithelium, ischemic myocardium, and cholinergic synapses. The hypothesis that CD73 produces adenosine that is important for T cell development is presented.

5'-Nucleotidase↗
PubMed1996

Failure to wean: exploring the influence of age-related pulmonary changes.

Half of the patients admitted to intensive care units are elderly and may require mechanical ventilation for respiratory failure. Although age is not an independent factor for determining weaning outcomes, it may play a role. Elderly patients are predisposed to hypoxic-hypercapnia failure caused by age-related pulmonary changes and coexisting diseases. These age-related changes limit the respiratory reserve, which affects the body's ability to provide an adequate oxygen supply to meet the demand. The consequence is that during weaning there may be an imbalance between supply and demand and weaning failure may result. More research is needed regarding the weaning of elderly patients. Some specific areas that require further investigation are the relationship between age and dyspnea, the influence of age on weaning, and the relationship between age and the work of breathing.

Aged↗
PubMed1995

Isolation and characterization of the promoter of the human 5'-nucleotidase (CD73)-encoding gene.

Ecto-5'-nucleotidase (NT, CD73) is a purine salvage-pathway enzyme located on the surface of various cell types, including subsets of human lymphocytes and certain leukemias and lymphomas. In addition to purine salvage, NT has proposed roles in lymphocyte maturation and activation, and its expression has been associated with the resistance of some tumor cell lines to chemotherapeutic agents. To better understand the regulation of NT gene expression in normal lymphocyte development and the elevated expression seen in some drug-resistant tumor cell lines, we isolated NT genomic clones containing the promoter region. The genomic DNA upstream from the NT start codon is high in G+C content, with one cAMP-responsive element and five consensus Sp-1 binding sites, but no TATAA box. RNase protection assays identified a cluster of potential transcription start points (tsp). One tsp, at -63 bp relative to the start codon, was confirmed as authentic by 5'-RACE (rapid amplification of cDNA ends) cloning. Transient transfection experiments utilizing luc as a reporter gene have demonstrated that a 155-bp NT genomic DNA segment inclusive of the tsp functions as a promoter in both NT+ (WI-L2 and MG) and NT- (Jurkat, Hela and Raji) cell lines. The addition of 5'-flanking sequences extending as far as -1.9 kb did not confer cell-type-specific expression to the core promoter. However, nuclear run-on analysis of nascent NT transcripts suggested that differential transcription initiation is at least partially responsible for the regulation of NT expression. Thus, additional information is necessary, either at the chromatin level, or within elements outside of the promoter region, to direct tissue-specific expression of NT.

5'-Nucleotidase↗
PubMed1995

Antigen recognition by CTL is dependent upon ectoATPase activity.

Alloantigen-specific and OVA-specific CD8+ CTL were shown here to express an ectoATPase. These CTL also express an ectoADPase, but do not express detectable levels of an ectoAMPase. CD8+ CTL transported adenosine into their cytoplasm at a rate of 2.3 x 10(-11) mmol/min/10(5) cells. In contrast, adenosine uptake was 34-fold lower when ATP was used as the source of the nucleoside. This was consistent with the lack of ectoAMPase and suggests that the role of ectoATPase is not in the salvage of extracellular nucleotides. 5'-p-(fluorosulfonyl)benzoyl adenosine (5'-FSBA) is an ATP analogue affinity label that irreversibly inhibits CTL ectoATPase. Cells made ectoATPase activity deficient by modification with 5'-FSBA were not susceptible to potential lytic effects of extracellular ATP with less than 20% specific lysis at 20 mM of exogenous ATP. However, cells modified by 5'-FSBA were unable to kill their respective target cells. Complete inhibition of cell-mediated killing was observed with 1 mM 5'-FSBA. CTL modified by 5'-FSBA also failed to secrete TNF-alpha and IFN-gamma after activation by the appropriate Ag. Killing was also inhibited by 5'-adenylylimidodiphosphate (a nonhydrolyzable ATP analogue), but not by ATP, ADP, alpha, beta-methylene ADP (a nonhydrolyzable ADP analogue), AMP, or adenosine. Blockage of CTL activity by 5'-FSBA was not reversed by addition of ADP, suggesting that hydrolysis of ATP is an essential ectoATPase-mediated signal for CTL activation. These results suggest that ectoATPase is essential for Ag recognition and/or effector activities of CTL.

Adenosine↗
PubMed1995

Ecto-5'-nucleotidase activity is not required for T cell activation through CD73.

It had previously been shown that CD73 (ecto-5'-nucleotidase) expressed on 30% of normal human peripheral T-lymphocytes can mediate costimulatory signals in T cell activation. To check for a possible contribution of the catalytic property of CD73 to this function transfected cell lines were created. Using site-directed mutagenesis on human CD73 cDNA the codons of His92 and His194 were exchanged for alanine codons. Wild type and mutant cDNAs were cloned into an expression vector and stably expressed in the T cell line Jurkat. Surface expression as quantitated by immunofluorescence with anti CD73 mAbs was comparable in wild type and mutant transfectant clones, whereas in contrast to the wild type both His to Ala mutants completely lacked enzymatic activity. The Jurkat transfectants were stimulated with a combination of soluble anti-CD73 mAb and PMA and their response was quantitated by IL-2 production. The responses of different clones of wild type and mutant transfectants varied within a wide range; however, the ranges of wild type and mutants overlap. It is concluded that in order to transmit costimulatory signals CD73 does not require its catalytic activity.

5'-Nucleotidase↗
PubMed1995

Production and characterization of multiple antigenic peptide antibodies to the adenosine A2b receptor.

A polyclonal antibody to the human adenosine A2b receptor (A2bR) was produced by immunizing a chicken with a multiple antigenic peptide consisting of eight copies of a 16-amino acid peptide, corresponding to the presumed second extracellular loop of the A2bR, linked to a branched lysine core. Western blotting with affinity-purified antibody revealed the human A2bR to be a protein of approximately 50-55 kDa, found in a variety of tissues including thymus, colon, and small intestine. The antibody also recognized mouse and rat A2bRs and revealed heterogeneity in size, with a 35-kDa protein being detected in small intestine in addition to the larger 50-52-kDa species in thymus, colon, and placenta. The chicken anti-human A2bR peptide antibody recognized the receptor in both frozen and formalin-fixed tissue sections. In human colon, the A2bR was highly expressed in epithelial cells of the crypts. A2bR immunoreactivity was also apparent in syncytiotrophoblast cells of human placental villi and in the basal zone of murine chorioallantoic placenta. These cell type-specific patterns of expression are consistent with the hypothesized roles of the A2bR in mediating electrogenic Cl- secretion and the resulting secretory diarrhea caused by colonic crypt abscesses and in regulating morphogenesis of the placenta. Insight into the multiple physiological consequences of A2bR engagement will be forthcoming from an analysis of the cell type-specific expression of this receptor in additional tissues.

Amino Acid Sequence↗
PubMed1994

Glycosyl phosphatidylinositol membrane anchor is not required for T cell activation through CD73.

Ecto-5'-nucleotidase (5'-NT,) CD73 is a glycosyl phosphatidylinositol (GPI)-anchored differentiation Ag and purine salvage enzyme expressed on the surface of subsets of human lymphocytes. CD73 mAbs, in combination with submitogenic PMA or OKT3, activate human T cells to proliferate, secrete IL-2, and express IL-2 receptors. For several GPI-anchored proteins implicated in T cell activation, activation is thought to occur through a mechanism that requires the GPI anchor. To investigate the role of the GPI anchor in CD73-mediated signal transduction, CD73 cDNA was transfected into the human T cell leukemia line Jurkat. Transfectants were stimulated to secrete IL-2 by immobilized OKT3 + PMA and by soluble CD73 mAb + PMA. The amount of IL-2 synthesized by individual clones in response to CD73 mAb + PMA was proportional to the IL-2 response to immobilized OKT3 + PMA. CD73 transfectants of Jurkat mutants defective in the TCR, in p56lck, or in the tyrosine phosphatase CD45 did not secrete IL-2 in response to CD73 mAb + PMA, suggesting a role for the CD3/TCR-signaling complex in CD73-mediated signal transduction. A transmembrane form of CD73 was expressed in Jurkat cells by fusing the extracellular portion of CD73 to the transmembrane domain of human tissue factor. Although as a group, clones expressing transmembrane CD73 synthesized less IL-2 in response to CD73 mAb + PMA than clones expressing the GPI-anchored form of the molecule, the responses of the two groups overlapped considerably. In contrast to previous studies with Ly-6, Qa-2, and CD55, the GPI anchor is not critical for activation through CD73.

5'-Nucleotidase↗
PubMed1993

Murine ecto-5'-nucleotidase (CD73): cDNA cloning and tissue distribution.

The murine cDNA, encoding the purine catabolic enzyme, ecto-5'-nucleotidase (NT), was cloned and the tissue-specific distribution of both the mRNA and enzyme activity was examined. Starting with kidney RNA and primers based on the known rat sequence, reverse transcriptase-polymerase chain reaction (RT-PCR) was utilized to obtain the complete sequence for the translated portion of the murine cDNA. Murine NT is 94% identical to human NT at the amino acid (aa) level and 86% identical at the nucleotide (nt) level. NT enzyme assays revealed greater than tenfold more NT activity in mature vs. immature murine T- and B-lymphocytes. A similar increase in NT activity was also found when the pre-B-cell line, 70Z/3, was induced to produce surface kappa light chains with lipopolysaccharide (LPS) and gamma-interferon (gamma-IFN). Thus, culture systems in which murine lymphocytes mature may be useful for examining the mechanisms of NT gene regulation, as well as the function of NT in the immune system. In tissues, enzyme activity varied over 30-fold, from the lowest levels in skeletal muscle, thymus and spleen to highest in placenta, kidney and forestomach. Levels of mRNA, as determined by RNase protection assay, showed increased NT expression in the early gestation site, as compared to non-pregnant uterus, and in day-19.5 placenta, as compared to day-13 chorioallantoic placenta. Messenger RNA levels were in general proportional to enzyme activity, except in the lung and glandular stomach where mRNA levels were higher than expected, based on enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

5'-Nucleotidase↗
PubMed1993

Co-stimulatory signal delivered by CD73 molecule to human CD45RAhiCD45ROlo (naive) CD8+ T lymphocytes.

CD73 is a molecule expressed by a subset of CD8+ human T lymphocytes and is involved in T cell activation. CD73 expression and function were analyzed in peripheral blood CD45RAhiCD45ROlo (naive) and CD45RAloCD45ROhi (memory) CD8+ cells. We found that CD73 was expressed by a majority of naive cells (74 +/- 12%), whereas fewer memory cells were CD73+ (29 +/- 10%). Moreover, CD73 was selectively expressed by the CD11b- subset of naive CD8+ cells, which were almost all CD73+. The same result was found on CD8+ cord blood lymphocytes, which prevalently display the naive phenotype. Naive CD8+ CD11b- cells were almost unresponsive to CD3 engagement, but this apparent anergy was completely overcome when CD3 and CD73 were simultaneously cross-linked by plastic-immobilized CD73 and CD3 mAb, showing that CD73 delivers an accessory signal that allows their activation via the CD3/TCR. This costimulatory signal was tenfold more potent than that induced by CD28 ligation. A phosphotyrosine analysis by Western blotting showed that cross-linking of CD73 induced the phosphorylation of two proteins with a molecular mass of approximately 28 and 100 kDa respectively, whereas ligation of CD3 induced phosphorylation of many substrates. When CD3 and CD73 were simultaneously triggered these substrates were hypophosphorylated. Because CD73 is linked to the cell surface by a GPI anchor, the transduction of this signal is probably mediated by a lateral interaction with transmembrane molecules. This hypothesis was assessed by cocapping, which showed that CD73 associates strongly with CD45RC, moderately with CD8, and weakly with CD3. These data suggest that CD73 signaling is coupled to both tyrosine kinase and phosphatase activities.

5'-Nucleotidase↗
PubMed1992

P-selectin (CD62) binds to subpopulations of human memory T lymphocytes and natural killer cells.

P-selectin (CD62) is a Ca(2+)-dependent lectin expressed on activated platelets and endothelium. Although P-selectin is known to function as a receptor for myeloid cells, previous studies indicated that P-selectin also bound to a subset of lymphocytes. Using a multi-color immunofluorescence assay we found that purified P-selectin bound to 12.2 +/- 4.1% of peripheral blood lymphocytes and that P-selectin could mediate adhesion of activated platelets to lymphocytes. A subpopulation of CD4+, CD8+, and CD16+ lymphocytes bound P-selectin. There was a marked preference for P-selectin binding to memory cells (CD45RO+) in both the CD4+ and CD8+ populations. Binding to all cell types was Ca(2+)-dependent and blocked by pretreatment of the cells with sialidase. These data suggest that P-selectin may play a role in the recruitment of specific lymphocyte populations to sites of inflammation.

Antigens, CD↗
PubMed1992

Adenosine levels in the postimplantation mouse uterus: quantitation by HPLC-fluorometric detection and spatiotemporal regulation by 5'-nucleotidase and adenosine deaminase.

Extracellular adenosine has the potential to influence many aspects of target cell metabolism. The present study has determined the endogenous levels of adenosine in the pregnant mouse uterus and developing embryo-decidual unit with respect to the expression of two key enzymes of adenosine metabolism, 5'-nucleotidase (5'-NT; EC 3.1.3.5) and adenosine deaminase (ADA; EC 3.5.4.4). To measure adenosine levels, nucleoside extracts were etheno-derivatized and quantitated by high-performance liquid chromatography-fluorescence detection (0.03 pmol/mg protein sensitivity). Adenosine levels were determined to be 0.18 nmol/mg protein in the nonpregnant uterus; however, two statistically significant changes were identified in the pregnant uterus: (1) a periimplantation surge between day 3 (0.24 nmol/mg protein) and day 5 (0.59 nmol/mg protein) of gestation (plug day 0; implantation day 4); and (2) an early postimplantation decline between day 6 (0.54 nmol/mg protein) and day 7 (0.10 nmol/mg protein). The periimplantation adenosine surge coincided with uterine expression of 5'-NT, an enzyme which catalyzes the irreversible dephosphorylation of 5'-AMP to adenosine. 5'-NT expression was shown by Northern blot analysis to peak in the embryo-decidual unit on day 5 of gestation and then to decline through day 9; transcripts remained elevated in the placenta between day 9 and day 13 (the latest day examined in this study). By use of specific enzyme histochemistry, most 5'-NT activity was localized to the primary decidual zone on day 5. This expression subsequently declined during regression of the primary decidua; however, 5'-NT appeared on giant trophoblast (days 7-13) and the metrial gland (days 11-13). Other purine catabolic enzymes degrading AMP (adenylate deaminase) or generating adenosine (S-adenosylhomocysteine hydrolase) were not detected in the embryo-decidual unit suggesting that the net flux of utero-placental AMP catabolism proceeds with adenosine as an intermediate, this being the major pathway of adenosine formation. The sharp drop in adenosine levels between day 6 and day 7 coincided with a rise in the activity and mRNA expression of ADA, an enzyme which catalyzes the irreversible deamination of adenosine to inosine. ADA was previously localized to the secondary decidual zone (days 6-11), secondary giant cells (days 7-13), and spongiotrophoblasts (days 8-13) in the mouse (Knudsen et al., 1991). Results of developmental Northern blot analysis demonstrated a direct correlation of relative 5'-NT/ADA mRNA band intensity to adenosine content between day 4 and day 9 of gestation, suggesting that the local availability of adenosine in the antimesometrium is dependent upon the distribution of these enzymatic activities.(ABSTRACT TRUNCATED AT 400 WORDS)

5'-Nucleotidase↗
PubMed1992

Distribution and quantitative expression of the complement receptor type 1 (CR1) on human peripheral blood T lymphocytes.

The complement receptor, type 1 (CR1) is expressed on a variety of cell types including primate erythrocytes, phagocytic cells, and B lymphocytes. On these cells, CR1 plays a role in a diverse spectrum of biological activities including the clearance of immune complexes from the circulation, down-regulation of the complement system, recognition of complement-coated microorganisms, and cellular activation. CR1 is also expressed by some, but not all, T lymphocytes. The present study was undertaken in order to examine the distribution of CR1 on normal human T cell subsets by flow cytometry and to quantify the expression of T cell CR1 by radioimmunoassay. Data presented here indicate that, in a panel of 19 normal individuals, a mean of 9.7% of the overall peripheral blood lymphocyte population expressed CR1 and that, as assessed by two-color flow cytometry, 12.0% of CD3+, 13.0% of CD4+, and 20.0% of CD8+ cells expressed CR1. While single peaks of CR1 staining were observed within the CD3 and CD4 subsets, a biphasic pattern of staining was evident within the CD8 subset in which relatively high-intensity CR1 staining was detected within the subpopulation of "dull" CD8+ cells, whereas a lower intensity of CR1 staining was observed within the subpopulation of "bright" CD8+ cells. Duplicate analyses performed over a relatively short time frame suggested that, while the overall percentage of cells that expressed CR1 varied considerably among normal individuals, in at least some individuals the percentage of cells expressing CR1 was relatively stable, especially within the CD4 subset. In cell suspensions enriched for T lymphocytes by rosetting with sheep erythrocytes, 10.0% of the cells were CR1+ and a mean of approximately 3700 CR1 were expressed per CR1+ cell. There was no apparent correlation between the number of CR1 per T cell and the number of CR1 expressed per erythrocyte in the same blood sample. The expression of CR1 on subpopulations within the CD3+, CD4+, and CD8+ lymphocyte subsets may play a role in both normal cell function and in the pathophysiology of disease states including the acquired immune deficiency syndrome (AIDS).

Adult↗
PubMed1991

Expression of 5'-nucleotidase (CD73) related to other differentiation antigens in leukemias of B-cell lineage.

Ecto-5'nucleotidase (5'NT; CD73) expression was studied with a monoclonal antibody (7G2) and a radiochemical assay and compared with the expression of other antigens in B-cell-lineage leukemias on cells from 100 leukemic patients and two cell lines. A B-cell origin was confirmed by the expression of CD19 and HLA-DR. Four stages of B-cell leukemias were defined: stage I (pro-B) as CD10-, cytoplasmic mu- (c mu-), surface Ig- (sIg-); stage II (cALL) as CD10+/c mu-/sIg-; stage III (pre-B) as CD10+ or -/c mu+/sIg-; and stage IV (B) as CD10-/c mu-/sIg+. A linear correlation was found between immunohistochemical and radiochemical determination of 5'NT (r = .86). 5'NT expression was low in T-cell leukemias and stage I, high in stages II and III, and low again in stage IV of B-cell leukemias. 5'NT expression was not related to c mu, CD20, CD21, CD22, CD34, and terminal deoxynucleotidyl transferase (TdT) expression, but was significantly related to CD10 and inversely related to kappa/lambda expression. However, the 5'NT activity in CD10+ leukemias (stages II and III) shows a very wide range. Within the group of CD10+ leukemias no differences were detected between 5'NT+ and 5'NT- cells in their expression of other B-cell antigens. We conclude that the place of 5'NT in leukemias corresponding to early stages of B-cell development has been characterized. 5'NT is expressed in CD10+ stages and decreases before the expression of sIgs. Future studies should make clear whether a high expression of this enzyme in CD10+ stages is a normal maturation phenomenon or a malignant phenomenon.

5'-Nucleotidase↗
PubMed1991

Expression of ecto-5'-nucleotidase (CD73) in normal mammary gland and in breast carcinoma.

Ecto-5'-nucleotidase (ecto-5'-NT) is a phosphatidylinositol anchored membrane structure recently defined as the lymphocyte differentiation antigen CD73. Using CD73 (1E9.28.1) monoclonal antibody, normal mammary gland and breast carcinoma were immunohistochemically investigated for ecto-5'-NT expression. In normal breast epithelium, CD73 was differentially expressed in lobular, ductal and myoepithelial cells and was most frequently detected in the myoepithelial compartment. The glandular stroma contained fibrocytes, a subset of which was also CD73-positive. Among 102 unselected breast carcinoma primary lesions, only 9 contained CD73-positive tumour cells, whereas in 95 cases, stromal fibroblasts and fibrocytes showed variable degrees of CD73 expression. The extent of stromal CD73 expression correlated positively with the estrogen receptor (ER) status of the tumour (P less than 0.038). We conclude that ecto-5'-NT-expression reflects a still unknown state of activity of normal breast epithelium which is lost in the majority of carcinomas derived therefrom. It may also be indicative of some functional activity of stromal fibroblasts which is significantly enhanced in ER-positive carcinomas.

5'-Nucleotidase↗
PubMed1990

Characterization of soluble vs membrane-bound human placental 5'-nucleotidase.

Three forms of 5'-nucleotidase purified from human placenta (two membrane-bound forms, one sensitive and one resistant to cleavage by phosphatidylinositol-specific phospholipase C, as well as a soluble form) had the same molecular weight before (73,000 Da) and after (56,000 Da) digestion with N-glycosidase F and showed similar amino acid compositions, N-terminal amino acid sequences, and KMs for IMP (9.6 to 11.9 microM). Thus, these three forms of 5'-nucleotidase appear to have very similar structures. The form sensitive to phosphatidylinositol-specific phospholipase C contained nearly 1 mol myo-inositol/mol of protein as determined by mass spectrometry, indicating a glycosyl phosphatidylinositol membrane anchor. Soluble 5'-nucleotidase contained a similar quantity of myo-inositol, suggesting that it was previously membrane-anchored via glycosyl phosphatidylinositol. The form resistant to phosphatidylinositol-specific phospholipase C contained less myo-inositol, leaving open the possibility of a third form of 5'-nucleotidase with a conventional transmembrane anchor.

5'-Nucleotidase↗