Randall W. Barton

A cell adhesion molecule, ICAM-1, is the major surface receptor for rhinoviruses

Rhinoviruses, which cause common colds, possess over 100 serotypes, 90% of which (the major group) share a single receptor. Lymphocyte function associated molecule 1 (LFA-1) mediates leukocyte adhesion to a wide variety of cell types by binding to intercellular adhesion molecule 1 (ICAM-1). We demonstrate identity between the receptor for the major group of rhinoviruses and ICAM-1. A major group rhinovirus binds specifically to purified ICAM-1 and to ICAM-1 expressed on transfected COS cells, and binding is blocked by three ICAM-1 monoclonal antibodies (MAb) that block ICAM-1-LFA-1 interaction, but not by an ICAM-1 MAb that does not block ICAM-1-LFA-1 interaction. This suggests that the ICAM-1 contact site(s) for LFA-1 and rhinoviruses is proximal or identical. In addition, ICAM-1 MAb block the cytopathic effect in HeLa cells mediated by representative major but not minor group rhinoviruses. ICAM-1 is induced by soluble mediators of inflammation, suggesting that the host immune response to rhinovirus may facilitate spread to uninfected cells.

Nucleotide-metabolizing enzymes and lymphocyte differentiation

SummaryInherited deficiencies of adenosine deaminase and purine nucleoside phosphorylase have been found to be associated with certain immunodeficiency syndromes which are characterized by deficiencies of mature peripheral lymphocytes. The immunodeficiency states associated with these enzyme deficiencies are thought to arise from blocks in lymphocyte differentiation. Deficiencies of these enzymes have profound and apparently selective effects on lymphocyte differention. Their discovery has focused attention on previously unknown relationships between purine nucleotide metabolism and lymphocyte development and function. In this article three aspects of nucleotide-metabolizing enzymes and lymphocyte differentiation will be discussed: 1) the distribution of the enzymes among lymphocyte populations at differing stages of differentiation; 2) the possible biochemical mechanisms which give rise to the immunodeficiencies; 3) the stages of lymphocyte differentiation which are affected by the enzyme deficiencies.

Anti-idiotypic immunity and autoimmunity: II. Idiotypic determinants of autoantibodies and lymphocytes in spontaneous and experimentally induced autoimmune thyroiditis

In a previous report, it was demonstrated that heterologous anti-idiotypic antibodies to autoantibodies against rat thyroglobulin (ART) were capable of inhibiting the in vitro binding between ART and rat thyroglobulin. It has also been shown that repeated injections of anti-idiotypic antibodies into Buffalo (BUF) rats with spontaneous autoimmune thyroiditis were followed by a significant decrease in the levels of circulating ART. In this report, cross-reacting idiotypic determinants, detectable by rabbit anti-idiotypic antibodies to ART, are shown to also be present on ART from rats with experimentally induced autoimmune thyroiditis. In addition, antibodies to rat thyroglobulin from animals of various strains and species are shown to also express idiotypes cross-reacting with those of spontaneous ART of BUF rats. Finally, it is reported that idiotypic determinants similar to those of circulating ART are present on spleen lymphocytes from rats with autoimmune thyroiditis.

Binding of the T cell activation monoclonal antibody Ta1 to dipeptidyl peptidase IV.

The monoclonal antibodies, Ta1 and IOT15, define T cell activation cell surface markers and have been assigned to the CD26 leukocyte differentiation antigen cluster. Dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) is an exoaminopeptidase that, among leukocytes, is expressed almost exclusively on activated T cells. Comparative binding studies showed that the Ta1 mAb binds to DPP IV purified from human placenta as well as in extracts of the human YT lymphoid cell line and of CD3 stimulated normal human peripheral blood mononuclear cells. The mAb IOT15 did not bind to DPP IV from any source even upon repeated incubations. Western blot analysis of YT cell extracts revealed that Ta1 and IOT15 bound to distinctly different molecular weight molecules. Immunofluorescent cell surface capping experiments showed that capping of the IOT15 did not alter the surface distribution of the Ta1 fluorescence. The capping results combined with the DPP IV binding results indicate that IOT15 and Tal mAbs bind to different, apparently unassociated, molecules on the surface of T cells and that only Ta1 binds the T cell surface enzyme DPP IV.

Long-Term Erythropoietin Gene Expression from Transduced Cells in Bioisolator Devices

Recombinant erythropoietin (EPO) is widely administered for long-term treatment of anemia associated with renal failure and other chronic diseases. The ability to deliver EPO by gene therapy would have clinical and economic benefit. We compared autologous and allogeneic transduced primary vascular smooth muscle cells for their ability to provide sustained EPO gene expression when encapsulated in TheraCyte devices implanted subcutaneously (SQ) or intraperitoneally (IP) in rats. Cells were transduced with retrovirus vector LrEpSN encoding rat EPO cDNA. Rats that received either autologous or allogeneic transduced cells showed elevated hematocrits (HCTs) ranging from 50 to 79% that were sustained for more than 12 months. The HCT of control rats remained at baseline (45.8%). Rats that received second SQ implants of either autologous or allogeneic cells showed elevations in hematocrit that were sustained for up to 12 months, suggesting the absence of immunological responses to transduced cells or implant material. All experimental groups had statistically significant elevated HCT (p < 0.001) when compared with controls. Both SQ and IP implantation were equally effective in delivering EPO long term. There were no significant differences in white blood cell (WBC) or platelet (PLT) values between treated and control animals. Implantation of TheraCyte devices was well tolerated and histological evaluation of the devices up to 12 months after surgery revealed a high degree of vascularization and no evidence of host immune response. TheraCyte devices offer a simple and safe gene delivery system that provides sustained therapeutic gene expression, permit removal and implantation of new devices, and do not require immunosuppression of the host.

Disparate cleavage of poly-(ADP-ribose)-polymerase (PARP) and a synthetic tetrapeptide, DEVD, by apoptotic cells.

In the present investigations, we have shown differential cleavage of cellular PARP and a caspase 3-selective synthetic tetrapeptide substrate, Z-DEVD-AFC or Ac-DEVD-AMC using a T lymphoblastoid cell line Jurkat, and its variant clone E6.1(J-E6). Anti-Fas antibody-mediated apoptosis resulted in DNA fragmentation and PARP cleavage in both Jurkat and J-E6 cells. However, unlike Jurkat, J-E6 cells did not cleave a synthetic tetrapeptide substrate efficiently. The failure to cleave the DEVD tetrapeptide by apoptotic J-E6 cells was not due to insufficient expression or processing of caspase 3 in J-E6 cells. Interestingly, when the J-E6 cells were transiently transfected with a cDNA encoding caspase 3, efficient cleavage of Z-DEVD-AFC was achieved. The observations that apoptotic J-E6 cells barely cleaved a synthetic DEVD tetrapeptide, but efficiently cleaved endogenous PARP, potentially at the most preferred DEVD site, suggest that active caspases may have disparate characteristics to recognize substrates presented in different context.

Binding of human lymphocyte-specific monoclonal antibodies to common marmoset lymphoid cells

Commercially available monoclonal antibodies which bind to human lymphocyte subsets were screened for their ability to bind to lymphoid cells from the common marmoset Callithrix jacchus. Anti-Leu-5 and T11 were the only pan T-cell antibodies which reacted strongly. None of the antibodies which bind human lymphocytes of the helper/inducer subpopulation reacted with C. jacchus cells and only one antibody, T8, specific for the cytotoxic/suppressor subset, bound to the marmoset cells. The two antibodies tested which bind human B cells, B1 and anti-HLA-DR, were also reactive with marmoset cells. The cellular specificity of the T11, T8, and B1 antibodies was determined by dual binding studies on the fluorescence-activated cell sorter. The B1 antibody bound only Ig+ cells and all Ig+ cells were B1+. The T11 and T8 antibodies bound only to Ig- marmoset lymphoid cells and, as in the human, all T8+ marmoset cells were also T11+. Thus, using these monoclonal antibodies in the common marmoset one can identify three populations of lymphoid cells: (1) T11+, T8+ cells; (2) T11+, T8- cells; (3) B1+ cells.

An analysis of clinical outcomes and costs of a long term acute care hospital.

Abstract Objective: Compare clinical outcomes and costs in a study group of long-term acute care hospital (LTCH) patients with a control group of LTCH-eligible patients in an acute care hospital. LTCHs were created to provide post-acute care services not available at other post-acute settings. This is based on the premise that these patients would otherwise have stayed at acute care hospitals as high-cost outliers. The LTCH hospital is intended to deliver care to patients more efficiently, however, there are little documented clinical and financial data regarding the comparative clinical outcomes and costs for patients. Methods: Retrospective medical and billing record review of patients from the following groups: (1) LTCH study comprising patients admitted directly from an acute care hospital to the study LTCH and discharged from the LTCH from September 2004 through August 2006; (2) a control group of LTCH-eligible, medically complex patients treated and discharged from an acute care hospital in FY 2002. The control group was selected from approximately 500 patients who had at least one of the ten most common principle diagnosis DRGs of the study LTCH with >30-day length of stay at the referring hospital and met NALTH admitting guidelines. Results: Discharge disposition is an important outcome measure of the quality of care of medically complex patients. The in-hospital mortality rate trended lower and home discharge was 3 times higher for the LTCH study group than for the control group. As a possible result, SNF discharge of LTCH patients was approximately half that of the control group. Both mean patient cost per day and mean total cost per patient were significantly higher in the control group than in the LTCH study group. Conclusions: The patients in the LTCH study group had both better clinical outcomes and lower cost of care than the control group.

Effect of deoxycoformycin and Val-boroPro on the associated catalytic activities of lymphocyte CD26 and ecto-adenosine deaminase.

CD26 and ecto-adenosine deaminase (ADA) are found associated on the plasma membrane of T lymphocytes and each possess distinct catalytic activities. CD26 has a proteolytic activity identical to dipeptidylpeptidase IV (DPPIV; E.C. 3.4.14.5), and ecto-ADA (E.C. 3.5.4.4) degrades extracellular adenosine. The cell surface expression of CD26 and ecto-adenosine deaminase (ecto-ADA) is regulated on stimulated T lymphocytes, and ADA binding to CD26 produces a synergistic costimulatory response with T cell receptor activation. This study addresses the potential regulation by allosteric interactions of the catalytic activities of CD26 associated with ecto-ADA, which could define the mechanism of the synergism observed in T cell signaling. Cell lines genetically deficient in ADA, ligands for ADA such as adenosine, and a specific inhibitor of ADA, deoxycoformycin, were used to define the effect of ADA activity on CD26 DPPIV activity and affinity for dipeptide substrate. Conversely, a recombinant Chinese hamster ovary cell line expressing human CD26 with or without a mutation in the DPPIV catalytic domain, and the boronic acid inhibitor Val-boroPro, were used to determine the effect of DPPIV activity on ecto-ADA activity and association with CD26. These studies found no significant allosteric interaction between the catalytic activities of CD26 and ecto-ADA when associated. Therefore, signaling events in T cells involving costimulation with CD26 and ecto-ADA and the synergism observed upon ADA binding to CD26 occur independently of the catalytic activities of these cell surface molecules.

The binding of Maclura pomifera lectin to cells of the T-lymphocyte lineage in the rat☆

Abstract The tissue, anatomic, and developmental distribution of Maclura pomifera (MP) lectin binding to rat lymphoid cells was examined. Analysis was performed by immunofluorescence microscopy and by the fluorescence-activated cell sorter. Comparison with anti-rat Ig to detect B cells and the monoclonal antibodies W3/13, W3/25, and OX 8 to detect T cells revealed that the MP lectin reacted with all T cells and not B cells in spleen and lymph node of young adult rats. The lectin also bound selectively to the thymus-dependent areas in frozen sections of spleen and lymph node. Using the MP lectin in conjunction with anti-Thy1 antibody and the monoclonal antibodies, W3/25 and OX 8, four T-cell subpopulations in spleen and lymphnode were identified on the basis of their cell surface antigenic phenotype. The T-cell developmental distribution of MP binding revealed that 100% of normal and neoplastic thymocytes bound the lectin whereas approximately 25% of TdT + bone marrow cells, putative thymocyte progenitors, were MP + . Thus, the MP lectin is a nonimmunoglobulin reagent which binds to prethymic, thymic, and post-thymic cells of the T-lymphocyte lineage. Affinity chromatography experiments indicated that the MP lectin binds, at least in part, to the major thymocyte cell surface glycoprotein which is recognized by the W3/13 monoclonal antibody.