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J W Semple

Publications and source records attributed to J W Semple.

At least 37 records · Page 2Linked to original sources
PubMed1996

Differences in serum cytokine levels in acute and chronic autoimmune thrombocytopenic purpura: relationship to platelet phenotype and antiplatelet T-cell reactivity.

Patients with both acute and chronic autoimmune thrombocytopenic purpura (AITP) have in vitro lymphocyte defects in the form of platelet-stimulated proliferation and cytokine secretion. A blinded study was performed to determine if these defects are related to serum cytokine levels and/or platelet antigen expression. Compared with controls, 53% of children with chronic AITP, but only 9% of those with acute AITP, had increased serum interleukin-2 (IL-2), interferon-gamma, and/or IL-10; however, none of the patients had detectible serum levels of IL-4 or IL-6, cytokine patterns suggesting and early CD4+ Th0 and Th1 cell activation. In children with chronic AITP, the levels of serum IL-2 correlated with in vitro platelet-stimulated IL-2 production. Few (17%) patients with AITP showed platelet activation, as measured by CD62 expression, or abnormal expression levels of platelet membrane glycoprotein (GP) IIbIIIa, but abnormal GPIb levels were observed in one-third of children with AITP. In contrast to normal controls and patients with nonimmune thrombocytopenia, a significant number of children with acute (80%), chronic (71%), or chronic-complex (55%) AITP and GPIb+ peripheral blood cells expressing HLA-DR. HLA-DR was variably coexpressed on distinct smaller and larger-sized GPIb+ cell populations with CD41, CD45, CD14, CD80, and/or glycophorin molecules. GPIb+ cells isolated from spleens of patients with chronic AITP had high expression (49% +/- 30%) of HLA-DR and splenic T cells had a high level of in vitro platelet-stimulated IL-2 secretion compared with controls. Platelet HLA-DR expression correlated inversely with platelet count, but not with therapy, serum cytokines, or in vitro lymphocyte antiplatelet reactivity. The results indicate that platelet HLA-DR expression is a common occurrence in patients with immune thrombocytopenia, whereas a large subpopulation of children with chronic AITP can be identified by increased serum cytokine levels and in vitro platelet-stimulated IL-2 secretion by lymphocytes, suggesting that differences exist in the immune pathogenesis of acute and chronic AITP, particularly at the level of platelet reactive T cells.

Acute Disease↗
PubMed1996

Characterization of platelet-reactive antibodies in children with varicella-associated acute immune thrombocytopenic purpura (ITP).

Biochemical analyses were performed on blood samples obtained from two children (P1, P2) who presented with acute immune thrombocytopenic purpura (ITP) following a recent varicella zoster virus (VZV) infection. Patient sera had antibodies that were reactive with normal blood-group O platelets as measured by flow-cytometric assay. Western blot analysis of electrophoretically separated normal blood-group O platelets under reducing and non-reducing conditions demonstrated that these sera were reactive with platelet antigens of approximately 50 and approximately 110 kD, respectively. These 50/110 kD antigens were not reactive with seven sera from acute ITP patients whose illness was not preceded by VZV infection, with serum from a patient with a prior history of VZV and no thrombocytopenia, nor with normal healthy control sera. VZV antibodies (IgG and IgM), isolated from patient sera by affinity chromatography using immobilized purified VZV glycoproteins, were found to bind to gel-filtered autologous platelets and with normal blood-group O platelets, as analysed by flow cytometry. No binding was observed using antibodies similarly prepared from healthy volunteer sera. To investigate their ability to sensitize platelets to complement activation, affinity-purified VZV antibodies were incubated with platelets and then with purified complement components C1 and 125 I-labelled C4. Platelets reacted with VZV-specific antibodies from the two patients and showed increases of 2.3-2.4-fold of platelet-surface deposition of 125 I-C4b, compared to controls. These data provide evidence that virus-specific antibodies occurring in children with varicella-associated acute ITP cross-react with normal platelet antigens, and may contribute to platelet clearance.

Antibodies, Viral↗
PubMed1996

Platelet-surface glycoproteins in healthy and preeclamptic mothers and their newborn infants.

Preeclampsia, a common complication of pregnancy, contributes significantly to maternal and fetal morbidity and mortality. It may lead to both quantitative and qualitative defects of maternal and neonatal platelets. In this prospective study, flow cytometry has been used to study expression of platelet-surface glycoproteins (GPs) on maternal and neonatal platelets of both healthy and preeclamptic subjects. We studied 15 preeclamptic women, 20-44 y of age, and their newborns (median gestational age, 32 wk; range, 26-38) and seven healthy women (aged 26-41 y) and their healthy newborns (median gestational age, 38 wk; range, 38-42). Compared with their healthy and preeclamptic mothers, resting platelets from neonates expressed significantly less CD41 and CD9. Thrombin activation resulted in significant increases in platelet-surface expression of CD62P, CD63, CD41, CD9, and CD36 in neonates and their healthy mothers. Compared with neonates of healthy mothers, platelets from neonates of preeclamptic mothers expressed lower levels of CD62P, CD63, CD9, and CD36 on activated platelets. These findings suggest that preeclampsia influences the expression of platelet-surface GPs on neonatal and maternal platelets, which may affect platelet function, leading to an additional risk for bleeding in thrombocytopenic neonates of mothers with preeclampsia.

Adult↗
PubMed1995

Indirect allorecognition of platelets by T helper cells during platelet transfusions correlates with anti-major histocompatibility complex antibody and cytotoxic T lymphocyte formation.

To study the cellular immunology of platelet-induced alloimmunization, a murine transfusion model was developed. BALB/c (H-2d) recipient mice were transfused weekly with 2 x 10(8) platelets or 10(3) leukocytes from C57BL/6 (H-2b) donor mice. Recipient antidonor major histocompatibility complex (MHC) class I alloantibodies could be detected in flow cytometric assays by the fifth platelet transfusion. In contrast, when leukocytes only were transfused, alloantibodies were not detected. In vitro assays demonstrated that murine H-2b platelets were positive for MHC class I expression but lacked MHC class II molecules on their membranes and were unable to stimulate proliferation or cytokine production when incubated with naive H-2d spleen cells. In vivo, however, platelet transfusions induced two distinct patterns of cell-mediated reactivity. First, during the initial transfusions and before alloantibody formation, there was induction of T-cell anergy, characterized by the inability of recipient T cells to respond to Concanavalin A (ConA) or to proliferate in an antidonor mixed lymphocyte reaction (MLR), together with suppressed natural killer (NK) cell activity. This unresponsiveness was associated with a transient increase in nitric oxide (NO)-dependent cytotoxicity and interleukin-1 (IL-1) production. Second, once alloantibodies developed, significantly increased antidonor CD8+ cytotoxic T lymphocyte (CTL) and NK cell responses were observed. At this time, when recipient spleen cells were depleted of CD8+ T cells and incubated with only donor platelets in 7-day antigen-presenting cell (APC) assays, enhanced proliferation and IL-2 production occurred. These cellular responses were not seen when 10(3) allogeneic leukocytes were transfused. Thus, the results suggest that leukoreduced platelet transfusions induce antidonor MHC antibodies and CD8+ CTL responses in recipient mice. At the same time, the transfusions induced recipient CD4+ T-cell activation when incubated with donor platelets in the presence of syngeneic APCs, an indirect recognition pathway that correlates with the time of alloantibody production.

Animals↗
PubMed1994

Naturally processed heterodimeric disulfide-linked insulin peptides bind to major histocompatibility class II molecules on thymic epithelial cells.

We determined whether disulfide-linked insulin peptides that are immunogenic in vitro for CD4+ T cells bind to major histocompatibility complex class II in vivo. Radiolabeled recombinant human insulin (rHI) was injected into BALB/c mice, and processed rHI peptides bound to I-Ad molecules on different thymic antigen-presenting cells were characterized. The A6-A11/B7-B19 and A19-A21/B14-B21 disulfide-linked I-Ad-bound rHI peptides were isolated from thymic epithelial cells but not dendritic cells. While both thymic epithelial cells and dendritic cells present rHI to HI/I-Ad-specific T cells, these antigen-presenting cells do not present the reduced or nonreduced forms of the disulfide-linked rHI peptides. Thus, a naturally processed disulfide-linked peptide can bind to major histocompatibility complex class II in vivo. The potential role of these peptides in immunological tolerance is discussed.

Amino Acid Sequence↗
PubMed1993

Downregulation of the anti-HLA alloimmune response by variable region-reactive (anti-idiotypic) antibodies in leukemic patients transfused with platelet concentrates.

Approximately 30% to 40% of patients with acute leukemia receiving repeated pooled random-donor platelet transfusions develop anti-HLA alloantibodies. Over time, however, serum anti-HLA concentrations decrease in approximately 50% of these patients, despite continued exposure to platelet and/or red blood cell transfusions. Using an enzyme-linked immunosorbent assay to measure serum Igs, the present study demonstrates that the sera of 67% of 82 transfused patients exhibiting a decrease in anti-HLA contain antibodies (anti-idiotypes) that react with the variable (V) region of anti-HLA antibodies. Anti-HLA binding to platelet membranes could be inhibited by these serum antibodies in 36% of the patients, indicating they had paratope-related reactivity. Protein G sepharose absorption showed that the anti-HLA V region-reactive antibodies were IgG. Of the 43 patients who had a decrease in anti-HLA levels, that were 16 whose anti-HLA decreased to undetectable levels; 7 (44%) developed anti-idiotypic antibodies that could specifically inhibit their own previously anti-HLA-positive serum. In contrast, antibodies with reactivity to the V region of anti-HLA antibodies (anti-idiotypes) were not demonstrable in patients who developed anti-HLA that did not decrease or disappear. The findings suggest that the development of anti-HLA V region-reactive antibodies (anti-idiotypic antibodies) correlates with a decrease in anti-HLA antibody formation in patients multiply transfused with platelet concentrates. The observations indicate that anti-idiotypic antibodies may downregulate alloimmune responses in patients undergoing repeated allostimulation during platelet transfusion therapy.

Acute Disease↗
PubMed1993

Rapid separation of CD4+ and CD19+ lymphocyte populations from human peripheral blood by a magnetic activated cell sorter (MACS).

Rapid purification of human lymphocyte subpopulations is an essential step in order to elucidate their interactions and/or contributions in various disease states. Cell purification using a Magnetic Activated Cell Sorter (MACS) is a relatively new technology which has been shown to be rapid and yield highly purified populations of cells. This report describes both a simple one-step positive selection method using the MACS to purify either human CD4+ or CD19+ lymphocytes from PBMC and a sequential separation of both CD4+ and CD19+ cell populations. These methods can separate the cell populations in approximately 4 h with yields > 90% and purity of 97 +/- 3% for CD4+ T cells and 92 +/- 5% for CD19+ B cells. In functional studies, purified CD19+ B cells secreted 13- and 24-fold more IgM and IgG, respectively, than the CD19- cell fraction in 10 day B cell stimulation assays. Purification of the two cell types did not cause any significant activation as shown by proliferation. Both cell types, however, were able to proliferate upon stimulation with interleukin-2.

Antigens, CD↗
PubMed1992

Processing and presentation of insulin. III. Insulin degrading enzyme: a neutral metalloendoproteinase that is non-homologous to classical endoproteinases mediates the processing of insulin epitopes for helper T cells.

Presentation of a protein antigen to T cells generally requires that the antigen be enzymatically processed into an immunogenic peptide(s). The identification of a protease(s) and its mechanism of action in the proteolysis of such an antigen is therefore a primary goal in the study of antigen processing. We show here that insulin degrading enzyme (IDE), a neutral thiol metalloendoproteinase that is structurally non-homologous to the classical metallo, thiol, acid, or serine proteinases, is relatively specific in its proteolytic activity for insulin and digests human insulin (H(I)) into peptides that are presented by murine TA3 B cell antigen presenting cells (APCs) to HI/I-Ad-reactive T cells. These peptides are, however, not presented by fixed TA3 APCs. Anti-IDE mAbs, after their internalization by TA3 cells, significantly inhibit the presentation of H(I) by these APCs. Immunoblotting experiments demonstrate that this inhibition is mediated by the reactivity of these mAbs with a 110 kDa protein, the known M(r) of IDE. These data show that IDE is an endoproteinase that is involved in the processing of insulin and that this IDE-mediated proteolysis is necessary but not sufficient for the recognition of insulin by T cells. Furthermore, we demonstrate that reduction of the disulfide bonds of a pre-processed A-loop containing heterodimeric insulin peptide is required to further process insulin into a T cell epitope.

Animals↗
PubMed1992

Cellular immune mechanisms in chronic autoimmune thrombocytopenic purpura (ATP).

Chronic autoimmune thrombocytopenic purpura (ATP) is a common autoimmune-mediated bleeding disease in which autoantibodies are directed against platelets, resulting in their enhanced Fc-mediated destruction by macrophages in the spleen. While there has been extensive studies relating to the autoantibodies in this autoimmune disorder, relatively few have dealt with cell-mediated immunoregulation of the anti-platelet autoantibody response. Nonetheless, there is accumulating evidence that suggests the production of these anti-platelet autoantibodies is under the influence of several abnormal lymphocyte-mediated mechanisms, i.e. enhanced anti-platelet T helper cell activity with concomitant reduced T suppressor cell activity. This review focuses on these cellular events and presents a working model which attempts to explain their close interrelationships.

Autoantibodies↗
PubMed1991

Increased antiplatelet T helper lymphocyte reactivity in patients with autoimmune thrombocytopenia.

Chronic autoimmune thrombocytopenic purpura (ATP) is a common hematologic disorder in which platelet-specific autoantibodies bind to platelets and enhance their destruction by the reticuloendothelial system. While there has been considerable investigation of the humoral immune abnormalities in ATP, little work has been performed on the cellular immunoregulatory aspects of this autoimmune disorder. We describe here that patients with ATP have lymphocytes that proliferate normally when stimulated by mitogens. However, when stimulated by normal control platelets in 7-day antigen-presenting cell cultures, peripheral blood mononuclear cells (PBMC) from patients with ATP proliferate at significantly higher levels (P less than .001) and their lymphocytes secrete significantly higher amounts of interleukin-2 (IL-2) (P less than .001) than do lymphocytes from control subjects. Depletion studies with monoclonal anti-CD8 and complement did not reduce the proliferative capacity of the responding PBMC population, indicating that CD4+ T-helper cells may be responsible for the response. Phenotypic analysis of peripheral blood lymphocyte subsets from patients with ATP showed that there was a significant reduction in CD4+Leu8+ T suppressor-inducer cells (P less than .001) and a concomitant increase in CD3+DR+ activated T cells (P less than .001) and CD19+ B cells (P less than .05). These data indicate that CD4+ T-helper cells from patients with ATP are stimulated by normal platelet antigen(s) to secrete IL-2 and may modulate the enhanced antiplatelet autoantibody response.

Adult↗
PubMed1991

Suppressed natural killer cell activity in patients with chronic autoimmune thrombocytopenic purpura.

Natural killer (NK) cell activity was studied in 17 patients with primary chronic idiopathic autoimmune thrombocytopenic purpura (ATP). Fifteen of 17 patients had a significantly reduced NK cytotoxicity against 51chromium labeled K562 target cells (mean LU20% = 18 +/- 20 in patients versus 65 +/- 25 in controls, P less than 0.001). NK activity was also significantly reduced in all of six patients with secondary ATP as compared with normal controls (LU20% 28 +/- 15, respectively, P less than 0.005). The NK activity in both patient groups correlated with the duration of therapy being received (r = 0.60, P less than 0.001). Immunophenotypic analysis of peripheral blood mononuclear cells from patients with ATP revealed that CD8- cells bearing CD57 (HNK-1, Leu 7) and CD3- cells bearing CD56 (Leu 19) were quantitatively within the normal range. These findings indicate that patients with ATP have a functional defect in NK cytolytic activity.

Adolescent↗
PubMed1991

Altered processing of human insulin by B lymphocytes from an immunologically insulin-resistant type I diabetic patient.

Immunologically insulin resistant (IIR) type I diabetic patients possess significantly elevated levels of anti-insulin serum autoantibodies. We investigated whether altered insulin processing by B lymphocytes contributes to this form of insulin resistance. A comparison was made of the 125I-labelled human insulin (HI) peptides processed by Epstein-Barr virus (EBV)-transformed B lymphocytes derived from HLA-identical and nonidentical IIR with non-IIR type I diabetic patients on insulin therapy and healthy non-diabetic individuals. Several peptides detected in the extracellular, membrane-associated and intracellular compartments of B cells from an IIR type I diabetic patient differed from those found in the corresponding compartments of B cells from two non-IIR type I diabetic patients and two normal individuals. These data suggest that HI is processed differently by B cells from an IIR type I diabetic patient compared with B cells from non-IIR type I diabetic patients and normal individuals. Further, we found that two of the five plasma-membrane associated processed HI peptides on the IIR patients' B cells were absent from the membrane compartments of the other B cell lines examined. Thus, it is possible that one or both of these peptides, unique to the IIR patient's B cells, consist of an immundominant epitope(s) that stimulates the production of insulin autoantibodies which mediate the onset of IIR in type I diabetes.

Antigen-Presenting Cells↗
PubMed1989

Processing and presentation of insulin. II. Evidence for intracellular, plasma membrane-associated and extracellular degradation of human insulin by antigen-presenting B cells.

To study the biochemistry of processing of a soluble protein Ag by an APC, we investigated how 125I-labeled human insulin (HI) is processed in situ by TA3 mouse hybridoma B cells. Fractionation of TA3 cells into their extracellular, plasma membrane-associated and intracellular compartments coupled with the use of HPLC enabled us to analyze several peptides derived from each compartment. One HI peptide found in all three compartments is composed of residues A1-A14 disulfide-linked to B7-B26 (A1-A14/B7-B26). The presence of this peptide in the extracellular compartment likely resulted from digestion of HI by an enzyme(s) released from the APC. Extracellular processing of radiolabeled HI was inhibited completely by unlabeled HI and N-ethylmaleimide, an inhibitor of a previously described insulin-specific protease, partially by lysozyme but not by BSA or OVA. This suggests that the enzyme involved in the extracellular processing of insulin is relatively insulin-specific and gives rise to the A1-A14/B7-B26 peptide. The processing of HI both at the plasma membrane and intracellularly was inhibited by chloroquine, monensin, and NH4Cl, suggesting that both intracellular pH changes and endocytic and exocytic events may be required for these compartments to process insulin. Kinetic analyses revealed that the processing of insulin into the A1-A14/B7-B26 peptide is first detected at the plasma membrane then intracellularly and finally in the extracellular compartment. This unlabeled A1-A14/B7-B26 peptide was purified from the extracellular compartment of TA3 APC by HPLC; when presented by TA3 APC this peptide effectively stimulated pork insulin (PI/I-Ad) specific Th cells to secrete IL-2. These data, taken together with the identification of another processed insulin peptide, A7-A11/B7-B26, have enabled us to elucidate the first steps in the biochemical pathway(s) of processing of insulin as an Ag in a B cell APC.

Animals↗
PubMed1989

Sulfated beef insulin treatment elicits CD8+ T cells that may abrogate immunologic insulin resistance in type I diabetes.

The in vitro responses of T cells from 13 insulin-nonresistant and 1 immunologically insulin-resistant (IIR) type I diabetes patients to sulfated beef insulin (SBI) were analyzed. Insulin A-loop specific CD4+ T cells from these patients did not respond to SBI. After 1 yr of treatment with SBI the IIR patient's T cell and antibody responses to beef, pork, and human insulin progressed from very high to nondetectable levels. This occurred in parallel to the appearance of her insulin-specific CD8+ T cells, which inhibited the response of her A-loop-specific CD4+ T cells to insulin. A transient increase in her CD8+ anti-insulin antibody activity coincided with a relative lack of her CD8+ T cell activity. CD8+ T cells that regulate T cell responsiveness to insulin are probably present but difficult to detect in most type I diabetes patients. These T cells were identified in only 2 of 13 insulin-nonresistant patients who presented with lipoatrophy and insulin allergy, respectively, and who possessed high-titered, anti-insulin antibodies. Our data demonstrate that CD8+ T cells play an important role in controlling peripheral tolerance to insulin and may abrogate IIR in a diabetic patient treated with SBI.

Adult↗
PubMed1988

Purification and characterization of radiolabelled biosynthetic human insulin from Escherichia coli. Kinetics of processing by antigen presenting cells.

An Escherichia coli strain transfected with a plasmid containing four linked human proinsulin genes was grown in the presence of 35S and 3H labelled amino acids to gain access to human insulin that was radiolabelled at 19 evenly distributed sites throughout the amino acid sequence. The multi-proinsulin precursor was cleaved at methionine residues with cyanogen bromide, then the individual proinsulin units were folded via their S-cysteine sulfonate derivative and converted to insulin by enzymatic digestion. Purification steps were carried out by ion-exchange and reverse-phase HPLC techniques. The final radiolabelled biosynthetic human insulin was produced at a specific activity of up to 300 Ci/mmol, and was shown to be indistinguishable from commercially available human insulin according to HPLC behavior, amino acid analysis, immunoreactivity and biological activity. A comparison of the kinetics of processing of 35S/3H-labelled biosynthetic human insulin and 125I-labelled commercial human insulin by murine TA3 hybridoma antigen presenting cells demonstrated that radiolabelled biosynthetic insulin was processed approximately 16 times slower than its iodinated counterpart. Measurable 125I TCA soluble radioactivity was detected extracellularly within 15 min whereas the same amount of extracellular TCA soluble 3H/35S radioactivity was not seen until 240 min. These results begin to address the importance of using a biosynthetically labelled protein as opposed to an iodinated protein to study how an APC handles antigen in a physiological manner.

Antigen-Presenting Cells↗