Monitoring unfractionated heparin in pediatric patients with congenital heart disease having cardiac catheterization or cardiac surgery.

Determine the effect of age and congenital heart disease (CHD) on whole blood tests for monitoring unfractionated heparin (UFH) in children. Determine correlation with anti-Xa levels in children undergoing cardiac catheterization or cardiac surgery. A prospective cross-sectional study of 211 healthy children about to have minor surgery (median age 3.5 years) and 110 CHD patients (median age 2.1 years) undergoing cardiac catheterization or cardiac surgery. Commonly used whole blood tests (two activated clotting times and an activated partial thromboplastin time; ACT+, ACT-LR, and APTT, respectively) were obtained before procedures and after UFH in CHD patients. Data were analyzed for effect of age and CHD and correlation with anti-Xa levels. In healthy subjects the ACT+ was lower in younger (<3 years) patients while the ACT-LR and APTT were unaffected. CHD patients exhibited an opposite trend with higher values in the younger patients. After bolus heparin the ACT+ exhibited the strongest correlation (r = 0.89) with anti-Xa levels in both locations (the APTT was too sensitive at post-bolus levels). When anti-Xa levels were below 1.0 IU/ml (range of thromboembolism therapy 0.35-0.7 IU/ml), the APTT correlation coefficient was 0.72. Some whole blood coagulation tests are affected by age in healthy children similar to laboratory tests and are variably influenced by the presence of CHD. ACT+ is the most reliable predictor of anti-Xa levels in both catheterization and surgery for pediatric patients. The APTT exhibited stronger correlation with anti-Xa than previous reports of laboratory APTT and warrants further evaluation for monitoring heparin thromboembolism therapy.

Identification of Bcd, a novel proto-oncogene expressed in B-cells.

A novel B-cell derived (Bcd) oncogene has been isolated from the peripheral blood lymphocytes of one B-cell chronic lymphocytic leukemia (B-CLL) patient using DNA transfer and a mouse tumorigenicity assay. The Bcd proto-oncogene was activated by a truncation in the 5' UTR. It predicts for two open reading frames (ORFs). ORF1 consists of 240 bp that would encode 80 amino acids, while the major ORF2 consists of 648 bp capable of coding for 216 amino acids. Predicted peptide sequence of ORF2 contained a zinc finger domain which showed significant homology to GC box binding proteins BTEB2 and SP1. Transfection of an expression vector containing ORF2 but not full length cDNA was able to transform NIH3T3 cells and induce tumors in nude mice. Bcd mRNA transcripts of < or = 2.6 kb were selectively expressed in PBL and testis of healthy individuals. Within the PBL, Bcd gene expression was restricted to CD19+ B-cells and absent from CD14+ monocytes and T-cells. Bcd transcripts were detected in all normal PBL samples tested but not in several malignant human B-cell lines and not in 50% of B-cells from B-CLL patients. However, stimulation of B-cells from B-CLL patients under conditions which induced differentiation into plasma cells was associated with induction of Bcd gene expression. The Bcd gene may therefore play an important role in B-cell growth and development.

Drug-induced apoptosis in B-cell chronic lymphocytic leukemia: relationship between p53 gene mutation and bcl-2/bax proteins in drug resistance.

We investigated the relationship among chemosensitivity to drug-induced apoptosis in vitro, the presence of p53 gene mutations, and the expression of bcl-2 and bax proteins in B-cells from B-cell chronic lymphocytic leukemia (B-CLL) patients. Apoptosis was induced with a camptothecin analogue, 9-amino-20(s)-camptothecin, or a purine analogue, fludarabine. Cell death was monitored by propidium iodide staining and FACS analysis. Drug-induced apoptosis in B-CLL cells was p53-independent. Immunoblot analysis of bcl-2 and bax expression revealed a correlation between drug-induced apoptosis and the ratio of endogenous levels of bcl-2 to bax proteins. B-CLL cells with none to low bcl-2/bax ratios were drug-sensitive as compared to cells with intermediate to high ratios that were drug-resistant (P = 0.015). Prior to drug treatment, bax protein migrated as a single species of 21 kDa. Following drug-induced apoptosis, anti-bax specific protein complexes of 36-42 kDa were up-regulated. Using two-dimensional gel electrophoresis, bax complexes were disrupted under reducing conditions to reveal homo- and heterodimers of 18 and 21 kDa suggesting that disulfide interactions were required for complex formation. The de novo appearance of the 18 kDa anti-bax specific protein together with its increased expression in drug-sensitive B-CLL B-cells undergoing cell death suggests a role for this protein in the regulation of apoptosis.

A unique spectrum of p53 mutations in B-cell chronic lymphocytic leukemia distinct from that of other lymphoid malignancies.

The spectrum and pattern of p53 mutations detected in 42 cases of B-cell chronic lymphocytic leukemia (B-CLL) were analyzed, and several interesting features were noted. Codon 209 in the p53 gene may be a new hot-spot for p53 mutation in B-CLL disease. Four of the 42 (10%) reported B-CLL p53 mutations occurred at codon 209 versus none in 214 cases of other lymphoid malignancies screened for p53 mutations (P = 0.0006). Transversion mutations predominated at codon 273 rather than the transition mutations that are known to occur at this CpG site. Four of six (67%) B-CLL cases had transversions at codon 273 compared with two of 17 (12%) of all other lymphoid tumors examined (P = 0.02). In addition, over 65% of the p53 mutations detected in B-CLL showed a strand bias for p53 mutations on the untranscribed DNA strand. This feature of DNA strand bias is notable in cancers of the lung, esophagus, and head and neck, which may result from high exposure to carcinogens. This spectrum of p53 mutations in B-CLL together with the high frequency of transversion mutations and DNA strand bias may implicate environmental carcinogens associated with p53 gene damage in some B-CLL patients.

Identification of melanoma cell surface antigens immunogenic in mice.

Active immunization to B16 melanoma cells or vaccines induces anti-melanoma immune responses in syngeneic mice. The immunogenic antigens stimulating immunity to this tumor have not been identified. In this study we detected several B16 melanoma antigens immunogenic in syngeneic mice using as probes antimelanoma antibodies induced by immunization to B16 melanoma vaccines. These antigens were identified by SDS-PAGE and autoradiographic analysis of specific immunoprecipitates. They were cell-surface components with approximate molecular weights of 41, 46, 50, 75, 80, and 104 KD. All these antigens were expressed by syngeneic and xenogeneic melanomas and by some unrelated syngeneic tumors but not by normal syngeneic cells, xenogeneic melanocytes, or by B16 melanoma cells obtained from fresh tumors or grown in defined medium. The antigens were distinct from murine viral antigens expressed by B16 melanoma cells and from components of the culture medium used to grow cells for vaccine production. These results indicate that several B16 melanoma cell-surface antigens are immunogenic in syngeneic mice. Expression of these antigens appears to be related to malignant transformation as they were found on all melanomas studied, and some other cancers, but not on normal cells.

p53 gene mutation in B-cell chronic lymphocytic leukemia is associated with drug resistance and is independent of MDR1/MDR3 gene expression.

We studied 53 patients with B-cell chronic lymphocytic leukemia (B-CLL) and found mutations of the p53 gene in 15%. Patients with p53 gene mutations were found to have an aggressive form of B-CLL disease characterized by advanced Rai stage, rapid lymphocyte doubling time (LDT), and resistance to chemotherapy. While 27 of 29 treated patients (93%) without p53 mutations achieved a partial remission, only one of seven treated patients (14%) with p53 mutations achieved a partial remission (P = .00009). Adjusting for prognostic factors (age, sex, race, and Rai stage), patients with p53 gene mutations had a 13-fold greater risk of death than patients without p53 mutations (P = .013). In addition to examining the clinical relevance of p53 gene mutations in B-CLL, we investigated the possible role of p53 gene regulation in the expression of the multidrug resistance genes MDR1 and MDR3. We quantitated MDR1 and MDR3 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR). Expression of both the MDR1 and MDR3 genes was independent of p53 gene mutation or prior drug treatment, and did not predict for clinical response. Our findings indicate that p53 gene mutations in B-CLL are associated with a poor clinical outcome and may be a prognostic indicator for drug resistance.

Comparison of the properties of the CsA analogs monoacetyl CyC (o-acetyl-threonine2 cyclosporin) and methyl-alanyl CsA (N-methyl-L-alanyl6 cyclosporin); monoacetyl cyclosporin is immunosuppressive without binding to cyclophilin.

Cyclosporin (CsA) is an immunosuppressant which binds to cyclophilin (Cyp). The relationship between Cyp binding and immunosuppression has been questioned since one of the analogs of CsA, N-methyl-L-alanyl6 cyclosporin (methyl-alanyl CsA) binds to Cyp but is not immunosuppressive. We compared the immunosuppressive properties of CsA, methyl-alanyl CsA and o-acetyl-threonine2 cyclosporin (monoacetyl CyC), since monoacetyl CyC does not bind to Cyp when tested in cell-free assays and its immunosuppressive properties had not been tested. Cyp is a peptidyl-prolyl isomerase which is abundant in all human tissues, yet the activities of CsA are mostly confined to inhibition of T cell and thymocyte activation, and to neuro- and nephro-toxicity and are independent of inhibition of the isomerase. Activation of thymocytes and of T cells is regulated by the binding of a nuclear factor(s) (NFs) to the NF-AT region (-285 to -255) of the IL-2 promoter. We studied inhibition of binding to the NF-AT region of NFs derived from primary cultures of thymocytes treated with CsA or its analogs. In addition, we compared the effect of CsA and its analogs on the expression of the IL-2 gene in a stably transfected Jurkat-cell line (Fgl 5) which contains three copies of NF-AT and the reporter enzyme beta-galactosidase; and on inhibition of proliferation induced by concanavalin A (Con A) or IL-2. We found that monoacetyl CyC which does not bind to Cyp is immunosuppressive by our criteria when tested in cultured cells due to either a different mechanism of action or to metabolic activation.

Repopulation potential of thymocytes forming rosettes with phagocytic cells of the thymic reticulum.

Thymocytes binding in vitro to phagocytic cells of the thymic reticulum (P-TR), termed 'rosetting thymocytes', were injected intravenously into irradiated congenic mice and their migration patterns were compared with those that do not bind to P-TR, called 'non-rosetting thymocytes', similarly transferred. Donor cells, C57BL/Ka Thy 1.2, were distinguished from recipient cells, C57BL/Ka Thy 1.1 by a direct immunofluorescence technique using an anti-Thy 1.2 monoclonal antibody. The results demonstrate that the rosetting thymocytes have a greater capacity for homing back to the thymus and for populating the mesenteric lymph node and the spleen. Intrathymic transfer assay revealed that the donor-derived cells detected in the peripheral organs were of thymic origin.

Control of helper-T-cell proliferation by recognition of Ia and Mac-1 antigens on phagocytic cells of the thymic reticulum.

Phagocytic cells of the thymic reticulum (P-TR) have been previously described as being Ia-positive, Mac-1-positive accessory cells which pursue a close relationship with thymocytes. They form rosettes with thymocytes, and these rosettes are inhibited by antibody directed against the complement receptor type 3 CR3 (anti-Mac-1). P-TR induce the proliferation of syngeneic thymocytes. In the present paper, we show that thymocytes enriched in mature medullary type are induced to proliferate in coculture with syngeneic P-TR, while the cortical type does not. After 5 days of culture, 85% of the thymocytes are of helper L3T4+Lyt-2- phenotype. As previously shown by others for syngeneic reactions, antibodies directed against related class II antigens (anti-I-A and anti-I-E) block this helper-T-cell syngeneic proliferation. A new finding is the blockage of helper-T-cell proliferation by anti-Mac-1 as well as with anti-LFA-1 antibodies, showing that accessory molecules may be as important as specific recognition of class II antigen molecules in the control of thymocyte proliferation and hence in thymocyte selection. Mac-1, like LFA-1, belongs to a novel family of differentiation antigens involved in cell interactions. The blockage of cell recognition and interaction between P-TR and thymocytes by either anti-Ia or anti-Mac-1 during the early induction phase of the syngeneic response leads to its inhibition. We demonstrate that P-TR/thymocyte interaction stimulates the enhanced expression of IL-2 receptors on thymocytes, a step which is necessary for helper-T-cell proliferation. The mechanism of syngeneic proliferation inhibition by anti-Ia, anti-Mac-1, and LFA-1 antibodies may be the prevention of IL-2 receptor expression on thymocytes, and/or the inhibition of IL-2 secretion. Although this is an in vitro model, which may not totally reflect in situ situation, our results indicate that thymic accessory cells may participate in a positive selection process which leads to helper-T-cell proliferation.

Control of prothymocyte proliferation by thymic accessory cells.

All thymocyte subpopulations derive from intrathymic precursors which are double negative (DN) for Lyt-2 and L3T4 differentiation antigens. Although nearly half of DN cells express a receptor for interleukin 2 (IL 2R), they respond poorly to IL 2. DN cell proliferation can be obtained in the presence of various exogenous stimuli, but the in vivo signal for DN cell response to IL 2 remains unclear. We show in the present report that phagocytic cells of the thymic reticulum are able to induce the proliferation of DN thymocytes in the presence of recombinant IL 2 (rIL 2). Cell-to-cell contact is needed for this effect. Antibodies directed against class I MHC antigens but not against class II can inhibit DN cell proliferation. DNA-synthetizing cells were labeled by incubation with 10 microM bromodeoxyuridine either before or at various times during the culture period. Bromodeoxyuridine was then detected in the DNA of proliferating cells and/or their progeny already stained with anti-Lyt-2 and L3T4 antibodies. During the initial 16 h and independently of culture conditions, 16-25% of the cells expressed surface antigens and 50-65% of them derived from DN cells which were in S phase just before culture; these differentiated cells had a very short life span. In the second culture period, the presence of both rIL 2 and thymic accessory cells was necessary for cell survival. In these conditions, DN cell number and proliferation rate were constant and a low number of Lyt-2+ and/or L3T4+ cells was continuously generated. Thymic accessory cells therefore appear to provide the signal(s) necessary for IL 2-induced proliferation of thymocyte precursors. Implications of these findings for normal in vivo intrathymic proliferation and differentiation are discussed.

Con A response and interleukin 2 receptor expression of thymocytes rosetting with phagocytic cells of the thymic reticulum.

Thymocytes bind to the phagocytic cells of the thymic reticulum (P-TR) and spontaneously forming rosettes were isolated and characterized. Rosetting thymocyte subsets included cells expressing different surface phenotype: most of them (91%) were of the double positive L3T4+ Lyt-2+ phenotype; a minor subpopulation (2.4%) expressed the helper L3T4+ Lyt-2- phenotype and finally a small subpopulation (6.3%) was negative for both L3T4 and Lyt-2 markers. Although the rosetting thymocytes were not enriched in mature single positive cells as compared to the non rosetting population, they showed a higher responsiveness to ConA plus recombinant IL-2. Moreover the rosetting thymocytes were induced to express IL-2 receptors just after stimulation with ConA whereas co-stimulation with ConA plus IL-2 was essential for the generation of IL-2 receptors on the non rosetting and total thymocytes. The possibility that the rosetting thymocytes acquired this activity during the rosetting process is discussed.

Thymic reticulum in mice. IV. The rosette formation between phagocytic cells of the thymic reticulum and cortical type thymocytes is mediated by complement receptor type three.

A phagocytic cell of the thymic reticulum (P-TR) with dendritic shape recently has been isolated and characterized. We have previously shown that P-TR have an important role to play in the constitution of the thymic microenvironment. Indeed, P-TR are able to produce interleukin 1 and prostaglandin E2, both of which regulate thymocyte activation and proliferation. They are able also to stimulate the proliferation of syngeneic thymocytes enriched in the medullary type. In the present paper, we analyze a close relationship which exists between P-TR and thymocytes of the cortical type. About 25% of P-TR are able to bind to thymocytes and to form rosettes. Rosetting thymocytes represent about 5% of the total population and are PNA+, Lyt 1+2+, H-2-, and sensitive to in vivo steroid treatment. Pretreatment of P-TR with anti-Mac-1, a monoclonal rat IgG antibody against mouse macrophages and specific for complement receptor type three (CR3), abolished rosette formation. Rosette formation also was found to be inhibited by zymosan-treated serum containing the CR3 ligand, C3bi, and by certain sugars, in particular, N-acetyl-D-galactosamine and L-xylose. Our results suggest that rosetting thymocytes bind to CR3 on the P-TR membrane and that sugar constituents of the carbohydrate moieties on the thymocyte surface may serve as a recognition site during the binding process.

Delayed hypersensitivity and migration inhibition in two lines of mice genetically selected for high or low responsiveness to phytohemagglutinin.

Delayed-type hypersensitivity (DTH) and cell migration inhibition (MI) were studied in two lines of mice genetically selected for the high (Hi/PHA) or low (Lo/PHA) in vitro response of their lymphoid cells to phytochemagglutinin (PHA). A rapid photoelectric procedure for reading cell migrations enabled the study of MI over a wide range (10 log) of antigen concentrations in vitro. Hi/PHA mice required immunization with a 10 times higher dose of ovalbumin (OVA) in Freund's complete adjuvant (FCA) than Lo/PHA mice for a comparable response in DTH (footpad swelling) and MI of their induced peritoneal exudate cells (PEC). Lo/PHA spleen showed marked bizonal MI on Day 5 after immunization with low doses (0.1 and 0.5 micrograms) of OVA in FCA, one peak being obtained in presence of in vitro concentrations of 10(-3) or 10(-2) micrograms/ml OVA and another peak at 1 or 10 micrograms/ml, whereas Hi/PHA spleen showed stimulation of migration. In contrast, MI in Lo/PHA spleen failed to persist beyond Day 19, whereas it appeared progressively in Hi/PHA spleen, being maximal by Day 27. Low-zone inhibition in Hi/PHA spleen and PEC was lacking or poor even after immunization with higher doses of OVA in FCA. The implications of these findings are discussed.