Lymphocyte transformation in vitro : V. Thymidine incorporation into unstimulated lymphocytes

In vitro conditions and kinetics of ¹⁴C-thymidine incorporation into unstimulated lymphocytes were studied. Lymphocytes cultured during 3 to 9 days displayed a progressive increase of thymidine uptake with time. The addition of varying numbers of autologous mitomycin-treated lymphocytes to cultures containing a fixed number of untreated lymphocytes markedly enhanced thymidine uptake per non-mitomycin-treated lymphocytes. In the latter cultures a sharp rise of thymidine uptake between the 7th day of culture was seen. Supernatants of non-stimulated lymphocytes, whether mitomycin-treated or not, showed no stimulating effect on autologous normal cells in these experiments. Although the mechanism of the enhancing effect of mitomycin-treated autologous lymphocytes remains unclear, it obviously may interfere in culture experiments in which the antigen specific stimulatory effect of mitomycin-treated cells is measured, which can only be detected by including the appropriate controls.     

Immunodiagnosis of platelet activation in immune thrombocytopenia through scFv antibodies cognate to activated IIb3 integrins

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by low platelet count and presence of IgG autoantibodies to platelet surface glycoproteins, such as αIIbβ3 and GPIb/IX. Our previous work has shown that platelets in ITP patients exist in an activated state. Two different marker-based approaches are used to study the course of platelet activation: (1) binding of PAC-1 antibody, signifying a change in αIIbβ3 conformation, and (2) expression of P-selectin, signifying alpha granule content release from platelets. Here, we describe the development of a new scFv antibody (R38) that, compared with PAC-1, appears to better distinguish between platelets of ITP patients and healthy controls. Notably, R38 was generated using commercially sourced resting-state integrin that was coated on a microtiter plate. Its ability to distinguish between ITP patients and healthy controls thus suggests that inadvertent integrin activation caused by coating involves a conformational change and exposure of a cryptic epitope. This report also describes for the first time the potential use of an scFv antibody in the immunodiagnosis of platelet activation in ITP patients.     

Immunodiagnosis of platelet activation in immune thrombocytopenia through scFv antibodies cognate to activated IIb3 integrins

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by low platelet count and presence of IgG autoantibodies to platelet surface glycoproteins, such as $$\alpha$$IIbβ3 and GPIb/IX. Our previous work has shown that platelets in ITP patients exist in an activated state. Two different marker-based approaches are used to study the course of platelet activation: (1) binding of PAC-1 antibody, signifying a change in αIIbβ3 conformation, and (2) expression of P-selectin, signifying alpha granule content release from platelets. Here, we describe the development of a new scFv antibody (R38) that, compared with PAC-1, appears to better distinguish between platelets of ITP patients and healthy controls. Notably, R38 was generated using commercially sourced resting-state integrin that was coated on a microtiter plate. Its ability to distinguish between ITP patients and healthy controls thus suggests that inadvertent integrin activation caused by coating involves a conformational change and exposure of a cryptic epitope. This report also describes for the first time the potential use of an scFv antibody in the immunodiagnosis of platelet activation in ITP patients.     

Biosynthesis of major platelet proteins in human blood platelets

We studied de novo protein biosynthesis in platelets of normal adult donors and in newly formed platelets isolated from splenectomized patients with idiopathic thrombocytopenic purpura (ITP). After metabolic labelling of platelet proteins, performed with different radiolabelled amino acids or carbohydrates, a tenfold increase in incorporation of radioactivity into trichloroacetic-acid-precipitable material was obtained with ITP platelets compared to control platelets. Electron microscopic studies of ITP platelets revealed the presence of rough endoplasmic reticulum and polyribosomes, providing morphological evidence for protein synthesis. SDS-PAGE of radiolabelled ITP platelet proteins followed by autoradiography showed that [35S]methionine and [3H]leucine were incorporated into almost all Coomassie-blue-stained proteins whereas [3H]carbohydrates only labelled a few bands. Using crossed-immunoelectrophoresis we identified some of the labelled platelet compounds and demonstrated that major membrane glycoproteins (GPIb, IIb, IIIa) and alpha-granule proteins, such as fibrinogen, thrombospondin, albumin and von Willebrand factor, were synthesized in newly formed circulating platelets.     

Studies on T cells in newborns. Higher reactivity of umbilical cord blood lymphocytes to PHA as measured by whole blood microtechnique.

The capacity of human foetal lymphocytes to respond to PHA and to form E-rosettes have been compared with data from adult individuals. For this purpose a microculture system that uses whole blood and avoids the problems of lymphocyte separation, has been developed. Foetal lymphocytes reached optimal stimulation with lower dosis of PHA (31,2 microgram/ml) as compared with adult cells (125-252 microgram/ml). However their quantitative response (measured by 14C-thymidine uptake) was equal in both groups. In addition, peripheral T cells (E-rosetting cells) reached values of 36.47 +/- 9% in newborn and 49.6 +/- 10% in normal adult controls. These results are discussed as to the status and development of cellular inmunity in human foetus.     

Platelet autoantigens: identification and characterization using immunoblotting

Antiplatelet autoantibodies are important in the etiology of idiopathic (or immune) thrombocytopenic purpura (ITP). Studies using immunoblotting techniques have been helpful in identifying the antigenic target proteins for the antibodies. Antibodies against the glycoprotein (GP) IIIa portion of the GPIIb/IIIa complex were the first to be demonstrated by this approach. Similar GPIIIa autoantigens have also been found to be the most frequent targets of ITP antibodies. Not all anti-GPIIIa antibodies are directed against the same epitope on GPIIIa. A subset of anti-GPIIIa antibodies found in patients with an acquired qualitative platelet dysfunction actually interfere with fibrinogen binding to normal platelets. Antibodies directed against targets on GPV have been found in patients with acute ITP of childhood. In patients with ITP associated with lupus erythematosus, antibodies which bind to intracellular proteins of apparent molecular weights of 66 and 108 kDa have been detected. Thus, ITP antibodies can have a variety of target antigens. Study of larger series of patients will determine whether identification of platelet autoantigens correlates with clinical course of ITP.     

Suppression of maturation of megakaryocyte colony forming unit in vitro by a platelet-released glycoprotein

The suppressive role of platelets on the growth of human marrow megakaryocyte colony forming units (CFU-M) in vitro was investigated by the use of a plasma clot assay. An inverse correlation was established between the number of megakaryocytic colonies grown and the platelet concentration of the plasma or the resultant serum used in the culture system. The suppressive effect of platelets on megakaryocyte colony formation reached a plateau at normal human blood platelet concentration and was specific for CFU-M growth, since marrow cell erythroid burst formation (BFU-E) and granulocytic-monocytic colony formation (CFU-GM) remained unaffected. The inhibitory activity was detectable in the supernatants of platelet suspensions aggregated by thrombin or ADP, and the inhibitory activity released from ADP-stimulated platelets was blocked by pretreatment of platelets with monoclonal antibody HuPl-m1. Partial purification of this activity was achieved by diethylaminoethyl (DEAE)-ion exchange and phytohemagglutinin (PHA)-E agarose affinity chromatography. This inhibitor is a glycoprotein with a molecular weight of 12-17K daltons. This platelet released glycoprotein does not affect the early proliferative phase of CFU-M in vitro but acts on a day 6-8 CFU-M-derived cell by adversely affecting its maturation into recognizable megakaryocytes. These findings demonstrate that a glycoprotein released from platelets suppresses the maturation of CFU-M into megakaryocytes.     

14 C-acetate incorporation and nucleic acid synthesis in human lymphocytes stimulated by PHA and tuberculin in vitro

Studies on the timing of incorporation of labeled acetate in relationship to other cellular events in phytohemagglutinin (PHA)-treated lymphocytes have suggested that acetylation of nuclear histones may constitute an important regulatory mechanism for gene activation. In the present investigation, it was shown that PHA stimulation of lymphocytes from a tuberculin-positive patient caused an early increased incorporation of ¹⁴C-acetate prior to RNA and DNA synthesis. Lymphocytes from the same patient, however, repeatedly showed no increased incorporation of ¹⁴C-acetate following exposure to the sensitizing antigen, tuberculin (PPD), even though RNA and DNA synthesis were markedly stimulated. These results suggest that regulatory mechanisms of DNA template activity other than acetylation may be operative in sensitized lymphocytes responding to specific antigen. One possible explanation for the differences in ¹⁴C-acetate incorporation is that the increased uptake of acetate exhibited by PHA-treated cells is an effect related to nonspecific membrane changes caused by the PHA. If this is the case, then template regulation in PHA and antigen-stimulated lymphocytes may be achieved via similar but yet to be defined mechanisms.     

Demonstration of significant differences in the proliferative capacity of lymphocytes from normal human subjects

Lymphocytes were obtained from six normal human subjects on three to five occasions. Lymphocyte response to pokeweed mitogen (PWM), phytohemagglutinin (PHA), and a pool of allogeneic lymphocytes was measured by incorporation of [³H]thymidine in cultures established in flat- and round-bottom multiwell plates. Unstimulated and mitogen-stimulated thymidine incorporation were not correlated. Therefore, incremental counts rather than stimulation indices were used to express data. Individual lymphocyte responses were more reproducible in round-bottom well plates. Using these data, correlation was found between the responses to PWM and PHA. Neither of these responses was correlated with the mixed lymphocyte reaction. There were no significant differences in the responses of these subjects to allogeneic lymphocytes. This response may, therefore, allow clear distinction between normal and abnormal lymphocyte reactivity. Among these normal subjects significant differences were found in their responses to both PWM and PHA. The biological import of these differences is not known.     

Stress platelets in normal individuals and patients with idiopathic thrombocytopenic purpura.

To test the hypothesis that stress platelets (SPs) described by Tong et al. in rats may be a parameter of young platelets in humans, we examined and characterized SPs in normal individuals and in patients with idiopathic thrombocytopenic purpura (ITP). Our results indicated that SPs comprise about 1.2% of the circulating platelets in normal individuals and 2.6% in ITP patients. The configuration of SPs as well as of various irregular forms of circulating platelets was found to be supported by synergism of both the platelet microfilaments and microtubules. SPs showed some segmentation, the degree of which was similar in normal individuals and ITP patients, and they underwent further segmentation during in vitro incubation, mainly promoted by microtubules, so that they sometimes appeared like discoid platelets in a chain. These observations suggest a new mode of production of discoid platelets in the circulation. Thus, identification and enumeration of SPs may be useful for evaluating thrombocytopoiesis in humans.     

Incorporation of hypoxanthine by PHA-stimulated HPRT-deficient lymphocytes

Phytohemagglutinin (PHA) markedly stimulates ³H-hypoxanthine incorporation by lymphocytes of normal subjects as revealed by radioautography. There is no corresponding increase in activity of hypoxanthine phosphoribosyltransferase (HPRT) in lysates but the level of phosphoribosylpyrophosphate (PRPP), the cosubstrate for the reaction, is higher. Lymphocytes from a patient with partial HPRT deficiency responded to PHA as did the normals, whereas the response in Lesch-Nyhan (LN) subjects was variable. PHA-stimulated lymphocytes from two LN patients showed some increase in ³H-hypoxanthine incorporation, while two others failed to respond. The observations provide further evidence of genetic heterogeneity among LN patients.     

Incorporation of 14C-thymidine in human platelets: possible relation with a diffuse infection of micrococci in the L form.

Following the results of previous researches suggesting that platelets might carry microbial forms, the incorporation of 14C-thymidine in suspensions of platelets from 500 normal human subjects has been taken under examination. The results have always yielded positive data even though with marked differences of a quantitative order from a case to another. The hypothesis that such an activity might be the consequence of a synthesis of DNA in the mitochondria had to be excluded. The peculiar relations linking the incorporation rate to the number of platelets and to the presence of plasma or serum in given amounts and the strong inhibition exerted by oxytetracyclines suggest that the detected metabolic activity may be attributed to the presence of bacterial L-forms carried by platelets. The results of cultural, optical and electron microscopical investigations, which will be published elsewere, confirmed such interpretation.     

Differential effect of cold agglutinins on lymphocytes from patients with Graves' disease and Hashimoto's thyroiditis

Peripheral leucocytes from healthy controls, patients with Hashimoto's thyroiditis or patients with Graves' disease were assayed for susceptibility to the cytotoxic activity of cold agglutinins. Lymphocytes from patients with thyroiditis were killed as readily as control cells, however, lymphocytes of patients with Graves' disease were clearly less susceptible to lysis by cold agglutinins. Lymphocytes from patients with Graves' disease cultured for 3 days in the presence or absence of PHA, were as susceptible to cold agglutinins as were control lymphocytes and lymphocytes from patients with thyroiditis. We suggest that the amounts of Ii antigens on the surface of Graves' lymphocytes may be modulated by autoantibodies which are present in many of these patients.     

Vitamin D receptor expression in human lymphocytes. Signal requirements and characterization by western blots and DNA sequencing.

The signals controlling the expression of the receptor protein for 1 alpha,25-dihydroxyvitamin D3 in normal human lymphocytes and the relationship of this protein to the classical vitamin D receptor were examined. Lymphocytes activated with the OKT3 antibody to the T-cell antigen receptor expressed fewer binding sites as compared to lymphocytes that were activated by the polyclonal activator phytohemagglutinin (PHA). However, combination of OKT3 and phorbol myristate acetate produced a concentration of binding sites similar to the PHA-activated cells. The receptor from OKT3 and OKT3 + phorbol myristate acetate-activated lymphocytes exhibited decreased binding to DNA-cellulose compared to PHA-activated lymphocytes. In lymphocytes activated either by PHA or OKT3 (but not in resting cells), a 50-kDa species cross-reacting with a monoclonal antibody against the intestinal vitamin D receptor was detected. Finally, RNA from activated lymphocytes was amplified by polymerase chain reaction using oligonucleotide primers flanking the 196 base pair long region encoding the DNA-binding domain of the human intestinal receptor. The amplified product showed an identical nucleotide sequence to the DNA-binding domain of the human intestinal receptor. These findings suggest that expression of the 1,25-(OH)2D3 receptor in lymphocytes is triggered by distinct and contingent signals, and that the protein and the mRNA encoding it are identical to the classical vitamin D receptor.     

Acute severe thrombocytopenia after c7E3 Fab (abciximab) therapy in a patient with unstable angina and stenting of the right coronary artery. Occurrence of subacute stent thrombosis and safe readministration of the GPIIb/IIIa inhibitor tirofiban

This paper reports a case of acute severe thrombocytopenia (platelet count: 1 x 10(9)/liter) occurring within minutes of an initial abciximab bolus during coronary angioplasty and stenting in a patient with unstable angina. After six days with platelets again in the normal range the patient developed stent thrombosis. The stent was reopened and the glycoprotein receptor inhibitor tirofiban (Aggrastat) was administered without any adverse effects on platelet count. Antibodies against heparin-platelet factor 4 complexes could be excluded. Allo- and autoantibodies (IgG, IgA, IgM) directed against platelets with and without binding of abciximab could not be detected by indirect and direct platelet fluorescence antiglobulintest. A possible activation or lysis of the platelets by abciximab could also be excluded by an in vitro bleeding test investigating the effect of abciximab on heparin and citrate blood of the patient and two healthy donors. The mechanisms of abciximab-induced thrombocytopenia in this case remain unclear. The possible mechanisms are discussed.     

实用淋巴细胞培养技术

采自人体的外周静脉血液,用淋巴细胞分离液进行分析,收集分离得到淋巴细胞,同时回收血液的血清成分,此血清不经灭活可直接用于培养。本实验对影响淋巴体外激少在、增殖的相关因子进行了详细的统计分析,实验结果表明,在基础培养液ＲＰＭＩ１６４０中添加１００μｇ／ｍｌ的ＰＨＡ、１００ｉｕ／ｍｌ的ＩＬ－２和１０％的自体或胎血清,可以有产地激活淋巴细胞并在营养条件允许的情况下长期处于增鱼的状态。     

Influence of mature and immature myeloid cells on lymphocyte reactivity in chronic myelocytic leukemia

Summary Earlier studies have shown anergy in chronic myeloid leukemia (CML), and it is known that myeloid cells influence lymphocyte responses. Therefore, lymphocytes from CML patients who had received no cytostatics for 2 weeks were stimulated in 89 tests with PHA and ConA. In 39 control tests, normal lymphocytes were used.Lymphocytes from CML patients were significantly less (p<0.05) markedly stimulated than normal ones. Lymphocytes from CML patients with more than 10×10⁹ white blood cells (WBC) per liter blood were inhibited to a greater degree than those from patients with a normal WBC count.When normal lymphocytes were stimulated with PHA in the presence of mononuclear cells from the blood of CML patients (mostly leukemic myelocytes), their response was significantly (p<0.05) inhibited. Inhibition with leukemic myelocytes was significantly (p<0.05) greater than that with mature granulocytes from CML patients. The latter did not seem to have an inhibitory effect.We suggest that patients with manifest CML are anergic to some extent because leukemic myelocytes have a suppressor effect.Visiting scientist and Anna Villa Rusconi Fellow, on secondment from Institute of Medical Pathology, University of Ferrara, Italy     

A comparative study of eicosapentaenoic acid metabolism by human platelets in vivo and in vitro

During long-term dietary n-3 fatty acid supplementation, eicosapentaenoic acid (EPA) is not incorporated into phosphatidylinositol or -serine of human platelets in vivo and is not detectable in phosphatidic acid upon stimulation with thrombin. However, EPA is released from platelet phospholipids and metabolized to thromboxane B3 (TXB3). In contrast, in vitro, platelets incorporate [14C]EPA into phosphatidylinositol, whether they contain endogenous EPA in their cellular lipids or not. Following platelet stimulation, [14C]EPA appears in phosphatidic acid, as free fatty acid, and is transformed to TXB3. We conclude that the fatty acid compositions of platelet phospholipid subclasses are regulated with a high degree of specificity in vivo. Qualitative differences exist between in vivo and in vitro uptake of EPA into platelet phospholipid subclasses. After in vivo incorporation, EPA is released by action of a phospholipase A2.     

Cellular cholesterol metabolism in mitogen-stimulated lymphocytes--requirement for de novo synthesis

The relationship between cholesterol synthesis and uptake in proliferating lymphocytes has been examined. [14C]Acetate incorporation into lymphocytes cultured under lipoprotein-deficient conditions increased initially in response to mitogen, decreased after 24 h, and increased rapidly between 72 and 96 h. Addition of LDL (10 micrograms/ml) to the culture during the 'trough' period caused [14C]acetate incorporation to return rapidly to baseline, while at peak periods LDL suppression of cholesterol synthesis was minimal. Lymphocytes cultured in the presence of the HMG-CoA reductase inhibitor, mevinolin, exhibited a time-dependent increase in their capacity to incorporate [14C]acetate into cholesterol, evident when mevinolin was removed by washing prior to assay. PHA enhanced 125I-labelled LDL receptor-mediated binding by lymphocytes cultured in lipoprotein-deficient medium over a 4 day period and mevinolin augmented the effect. [3H]Thymidine incorporation into mitogen-stimulated lipoprotein-deficient cultures was inhibited up to 75% by mevinolin (1 mumol/l). LDL (2.5-10 micrograms/ml) substantially reversed this inhibition in 72 h cultures, but only partially overcame inhibition in cells cultured for 96 h. Results suggest that endogenous cholesterol synthesis may be obligatory for lymphocyte proliferation after the initial round of cell division.     

Use of a monoclonal antibody to measure the surface expression of thrombospondin following platelet activation

The radiolabelled monoclonal antibody, 5G11, directed against native thrombospondin, has been used to assess the surface expression of secreted thrombospondin on human blood platelets. Emphasis has been placed on studying the role of fibrinogen in this process. Unstimulated platelets bound low amounts of 5G11 (about 2000 molecules/platelet). Binding increased 2-fold and 5-7-fold after stimulation of platelets with ADP or thrombin (or ionophore A23187) respectively. Unstimulated platelets from patients deficient in alpha-granule proteins (gray platelet syndrome) bound baseline levels of 5G11. However, binding was not increased after activation. Thrombospondin expression on thrombin-stimulated normal platelets was for a large part divalent-cation-dependent and was not affected by AP-2, a monoclonal antibody to GPIIb-IIIa complexes. However, binding of 5G11 was some 50% lower when platelets were stimulated in the presence of Fab fragments of a polyclonal rabbit antibody to fibrinogen. This suggested either a direct binding of thrombospondin to surface-bound fibrinogen or a steric inhibition due to a close proximity of the two proteins. The fact that binding of 5G11 was at the lower limit of the normal range to the stimulated platelets of an afibrinogenaemic patient specifically lacking detectable fibrinogen favoured the latter explanation. Thus, a major fibrinogen-independent pathway for thrombospondin expression must exist.