John Mendelsohn

The circulating "atypical" lymphocyte.

Atypical lymphocytes have been observed in the peripheral blood of patients in a large number of clinical situations, including immune reactions to transplantation and immunization, collagen diseases and other autoimmune disorders, malignant disease, drug reactions, and infectious mononucleosis, as well as other bacterial and viral infections. These cells are readily identified by their increased size and the presence of active DNA synthesis. In morphology, they closely resemble lymphocytes transformed into blasts by exposure to mitogens or antigens in vitro. They vary in morphologic detail as well as surface marker characteristics, indicating that they comprise a heterogeneous mixture of cell types. These data suggest that atypical lymphocytes may represent a polyclonal immune response to antigenic stimulation.

A direct radioimmunoassay for human epidermal growth factor receptor using 32P-autophosphorylated receptor

A simple and reproducible radioimmunoassay of the epidermal growth factor (EGF) receptor which uses 32P-labeled EGF receptor and anti-receptor monoclonal antibodies is reported. In vitro phosphorylation of A431 cell membranes with [gamma-32P]ATP in the presence of 20% dimethyl sulfoxide (which stimulates autophosphorylation of the EGF receptor) and 10 microM Na3VO4 (a potent inhibitor of phosphotyrosyl protein phosphatase) provides radiolabeled EGF receptor for radioimmunoassay without further purification. The most selective phosphorylation of the EGF receptor is achieved at ATP concentrations of 0.1-0.2 microM, which corresponds to the reported Km value for the autophosphorylation reaction of the EGF receptor (W. Weber, P.J. Bertics, and G.N. Gill, 1984, J. Biol. Chem. 259, 14631-14939). The incorporation of 32P into EGF receptors increases in proportion to the increase of ATP concentration up to 6 mol of labeled phosphate at 2.0 microM ATP. The label is entirely on tyrosine residues. The cell membranes can be stored at -70 degrees C for 3 months without loss of immunoreactivity and autophosphorylating activity. Standard curves for the radioimmunoassay were constructed employing either A431 cell membranes or whole cell homogenates containing a known amount of EGF receptor. The assay can detect 7 X 10(10) EGF receptor molecules or 20 ng of the receptor protein, and can quantitatively distinguish the difference in EGF receptor numbers between A431 cells and 29E2 and KB cells with 10-fold and 15-fold fewer receptors than A431 cells, respectively. 29E2 cells and KB cells express twofold more immunoreactive EGF receptors than EGF-binding sites. In contrast, A431 cells possess the same number of immunoreactive sites and receptor sites for EGF binding. To assess total EGF receptor expression, it is necessary to use a method which detects EGF receptors regardless of their intrinsic kinase activity, or capacity to bind EGF. This radioimmunoassay detects immunoreactive receptor molecules, even those which do not bind EGF.

Hodgkin's disease and anergy

ZusammenfassungDas Zustandekommen der klinischen, pathologischen und immunologischen Merkmale, die für den Morbus Hodgkin charakteristisch sind, könnte durch die folgende Hypothese erklärt werden: Eine lymphatische Zelle wird durch ein Virus transformiert und proliferiert als „Tumorzelle“. Die resultierende Immunantwort ist gekennzeichnet durch Lymphokinproduktion, B-Zellaktivierung, Suppression einer T-Zellaktivierung, aber ineffektive zelluläre Zytotoxizität gegenüber den Tumorzellen. Eine derartige Immunantwort könnte die chronische, reaktive Zellinfiltration in Nachbarschaft der Tumorzellen, eine gesteigerte Immunglobulin-Synthese und Anergie erklären. Gleichzeitig könnte die mangelhafte Zerstörung der Tumorzellen den chronisch fortschreitenden Charakter der Erkrankung erklären.SummaryThe clinical, pathologic, and immunologic features unique to Hodgkins disease can be explained by the following hypothesis. A viral transformation of a lymph node cell leads to proliferation of tumor cells and the generation of an immune response consisting of lymphokine production, B cell activation and concomitant suppression of further T cell activation, but ineffective cellular cytotoxicity against the tumor cells. The result of this interaction would be chronic infiltration around the transformed cells, increased immunoglobulin synthesis, and anergy. Failure to destroy the target cells would result in chronicity of these features and progressive disease.

KINETICS OF DNA SYNTHESIS AND CELL PROLIFERATION IN LYMPHOCYTE ACTIVATION: WORKSHOP REVIEW

Publisher Summary This chapter presents a summary of the workshop organized successively at the Third European Immunology Meeting (Copenhagen, August 23–26, 1976) and at the Eleventh Leukocyte Culture Conference . The workshop addressed the following topics: (1) culture circumstances that best promote lymphocyte transformation and proliferation, (2) kinetics of proliferation in response to antigens and mitogens, (3) the reliable quantitative assay methods for in vitro lymphocyte proliferation, and (4) solid information that exists on three categories of lymphocyte DNA with unusual or apparently specific characteristics. This chapter reviews various studies on this subject into context. The workshop focused on two highly relevant aspects of the lymphocyte response to stimulation, namely, initiation and termination of proliferation.

SPONTANEOUS LYMPHOCYTE PROLIFERATION IN HODGKIN'S DISEASE

Publisher Summary This chapter explores spontaneous lymphocyte proliferation in Hodgkins disease. A study described in the chapter attempted to determine whether a relationship exists between proliferation occurring spontaneously and that seen in response to stimulation with the mitogen, phytohemagglutinin (PHA). Proliferation was quantitated by the 3H-thymidine incorporation assay, utilizing 1 ml cultures containing 5 × 105 lymphs/ml. The study analyzed ten patients with active Hodgkins disease not under treatment at the time of study and five normal subjects. The patients as a group demonstrated significantly increased spontaneous 3H-thymidine incorporation on day 0 (the day of venipuncture) and significantly depressed 3H-thymidine incorporation on day 3 following stimulation with PHA. In general, those patients with more advanced disease had increased numbers of spontaneously dividing lymphocytes. When the individual values for unstimulated (day 0) and PHA-stimulated proliferation (day 3) were analyzed, a statistically significant inverse relationship between these values was demonstrated. It was also demonstrated that cellular immunity (the PHA response) in Hodgkins disease is quantitatively depressed in the presence of a heterogeneous group of dividing lymphocytes.

EVIDENCE FOR AND AGAINST UNUSUAL SPECIES OF DNA IN LYMPHOCYTE ACTIVATION

Publisher Summary This chapter presents a study in which it was found that PHA-stimulated cultures contained much fewer cells than expected from the amount of DNA synthesis. By determining the rate of entry into DNA synthesis (S) phase and by assuming every cell entering the S phase gives rise to a new cell, it was calculated that a concentrated (3 × l06 lymphocytes/ml initially) culture would after 6 days have contained 8 × 106 cells. However, only 2 × 106 cells were found. In the much better dilute culture circumstances, the culture expanded to 9.5 × 105 cells, but 13 × 105 were theoretically expected. The rate of entry into the S phase was always in excess of the rate of entry into mitosis. To calculate the cell death rate from the observable number of dead cells, a method to determine the disintegration time of dead lymphocytes was developed. Applying this, it was found that the vast majority of total cell and mitotic deficits were because of cell disintegration and cell death between entry in S and exit from mitosis, respectively. Thus, nearly all cells starting DNA synthesis eventually divide, unless they die underway.

ADENYLATE CYCLASE IN NORMAL AND LEUKEMIC HUMAN LYMPHOCYTES

Abnormalities in adenylate cyclase activity and cyclic AMP levels have been reported in studies of lymphocytes from patients with chronic lymphocytic leukemia (CLL). This chapter presents detailed characterization of adenylate cyclase activity in purified T and B lymphocyte subpopulations from normal donors. In a study described in the chapter, lymphocytes purified with Ficoll–Hypaque were obtained from healthy donors and 19 patients with B cell type CLL. T cells were identified by the E-rosette technique. B cells were identified with rabbit antihuman Ig and the EAC-rosette technique. Monocytes were detected by esterase staining. The results of assays for adenylate cyclase and cyclic AMP are shown in the chapter. The adenylate cyclase assay measured conversion of α 32 P labeled ATP to 32 P labeled cyclic AMP in freeze-thaw lysates of lymphocytes. Enzyme activity was 57 ± 4 pmoles 32 P Cyclic AMP/mg prot/min in normals, 39 ± 8 in CLL lymphocytes from patients with peripheral leukocyte counts 3 , and 20 ± 3 in patients with counts > 30,000/mm 3 .

IS THERE GENE AMPLIFICATION OR OTHER NONDUPLICATIVE DNA SYNTHESIS IN HUMAN LYMPHOCYTE ACTIVATION

Publisher Summary This chapter presents a few studies in which highly purified human peripheral lymphocytes were incubated in MEM containing 10% autologous serum, glutamine, and antibiotics, in the presence of optimal concentrations of phytohemagglutinin. Cultures were incubated at an initial cell concentration of 2 × 105 lymphocytes/ml, which is one log below the widely used concentration. Under these conditions, up to a fivefold increase in cell number was observed over a period of 7 days. DNA purified from cultures labeled with 5-bromodeoxyuridine between days 3 and 4 for 24 h, a period longer than one mean cycle time, was analyzed on cesium chloride equilibrium density gradients: the percentages of DNA in the heavy–heavy, heavy–light, and light–light regions were, respectively, 19, 56, and 25, providing direct evidence for replication of more than 50% of the DNA and reentry of some cells into a second proliferative cycle.

PITFALLS IN THE LYMPHOCYTE PROLIFERATION ASSAY: VARIATIONS IN PROLIFERATION KINETICS AND COLD THYMIDINE POOLS

Publisher Summary This chapter explores the variations in proliferation kinetics and cold thymidine pools. In a study described in the chapter, purified human peripheral blood lymphocytes were isolated, cultured, and PHA-stimulated. The use of an optimal dose of 1 μg/ml purified PHA, with low agglutinating potency, allowed accurate hemacytometer counting. DNA synthesis was assayed after 3H-TdR labeling (2 h, 2 μCi/ml, 6 Ci/mM) by autoradiography and liquid scintillation counting. The chapter presents two representative experiments that demonstrated a second difference between crowded and dilute PHA-stimulated cultures: variability in the kinetics of entry into proliferation. In the first experiment, a comparison of the fraction of cells labeled shows that the rates of entry into proliferation are similar for the two cell concentrations during the first 72 h. Thereafter, proliferation decreases in the crowded culture, whereas in the dilute culture, growth proceeds. The second experiment differs in that the entry into proliferation occurs more rapidly in the crowded culture. This would be expected if the cells produced additional recruitment activity that reached an effective level earlier in the concentrated than in the dilute cultures. In several such experiments described in the chapter, the kinetics of entry into proliferation varied unpredictably.