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This investigation studied the effects of cell preparation methods, different antibody panels and blood storage on antigen expression of abnormal B lymphocytes from patients with B-CLL. Blood specimens collected in Heparin de novo were processed by using conventional Hypaque-Ficoll density gradient centrifugation and whole blood lysis. These were stored for 3 days at 4 degrees C, 24 degrees C and 30 degrees C. Although clonal excess was detected by all antibody panels, significant differences could be observed in terms of molecules of equivalent fluorochromes (MEF/MESF units). Evaluation of 'weak and strong' staining is dependent on the antibody panel used. Immunofluorescent values for CD19 and CD45 were unchanged at 4 degrees C and 24 degrees C but immunoglobulin staining showed best results when blood was stored at 4 degrees C. Storage at 30 degrees C produced unreliable results. Abnormal B lymphocytes should be analysed immediately after the specimen is obtained. If shipment is necessary they should be kept at 4 degrees C. Surface immunoglobulins are the 'antigens' most sensitive to storage alterations. Sample alterations alone are sufficient to the correct classification of NHL, especially in the case of low-grade NHL.

Eine Steigerung der Cytarabin-Dosis auf das etwa 30fache (3 g/m2 Korperoberflache alle 12 Stunden in 500 ml 0,9%iger Kochsalzlosung als lstundige Infusion uber 6 Tage) der in den meisten ZytostatikaKo

Pure suspensions of human lymphocytes were separated from peripheral blood by means of nylon wool, homogenized in 0.34 M sucrose-0.01 M EDTA solution, and fractionated by differential centrifugation. The bulk of acid hydrolase activity was found to be concentrated in a 20,000 g x 20 min granular fraction, whereas nuclear, debris, and supernatant fractions contained lesser concentrations of hydrolases. Acid hydrolase activity present in the granular fraction showed appropriate "latency" as judged by its dose-dependent release into the 20,000 g x 20 min supernatant after exposure to membrane-disruptive agents such as streptolysin S, filipin, and lysolecithin. Heparin proved to be necessary in the suspending medium so that reproducible homogenization and cell fractionation could be obtained. Even excessive contamination of lymphocyte suspensions with platelets did not appreciably alter the acid hydrolase activity of lymphocyte homogenates or the distribution of enzymes in subcellular fractions. Discontinuous density-gradient centrifugation of a 500 g x 10 min supernatant, containing both acid hydrolase-rich organelles and mitochondria, resulted in partial resolution of hydrolase-rich organelles from mitochondria. Fine structural studies of the intact lymphocytes showed the presence of acid phosphatase-positive, membrane-bounded organelles. Electron microscopy of the "large granule" (20,000 g x 20 min) fraction of such lymphocytes demonstrated 80–90% mitochondria, 5–10% platelets, and 5–10% membrane-bounded acid phosphatase-positive structures. The data indicate the presence in human peripheral blood lymphocytes of acid hydrolase-rich granules which possess many of the biochemical and structural characteristics of lysosomes in other tissues.

Nuclei isolated from human peripheral blood lymphocytes stimulated by phytohaemagglutinin have a greater capacity to prime for nucleotide incorporation into RNA in the presence of exogenous bacterial RNA polymerase than nuclei from unstimulated lymphocytes.

Die Seren von insgesamt 189 Patienten mit Fettleber, akuter Hepatitis, chronischer Hepatitis, Leberzirrhose, primar-chronischer Polyarthritis und anderen internen Erkrankungen wurden mit dem Konsumptionstest unter Verwendung von Thymuskernen, Leberhomogenat, Gefashomogenat und Kollagen als Antigenen serologisch untersucht. Bei chronischer Hepatitis und Leberzirrhose waren mit dem gesamten Antigenspektrum haufig positive Reaktionen festzustellen; bei Patienten mit primar-chronischer Polyarthritis traten nur mit Thymuskernen, Kollagen und Gefashomogenat positive Reaktionen auf. Bei Fettleber, akuter Hepatitis und einer Kontrollgruppe mit anderen internen Erkrankungen waren positive Befunde selten. Serum samples of 189 patients with fatty liver, acute hepatitis, chronic hepatitis, liver cirrhosis, and chronic rheumatoid arthritis or other diseases of the internal organs were analysed with the antiglobulin consumption test and by employment of thymus nuclei, liver homogenate, vascular homogenate and collagen as antigens. Positive reactions frequently occurred with the entire antigen spectrum in chronic hepatitis and liver cirrhosis. In patients with chronic rheumatoid arthritis positive reactions only occurred with thymus nuclei, collagen and vascular homogenates. In fatty liver, acute hepatitis and other diseases of internal organs positive reactions occurred only rarely. La prueba de consuncion en la hepatopatias Los sueros de 189 pacientes con higado graso, hepatitis aguda, hepatitis cronica, cirrosis hepatica, poliartritis primaria cronica y otras afecciones internas fueron examinados serologicamente con la prueba de consuncion y utilizando nucleos timicos, homogenado hepatico, homogenado vascular y colageno como antigenos. En la hepatitis cronica y en la cirrosis hepatica se constataron con frecuencia reacciones positivas con el espectro antigenico global; en los pacientes con poliartritis primaria cronica se presentaron reacciones positivas solo con los nucleos timicos, colagenos y homogenado vascular. En el higado graso, hepatitis aguda y otras afecciones internas como controles se dieron resultados positivos raramente.

Seit Ende 1970 haben wir in der Hamatologischen Abteilung der Medizinischen Universitatsklinik Essen 32 Patienten mit aplastischem Syndrom beobachtet, bei denen der Markzustand nicht nur zytologisch, sondern auch histologisch beurteilt werden konnte. Die histologische Untersuchung ergab in allen Fallen eine Atrophie des Knochenmarks, die jedoch von unterschiedlichem Ausmas war, so das eine Unterteilung in Patienten mit totaler Knochenmarkatrophie (tA) (n = 9) und partieller Knochenmarkatrophie (pA) (n = 23) moglich wurde. Von diesen histologischen Gesichtspunkten ausgehend, versuchten wir in einer retrospektiven Studie Beziehungen zwischen dem initialen Ausmas der histologischen Veranderungen und dem klinischen Verlauf herzustellen.

We present a monoclonal IgG1 antibody, KiMy1R, which is specific for macrophages and their derivatives in the lymphatic tissue of the rat, and evaluate the distribution of subsets of mononuclear cells in popliteal and para-aortal lymph nodes of Wistar rats after injection of 50 mg DPH into the hindpads. Compared with resting lymphatic tissue and lymph nodes of animals treated with phenobarbital, DPH induced a significant increase (P less than 0.01) of the proportion of phagocytic cells. Furthermore, the soluble antigen alkaline phosphatase was traced after inoculation into the footpads of rats: in locoregional lymph nodes the percentage (mean 12.8/10(3] and the total number (mean 476 x 10(3) cells/lymph node) of cells with membrane-bound or intracytoplasmic alkaline phosphatase, as detected by a monoclonal anti-alkaline phosphatase antibody, were significantly higher (P less than 0.001) in animals pretreated with DPH than in rats pretreated with phenobarbital (mean 2.1/10(3); 31.2 x 10(3) cells/lymph node) and in untreated animals (mean 1.9/10(3); 4.1 x 10(3) cells/lymph node). If verified in humans, the effect of DPH in enhancing the number and function of macrophages and other antigen-presenting cells may exert favourable effects in patients with impairment of the mononuclear phagocytic system.

Es ist bekannt, das Phytohamagglutinin (PHA) und Pokeweed-Mitogen (PMW) zu einer „unspezifischen“ Stimulierung von Blutlymphocyten gesunder Menschen fuhren [1, 2, 3]. Obwohl in den letzten Jahren in den stimulierten Zellen zahlreiche morphologische und biochemische Veranderungen festgestellt wurden, ist der Mechanismus der Transformation bisher nicht geklart.

Die Lymphozyten der Wirbeltiere von den Vogeln an aufwarts in der Phylogenese sind keine homogene Population, sondern lassen sich auf Grund ihrer funktionellen Eigenschaften und charakteristischer Oberflachenmerkmale im wesentlichen in 2 Klassen einteilen. So unterscheidet man auch beim Menschen zwischen den thymusabhangigen T-Lymphozyten und den thymusunabhangigen B-Lymphozyten, die wahrscheinlich unter dem Einflus eines beim Menschen bisher noch unbekannten Aquivalentes des cloakalen lymphoiden Organs der Vogel, der Bursa Fabricii, stehen.

Kleine Blutlymphocyten werden in vitro durch Phytohamagglutinin (PHA) und andere „unspezifische“Wirkstoffe zu einer blastenartigen Transformation und erhohten Mitoseaktivitat angeregt [1]. Diese Transformation ist bei 50 bis 95% der Zellen zu beobachten, die hochste Mitoserate findet sich nach 2 bis 3 Tagen. Mit den morphologischen Veranderungen gehen erhebliche Stoffwechselalterationen einher, die z. T. bereits wenige Minuten nach Zusatz von PHA verifiziert werden konnen [2, 3, 4, 5]. Der Mechanismus der Lymphocytentransformation ist bisher unbekannt. Cyto- und biochemische Untersuchungen zeigten, das im Cytoplasma PHA-behandelter Lymphocyten wahrend der pramitotischen Phase vermehrt lysosomenartige Organellen und erhohte Aktivitaten lysosomaler Enzyme nachweisbar werden [6, 7, 8, 9]. Die vorliegenden Untersuchungen gingen von der Frage aus, ob auch wahrend der Fruhphase der Lymphocytenstimulierung Lysosomenveranderungen auftreten, die sich mit biochemischen Methoden erfassen lassen.

Resting blood lymphocytes in vitro undergo transformation into large, blastlike, dividing cells following incubation with phytohemagglutinin (PHA), a plant lectin from Phaseolus vulgaris (1). Farnes et al. (2) found another phytomitogen in saline extracts of the plant Phytolacca americana (pokeweed). Although both PHA and the pokeweed mitogen (PWM) induce nonspecific transformation of lymphocytes which differs markedly from specific transformation by antigens (3), the chemical composition and the biological effects of these agents are by no means entirely identical. PWM has been shown to possess a molecular weight much lower than that of PHA (4, 10), and to induce in vitro the formation not only of blasts like PHA-induced ones but also of a unique intermediate-sized cell type with cytoarchitectural features of plasmablasts (5). In previous studies (6, 7), we have demonstrated the presence in human peripheral blood lymphocytes of acid hydrolase-rich granules which display many of the biochemical and structural characteristics of lysosomes in other tissues. Between 30 and 120 min after addition of PHA to intact, purified lymphocytes, redistribution of acid hydrolases (beta-glucuronidase, acid phosphatase) from a 20,000 g X 20 min granular fraction into the corresponding supernatant was observed. These changes were associated with increased fragility of hydrolase-rich granules derived from PHA-stimulated lymphocytes upon subsequent incubation with membrane-disruptive agents in vitro. For these, and other reasons, it was suggested that redistribution of acid hydrolases may play a role in remodeling processes of lymphocytes stimulated by PHA (6, 7). As a necessary test of this hypothesis, we have, therefore, determined whether pokeweed mitogen could also produce changes in enzyme distribution among subcellular fractions of human lymphocytes. MATERIALS AND METHODS

AFTER addition of various mitogens such as phytohaemagglutinin (PHA) in vitro lymphocytes shift from a “resting” state to one of rapid enlargement, culminating in DNA synthesis and mitosis. The mechanism initiating transformation and proliferation is not yet fully understood. Some observations, however, suggest that alterations of cellular membranes might be involved in these processes. In their cytochemical studies, Allison and Mallucci1 observed increased permeability of lysosomes in normal human peripheral blood lymphocytes after exposure to PHA for 5 h. Hirschhorn et al.2 demonstrated that during the early phase of stimulation 30–120 min after addition of PHA in normal human blood lymphocytes a redistribution of lysosomal acid hydrolases from a sedimentable into a non-sedimentable form as well as an increased release by agents which disrupt membranes, such as streptolysin S and filipin, of lysosomal enzymes from a subcellular fraction rich in lysosomes are taking place. It has been suggested that these alterations, both reflecting enhanced fragility of lysosomal membranes, might be due to increased endocytosis induced by the mitogen and the hypothesis was raised that lysosomal hydrolases might be involved in intracellular processes leading to lymphocyte activation2. We have therefore studied the fragility of lysosomes in unstimulated and PHA-treated lymphocytes from patients with chronic lymphocytic leukaemia (CLL), because lymphocytes in most patients with this disease show a diminished and/or delayed or even no response to PHA3 and other mitogens4,5.

The investigation of 2 families with 9 informative siblings seems to give some evidence for linkage between the structural loci for AB0 and PGI. The recombination frequency is 1/9 or ∼0.1.