Familial occurrence of the May-Hegglin anomaly: is the accompanying renal failure part of a new subentity?

We present two cases of the May-Hegglin anomaly discovered in a patient and one of her two sons. The female patient was known to have proteinuria from the age of 14 and was hospitalized in 1980, at the age of 25 years, because of hypertension and proteinuria (1.5 g/day). Thrombocytopenia was found with an abundance of megakaryocytes in the bone marrow. Both steroid treatment and splenectomy failed to ameliorate the thrombocytopenia, thought to be due to idiopathic thrombocytopenic purpura. Progressive renal failure, secondary hyperparathyroidism and uremic osteodystrophy were diagnosed in 1995. In January 1996, when she was hospitalized because of high-grade fever, we saw giant platelets and prominent blue inclusion bodies in almost all granulocytes in the peripheral blood smear. Electron microscopy confirmed the diagnosis of May-Hegglin anomaly in this patient and one of her sons, who at that time showed thrombocytopenia but no renal disease. Three years later, however, at the age of 15, the affected son was found to develop proteinuria. Coexpression of the May-Hegglin anomaly and renal disease, reported previously in a few other patients, may in fact represent a new subentity.

[T-cell large granular lymphocytic leukemia associated with pure red cell aplasia, successfully treated with cyclophosphamide]. / T-sejtes, nagy granuláris lymphocyta leukaemiához társuló "pure red cell aplasia" eredményes kezelése cyclophosphamiddal.

Authors report effective treatment of T-cell large granular lymphocyte (LGL) leukaemia and secondary pure cell aplasia with cyclophosphamide. The current classification of LGL proliferations is presented, with emphasis on the issues of diagnosis, clinical course and treatment. LGL proliferations are not so rare that previously thought and should be involved in the differential diagnosis of neutropenia, pure red cell aplasia, Felty's syndrome and vasculitis of unknown origin.

Ultrastructural study on the in vitro interaction between haemopoietic cells and stromal cells. A new method using gel technique.

The ultrastructural study of the interaction between stroma and haemopoiesis is not an easy task because the loose attachment may be damaged during manipulation. This paper describes a technique by which the loose connection between preestablished stromal layer and attached haemopoietic cells (derived from blast colony forming cells) can be preserved and studied ultrastructurally. Stromal cultures were obtained from human bone marrow cells. Blast colony forming cells were studied by co-incubating the stroma with fetal calf serum supplemented McCoy's medium containing bovine plasma, thrombin and calcium to form a gel ('plasma clot'). Colony formers attached to the stroma formed myeloid colonies within 6 days. The semisolid plasma clot which solidifiers rapidly on the addition of glutaraldehyde or formaldehyde entraps the blastic colonies and haemopoietic cells in their position. Even the non-attached or mobile cells can be entrapped by this technique. The immature cells were found to be attached to the stromal surface and/or to the extracellular matrix, while the more mature cells migrated either to the surface of the colony or attached to the non-covered areas of the plastic surface. This method may offer a special technique to study dynamic interactions in other situations (e.g. chemotaxis etc.), too.

Formation of large, membrane skeleton-free erythrocyte vesicles as a function of the intracellular pH and temperature.

Vesiculation of intact erythrocytes can be induced by decreasing their intracellular pH and then heating the red cell suspension to a critical temperature value. While at intracellular pH 6 vesiculation begins at 45 degrees C, further decrease in the intracellular pH lowers the critical temperature. In addition, the critical temperature value can be modified by varying the length of the interval between titration and heating as well as by changing the temperature during this interval. The vesicles are large (1-3.5 micron in diameter), haemoglobin-containing and completely free of skeletal proteins. Pretreatment of the cells with diamide and 2,4-dinitrophenol had no substantial effect on vesiculation, while N-ethylmaleimide, chlorpromazine and wheat germ agglutinin proved to be inhibitory. Increasing the osmolarity of the incubation medium markedly decreased the critical temperature: red cells suspended in a solution of 600 mosM NaCl vesiculated at 42 degrees C instead of 45 degrees C when the intracellular pH was decreased to 6. We propose that the vesiculation is due to a purely physicochemical molecular mechanism which affects the state and dimension of the membrane skeleton. We also discuss the possible role of an altered haemoglobin-membrane interaction in preventing low pH-induced intramembrane particle aggregation in the membrane skeleton-free vesicles.

Structural differences between the Ca2+-ATPase enzymes of sarcoplasmic reticulum membrane from rabbit and carp muscles.

1. Structural features were compared in sarcoplasmic reticulum Ca2+-ATPase enzymes from carp (Cyprinus carpio L.) and rabbit muscles. 2. Both membrane preparations contained the 105,000 mol. wt Ca2+ pump protein in high local density. 3. The tryptic cleavage of the carp enzyme gave different peptide fragments from those observed from rabbit enzyme. 4. Addition of vanadate, Ca2+ or lanthanides did not cause two-dimensional Ca2+-ATPase crystal formation, in contrast to the rabbit enzyme, which forms extensive arrays under these conditions. 5. No differences were found in this respect between microsome preparations derived from warm and cold adapted fishes. 6. A different primary sequence as well as a different disposition of the enzyme in the membrane may stand behind the observed dissimilarities.

The distribution and aggregatability of intramembrane particles in phenylhydrazine-treated human erythrocytes.

Freeze-fracture analysis of phenylhydrazine-treated, unfixed human erythrocytes showed a random distribution of intramembrane particles both over membrane-bound Heinz-bodies and in the intervening areas when examined after fast freezing in liquid propane. The same results was obtained when unfixed, glycerinated red cells were frozen in liquid Freon. In contrast to previously published data (Low et al. (1985) Science 227, 531-533) these results indicate that binding of Heinz-bodies to the red cell membrane cannot cause morphologically detectable clustering of Band 3 in phenylhydrazine-treated red cells. Over numerous Heinz-bodies a decreased Acridine orange-induced particle aggregation was observed. The phenomenon of the oxidant-induced red cell fluorescence is described.

The mobility of intramembrane particles in non-haemolysed human erythrocytes. Factors affecting acridine-orange-induced particle aggregation.

It has previously been shown that reversible intramembrane particle aggregation can be induced in non-haemolysed human erythrocytes. This phenomenon, which can be induced by the cationic dye Acridine Orange, has been further investigated using different experimental conditions that are expected to influence the rate of aggregation of the particles. In addition to the concentration of the dye, the rate of aggregation was also found to be dependent on the extracellular and intracellular pH, as well as on the type of buffer used. While lowering the pH of the Acridine Orange solutions resulted in decreased particle clustering, low intracellular pH increased and elevated intracellular pH decreased particle aggregation. Furthermore, at a given dye concentration and a given pH, Acridine Orange caused more intense aggregation in Tris-buffered saline than in isotonic phosphate buffer or phosphate-buffered saline. Under appropriate conditions Acridine Orange caused significant particle aggregation at concentrations as low as 0.25 mM within 30 s. During this period only discocyte-stomatocyte transformation occurred; neither agglutination nor vesiculation of the erythrocytes could be detected. Treatment of the erythrocytes with Diamide (Serva), which cross-links spectrin via disulphide bridges and thereby reduces lateral diffusion of integral membrane proteins over large distances, had no inhibitory effect on Acridine-Orange-induced particle aggregation. Heating the erythrocytes to 50 degrees C, at which temperature denaturation of spectrin and fragmentation of the erythrocytes occur, and subsequently incubating them in Acridine Orange at room temperature, caused an almost maximal rate of particle aggregation within 10-30 s, without haemolysis. The possible mechanism and significance of the particle aggregation phenomenon are discussed.

Coated pit formation: a membrane function involved in the regulation of cellular iron uptake.

Diferric-transferrin induces a marked increase in the number of coated pits on the reticulocyte membrane. This increase is followed by a decline, often to below the initial number. Since a good correlation was found between the rate of iron uptake and the number of coated pits, but not between the rate of transferrin recycling and the coated pit count, it is likely that coated pit formation is necessary for the removal of iron from transferrin. The decline in the number of transferrin-induced coated pits was observed only when haem synthesis was undisturbed, indicating that the accumulation of intracellular haem inhibits coated pit formation. Based on these results we suggest that haem regulates the rate of iron uptake by inhibiting iron removal rather than receptor recycling.

A comparative study on the cellular processing of free and gold-conjugated transferrin.

The kinetics of the transferrin-reticulocyte interaction was studied using free and colloidal gold-conjugated double labelled transferrin (Tf and AuTf, respectively). Simultaneous biochemical and morphological experiments provided the following information: 1. The cellular recycling of Tf is significantly faster than that of AuTf. 2. AuTf induces a marked increase in the number of multivesicular elements (MVE) in rabbit reticulocytes. 3. The release of AuTf from the cells is very slow and accumulation of gold particles in MVEs can be observed during the process. The results suggest that the two postulated pathways of the transferrin-cell cycle (a fast, iron-donating and a slow, receptor-shedding cycle) are not similarly involved in the cellular processing of Tf and AuTf. While it has been suggested that in the Tf-cell interaction the fast recycling mechanism is dominating, it is likely that, probably due to steric effects, the majority of AuTfs are forced into the slower receptor-shedding pathway. These observations call attention to the possible limitations of the colloidal gold labelling technique in the determination of the kinetics and pathway of intracellular processing of free ligands.

Intercellular junctions of methylcholanthrene-induced rat skin basocellular and squamous carcinomas.

The occurrence of different intercellular junctions in epithelial rat skin tumours induced by methylcholanthrene was investigated using thin sections and freeze-fracture replicas examined by electron microscopy. Tumours which appeared first were basal cell carcinomas. Later, different tumours of hair follicle and of sebaceous gland origin were formed. Finally, in the majority of tumours a squamous component evolved. Metastases developed from the squamous carcinomas exclusively. Desmosomes and gap junctions were detected in basal cell carcinomas whereas, in squamous carcinomas, tight junctions were also seen. While all three types of junction were found in the primary squamous tumours, the tumour metastases in lymph nodes and lungs contained only desmosomes.

Oxidation and denaturation of hemoglobin encapsulated in liposomes.

A study was made of the in vitro stability of hemoglobin-containing liposomes ('hemosomes') prepared from phosphatidylcholines, equimolar cholesterol and red cell lysate by the hand-shaking and ether-injection methods. Absorption spectra indicated hemichrome formation in 'hemosomes' prepared by the ether-injection technique, and increased oxidation of hemoglobin in hand-shaken 'hemosomes'. The denaturation of hemoglobin in ether-injection 'hemoglobin' was increased if the initial methemoglobin content of the hemolysate, or the temperature of preparation was elevated. It was slower if liposomes were prepared under either N2 or CO, or if the radical scavenger 1,3-diphenylisobenzofuran was added with the ether. Egg phosphatidylcholine and synthetic saturated phospholipids gave the same results. With hand-shaken 'hemosomes' the oxidized product was primarily methemoglobin, and oxidation could be inhibited by using saturated phosphatidylcholines instead of egg phosphatidylcholine. Lysophosphatidylcholine levels were higher and arachidonic acid levels lower in egg phosphatidylcholine 'hemosomes' than in equivalent liposomes containing no hemolysate. The 'hemosome' seems to be a suitable model for the study of hemoglobin-lipid membrane interactions and the resulting hemoglobin denaturation process.

Intense, reversible aggregation of intramembrane particles in non-haemolyzed human erythrocytes. A freeze-fracture study.

Acridine orange, a strongly cationic, membrane-penetrating dye, caused intense aggregation of intramembrane particles in non-haemolyzed human erythrocytes at 5 mM concentration. Simultaneously with the particle aggregation, large, empty, intramembrane particle-free or particle-depleted vesicles were detached from the red cells. Washing the erythrocytes after Acridine orange treatment resulted in complete disaggregation of the intramembrane particles. Less cationic acridine dyes (9-aminoacridine, 5-aminoacridine and Quinacrine) caused much less conspicuous alterations. Rivanol (ethacrine lactate), on the one hand, caused intramembrane particle aggregation in human red cells as well as ribosome aggregation in rabbit reticulocytes when it was dissolved in lactate-buffered sucrose. On the other, Rivanol dissolved in phosphate-buffered saline failed to induce these alterations. Neuraminidase treatment had no effect on the intensity of Acridine orange-induced particle aggregation, but impeded disaggregation. Our results indicate that, in contrast to previous observations, intense and reversible clustering of intramembrane particles is certainly possible in non-haemolyzed erythrocytes. The intramembrane particle aggregation may be due primarily to perturbation of the inner red cell surface by strongly cationic dyes and the presence of sialic acid residues on the outer red cell surface seems to be essential for the reversibility of the process.

Electron microscopic structure of the human round window membrane.

The fine structure of the human round window membrane has been investigated. It was found to consist of three layers: the tympanic cavity layer, the middle connective tissue layer, and the scala tympani layer. The middle connective tissue layer is considered the most important part of the round window membrane as it permits the movements of the inner ear fluid caused by the movements of the stapedial foot plate. The strength and elasticity of the membrane is guaranteed by three-dimensionally arranged collagen and elastic fibres and elastic networks in the middle connective tissue layer. The rupture of the round window membrane seems to be due to the diminished number and elasticity of the elastic elements in the middle connective tissue layer of the round window membrane.

Freeze-fracture study of the red cells and red cell precursors of a patient with congenital inclusion body anaemia.

Comparison of thin sections and freeze fracture replicas of red cells and red cell precursors from the peripheral blood of a patient suffering from congenital inclusion body anaemia provided new morphological information on Heinz body substructure, Heinz body-membrane attachment, intracellular membrane formation, as well as on membrane fusion and orientation of intracellular membranes. It is suggested that the fusion of autophagic vacuoles with the plasma membrane may contribute to the increased haemolysis of red cells in inclusion body anaemia.

T acute lymphoblastic leukaemia with breast tumor. A brief report.

Acute lymphoblastic leukaemia of L-2 type was diagnosed in an 18-year-old female with breast tumour. Her peripheral leukaemic cells reacted with anti-human peripheral T cell serum and part of the cells expressed SRBC receptor indicating the T origin of her leukaemia. She had a tumoral mass in her breast consisting of the same leukaemic cells. Combination chemotherapy was applied and she went into complete haematological remission with simultaneous disappearance of the breast tumour.

Intracellular "labyrinths" in acute leukaemia. Do they indicate the maturational ability of leukaemic cells?

The occurrence and ultrastructure of the so-called "labyrinths" were investigated in the bone marrow cells from 13 patients with preleukaemia and acute leukaemia. Except for one patient, labyrinths were found in the myeloid cells from patients with preleukaemia and acute myelogenous leukaemia. Labyrinths could not be detected in lymphoblasts from patients suffering from acute lymphoblastic leukaemia or in plasma cells from a patient with plasma cell leukaemia. In patients with preleukaemia and smoldering leukaemia, labyrinths could be detected in all differentiation forms, from myeloblasts to mature polymorphonuclear granulocytes. Since it is improbable that labyrinths develop in consequence of exogenous effects, their presence in the mature granulocytes points to the in vivo maturational ability of leukaemic precursors in certain cases of acute myelogenous leukaemia and preleukaemia.

Erythrocyte alterations in hemoglobin H disease.

This study on erythrocytes in hemoglobin H (Hb H) disease reveals that unstable Hb H is bound to membranes to a greater extent, especially when it forms methemoglobin or is precipitated as inclusion body. The methemoglobin content of these erythrocytes is elevated in spite of a higher activity of NADH-methemoglobin reductase. The ATPase activity is doubled, and the ATP is presumably used for phosphorylation of membrane proteins, which leads to cross-linking of membrane proteins. This assumption could be supported by the observed decrease in non-electrolyte permeability, by increased binding of hemoglobin to the membrane and by polymerisation of membrane proteins detected by SDS-polyacrylamide gel electrophoresis. By means of electron microscopy, it could also be shown that the inclusion bodies are bound to the inner surface of membrane and cause its protrusion. This linkage might be responsible for the observed inhibition of the lateral movement of intramembrane particles.