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Multiple forms of lactoferrin (Lf) were detected in granulocytes isolated from normal individuals and patients with granulocytic leukemias. One class of Lfs bound iron; a second class did not bind iron but possessed potent ribonuclease activity. The different forms of Lf were similar, if not identical, in their physical, chemical, and antigenic properties. The multiple forms of Lf may relate to the various functions ascribed to the molecule.

To examine the role of DNA methylation in breakpoint location of chromosomal translocation, HpaII sites in and flanking the M-bcr on chromosome 22 were mapped in DNA from blood granulocytes and lymphocytes, bone marrow cells, thymic tissue, and spermatozoa from normal individuals. Allelic HpaII sites were identified clustered in a 600-base pair genomic area of the M-bcr. Bone marrow cells and blood granulocyte DNA showed identical allelic patterns. Thymic tissue and blood lymphocytes showed identical allelic patterns distinct from bone marrow cells and blood granulocytes. Spermatozoa showed a third methylation pattern. In all individuals, the HpaII sites were present within the BamHI/BglII fragment of the M-bcr, the same area associated with high breakpoint frequency in chronic myelogenous leukemia (CML). Three of 15 patients with chronic phase CML showed fully methylated rearranged BglII/BglII M-bcr restriction fragments not seen in normal bone marrow cells. These methylation patterns of the M-bcr may be important in CML breakpoint location and may be a marker for tissue differentiation.

We discovered that the common acute lymphoblastic leukemia antigen, CALLA (CD10), was identical to human neutral endopeptidase 3.4.24.11 (NEP), a Zn-binding glycoprotein with an extracellular active site capable of hydrolyzing several biologically active peptides. In this study we compare the expression of CALLA/NEP in terms of antigenic density and enzymatic activity at the cell surface and of messenger RNA (mRNA) levels on granulocytes, leukemic cells, and CALLA-transfected COS-1 cells. Mature granulocytes, the only readily available source of normal human CALLA, express relatively low but constant levels of antigen, NEP activity (3.5 pmol/min/10(6) cells), and mRNA. The two major CALLA-mRNA species of 6.5 kb and 3.8 kb, observed to date in a variety of cells and tissues, were also found in four independent granulocyte preparations. With leukemia cell lines, a correlation was established between the density of CALLA antigen and the level of enzymatic activity (3.4 to 21.0 pmol/min/10(6) cells). This paper constitutes the first report of NEP activity on blast cells derived from patients with non-T acute lymphoblastic leukemia (ALL); the levels of activity were variable (1.5 to 35.9 pmol/min/10(6) cells for six cases) but correlated with the level of CALLA assessed by flow cytometry. Heterogeneous levels of expression of the CALLA-mRNA species were also observed in non-T ALL cases that correlated with the level of CALLA expression at the surface of these cells. Very high levels of NEP activity were achieved by transfecting COS-1 cells with pSV-CALLA; 20% of the transfected cells were CALLA+ and expressed 550 pmol/min/10(6) cells. Extracts prepared from COS-1 cells transfected with pSV-CALLA (carrying human NEP cDNA) and pSVENK19 (carrying rabbit NEP-cDNA), respectively, gave Michaelis constant (Km) values of 50 mumol/L and similar inhibition curves with thiorphan. Thus the recombinant proteins encoded by these two genes have similar enzymatic properties, confirming the high degree of their structural relatedness. The expression of high levels of CALLA/NEP on COS-1 cells should allow the use of this system to test the effects of specific mutations on activity and might lead to the understanding of the role of CALLA in the onset and/or progression of leukemia.

Axons that normally will encounter either CNS or PNS glia have been shown to contain a powerful mitogen for both Schwann cells and oligodendrocytes. The normally nonmyelinated, nonglial ensheathed cerebellar granule cells have been shown to possess a proliferative signal for Schwann cells, suggesting that a glial mitogen is common to all axons. To determine if a glial mitogen capable of stimulating both Schwann cells and oligodendrocytes is colocalized on all types of axons we have (1) cocultured granule cells with oligodendrocytes, (2) incubated oligodendrocytes with granule cell membranes, and (3) evaluated the ability of heparin extracts of granule cell membranes, splenic nerve microsomes, and axolemma-enriched fractions isolated from rat and bovine CNS to stimulate mitosis of cultured oligodendrocytes. Neither the intact granule cells nor the granule cell membrane fraction stimulated cultured oligodendrocytes to divide. However, heparin extracts of the granule cell membranes were significantly mitogenic to the cultured oligodendrocytes. Heparin extracts of splenic nerve microsomes were more mitogenic than the comparable extract obtained from bovine CNS axolemma-enriched fractions. These results suggest that the neuronal mitogen for oligodendroglia is colocalized with the neuronal mitogen for Schwann cells.

This study was undertaken to evaluate the efficiency of flow cytometry in the detection of the serological H-Y antigen, and to survey expression of H-Y in the normal human population. Peripheral blood leukocytes (granulocytes) were reacted with monoclonal H-Y antibody, gw-16, and with FITC-conjugated goat anti-mouse IgG, and then were scored for fluorescence in the flow cytometer. Assigned H-Y phenotype was correlated accurately with sex phenotype in 33 out of 38 women and 62 out of 65 men. Data were evaluated by exploratory data analysis (EDA).

In these studies, we characterize an Fc receptor (FcR) for IgA that is present on human granulocytes, monocyte/macrophages, and their corresponding cell lines. Receptor expression appears to be constitutive but can be selectively upregulated on monocyte cell lines by stimulation with a phorbol ester and polymeric IgA. Both the induction requirements and ligand specificity of the IgA receptor differ from the IgG receptors, Fc gamma R I, II, and III, that are also expressed on monocytes and granulocytes. IgA binding to the cell surface receptor is mediated via the Fc alpha region. The Fc alpha R is a heterogenously charged, approximately 60-kD molecule with an isoelectric point of 4.5-5.6 that binds monomeric or polymeric IgA1 and IgA2 molecules. This transmembrane glycoprotein appears to be composed of 32- and 36-kD protein cores with multiple N-linked carbohydrate moieties. We conclude that this Fc alpha R represents a novel member of the FcR family that may have a distinctive role in host defense.

We analyzed the amounts and types of glycosphingolipids (GSLs) from peripheral blood lymphocytes, monocytes, and granulocytes isolated by counter-current elutriation. The three cell types contained different amounts of neutral and acidic GSLs. The highest amount of neutral GSLs (109 micrograms/10(8) cells) was found in granulocytes, with considerably less found in monocytes (11 micrograms/10(8) cells) and lymphocytes (4 micrograms/10(8) cells). The neutral GSLs were composed of four types of lipids, GL1 through GL4 (mono-, di-, tri-, and tetraosylceramide). The highest percentage of GL1 was detected in lymphocytes and the lowest percentage in granulocytes, with the reverse order observed for GL2. GL3 and GL4, which were minor components of the neutral GSLs, were highly cell specific, with lymphocytes containing GL3 and GL4 of the globo series, granulocytes containing GL3 and GL4 of the lacto or neolacto series, and monocytes containing GL3 and GL4 of both types. The acidic GSL, sialosyl hexaosylceramide (lacto-series), was abundant in granulocytes but not in monocytes or lymphocytes. Another ganglioside, GM3, although present in all three cell types, was most abundant in monocytes and lymphocytes, whereas sialosyl paragloboside was higher in granulocytes than in lymphocytes and monocytes. These results indicate that peripheral blood lymphocytes, monocytes, and granulocytes have distinct "GSL fingerprints."

Studies on cell-membrane-bound proteins in the human hematopoetic system revealed that the expression of certain peptides is restricted to the differentiation lineage. We applied discontinuous polyacrylamide gel electrophoresis of triton X-114 lysates to identify such proteins for a new diagnostic approach in human leukemia. A polypeptide with an apparent molecular mass range of 24 kd (p24) was found predominantly in cells of chronic granulocytic leukemia (CGL), myeloic type of blast crisis, and normal granulocytes. The data presented here suggest a role of this protein in the biology of malignant cells in chronic granulocytic leukemia throughout the course of the disease.

Concentrations of free amino acids were measured concurrently in plasma, erythrocytes, granulocytes, and lymphocytes in umbilical cord blood, neonates, children, and adults. In each age group, the patterns of free amino acids were fairly similar in plasma and erythrocytes except for aspartic acid which was more abundant in erythrocytes. Of the amino acids in granulocytes, 71-77% was taurine; in lymphocytes taurine, aspartic acid, and glutamic acid comprised 35-44%, 18-24%, and 20-28%, respectively, of the total in all age groups. Leukocytes may contribute to the interorgan transport of amino acids to about 10% of the erythrocytes' contribution. Postnatally, the levels of glutamic acid and tyrosine in plasma; threonine plus glutamine, serine plus asparagine, and tyrosine in erythrocytes; histidine in granulocytes; and glutamic acid in lymphocytes were significantly increased (p less than 0.001); while the levels of phenylalanine and lysine in plasma; taurine in erythrocytes; valine and phenylalanine in granulocytes; and threonine plus glutamine, tyrosine, and phenylalanine in lymphocytes were significantly decreased (p less than 0.001). After the neonatal period concentrations of taurine and aspartic acid in erythrocytes, taurine and valine in granulocytes, and tyrosine and phenylalanine in lymphocytes increased gradually with age; while concentrations aspartic acid in plasma, histidine in granulocytes, and glycine in lymphocytes decreased gradually with age. The levels of glycine and valine in plasma, alanine and valine in erythrocytes, serine plus asparagine, glycine, alanine, and tyrosine in granulocytes, and aspartic acid, serine plus asparagine, and alanine in lymphocytes remained constant in all age groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Cultured adherent human macrophages and a promonocytic cell line, U 937, were previously shown to produce a Mr 95,000 gelatin-binding protein. The protein has no immunologic cross-reactivity with the well-characterized gelatin-binding protein fibronectin and the Mr 70,000 gelatin-binding protein produced by a variety of mesenchymal or epithelial cell types (T. Vartio et al, J Biol Chem 257:8862, 1982). In the present study the Mr 95,000 protein was found in Triton X-100 extracts of granulocytes purified from human blood buffy coat. The protein, as isolated by gelatin-agarose, was immunologically cross-reactive with the corresponding macrophage protein in immunoblotting assay. When peripheral blood and bone marrow cells were examined for the presence of the Mr 95,000 protein by indirect immunofluorescence, positive staining was detected only in differentiated granulocytes but not to any significant extent in metamyelocytes, myelocytes, promyelocytes, or in normal or leukemic blasts. In granulocytes the protein had a granular cytoplasmic distribution. In freshly prepared monocyte cultures, the Mr 95,000 protein was detected in low amounts in the cytoplasm, while along with differentiation of the cells into macrophages, the immunofluorescence increased in a reticular and vesicular cytoplasmic pattern and in a juxtanuclear cap, probably representing the Golgi complex. In conclusion, the Mr 95,000 gelatin-binding protein was specifically detected in macrophages and granulocytes and may thus serve as a differentiation marker for these phagocytic cells.

The antibiotic activity of a polypeptide fraction purified from bovine granulocyte granules was tested against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Bacillus subtilis, Bacillus stearothermophilus, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus faecalis, Streptococcus pyogenes, and clinical isolates of Staphylococcus and Enterobacter spp. All of these bacterial species were susceptible to the antibiotic polypeptide(s), with MICs ranging from 3 to 100 micrograms of protein per ml. The antimicrobial activity was resistant to boiling and abolished by proteinase treatment. Saccharomyces cerevisiae and human fibroblasts grew normally in the presence of 100 and 50 micrograms of antibiotic polypeptide(s) per ml, respectively. [3H]thymidine incorporation into bacterial, but not fibroblast, DNA was efficiently and promptly inhibited by the antimicrobial polypeptide preparation. This suggests that its main target is a component of the system, which catalyzes and regulates the biosynthesis of bacterial DNA.

Leukemia-associated inhibitory activity suppresses colony and cluster formation in vitro cells derived from granulocyte-macrophage progenitor cells of normal donors. It does not inhibit these same progenitor cells from patients with leukemia and it may contribute to the proliferative advantage leukemia cells appear to possess over normal hematopoietic cells during acute leukemia. The inhibitory activity was isolated by a combination of procedures including: ultracentrifugation, Sephadex G-200, carboxymethylcellulose, SDS-polyacrylamide gel electrophoresis, thin-layer and preparative isoelectric focusing and concanavalin A-Sepharose. Leukemia-associated inhibitory activity was characterized as a glycoprotein. it was inactivated by trypsin, chymotrypsin, pronase and periodate treatment. It bound to and was eluted by alpha-methylmannose from concanavalin A-Sepharose columns and had an apparent Mr range of 450-550 000 and an isoelectric focus value between pH 4.6 and 4.9. Crude leukemia associated inhibitory activity was temperature sensitive but the more purified preparations were heat stable.

We have investigated the nature of the antigens recognized by four classes of mouse anti-human monoclonal antibodies that characteristically reacted with neutrophilic granulocytes and their precursor cells, but not with monocytes or other normal hemopoietic cells. The antigenic targets of the majority (9/12) of the independently isolated monoclonal antibodies were present on two surface glycoproteins (Mr 145,000 and 105,000) and glycolipids. This antigen(s) was also detected on granulocyte precursor cells, including the bone marrow granulocyte/monocyte progenitor cells (CFU-GM). The same antigen(s) detected by these monoclonal antibodies was also present in non-hemopoietic cell lines (colon carcinoma and neuroblastoma). Three other antigens, defined by monoclonal antibodies AHN-8, L12.2, and L13.1 and present on granulocytes and their mid-late precursor cells, could not be identified as proteins but were detected in a protein-free glycolipid extract of these cells. The diversity of the antigens was confirmed by cross-competition experiments and by the identification of their different patterns of reactivity with cell lines and bone marrow cells.

This report describes a very simple, quick and effective method for in situ staining of granulocyte-macrophage colonies in agar cultures and for classifying various colony types. The procedure takes only one minute to fix and a few minutes to stain; a few additional minutes are required for preparation of the permanent whole plate. In this process the Riu stain, a modified Romanowsky stain, is used. Besides the ease and rapidity of this procedure, the identification of colony types appears to be enhanced. Thus, the method seems to be very beneficial in routine observations of colony types.

Human normal marrow cells from 5 donors have been separated via discontinuous density gradient according to the Dicke's technique and the morphology of granulocytic cells recovered from density fractions has been examined; peroxidase negative myeloblasts are concentrated in low density fractions (specific density below 1.063), while granulated myeloblasts (peroxidase positive) are concentrated at densities ranging between 1.063 and 1.073; promyelocytes and myelocytes have been observed in all fractions although their maximal concentration is at density 1.073; metamyelocytes and polymorphonucleated cells are concentrated in the highest density fraction (density 1.078); some differentiated cells which are peroxidase negative have been observed; they concentrate at a density lower than that of peroxidase positive cells which look morphologically identical.

The quantitative and qualitative characteristics of cells in the peritoneal dialysate from 12 patients were examined. The number of cells in each subsequent fraction of dialysate decreased, while the differential cell count remained relatively constant for each individual patient. Macrophages, lymphocytes, granulocytes and occasionally mesothelial cells were observed. In 1 patient, plasmocytes were also found. Evident differences in cellularity and cell composition were noticed in dialysate obtained from different patients, especially in 2 patients with bacterial peritonitis there was a rise in cellularity with neutrophilia. Cytochemical (peroxidase, nonspecific esterase activity) and functional (phagocytosis, receptor expression) tests revealed that macrophages form a heterogeneous population of cells.

HUMAN ERYTHROBLASTS IN CULTURE, IRRESPECTIVE OF THE ONTOGENIC STAGE OF THEIR PROGENITORS, ARE CHARACTERIZED BY: (i) the barely detectable amount of band 3 glycoprotein, (ii) the presence of two glycoproteins with molecular weights 105,000 and 95,000, (iii) the high concentration of glycophorin, and (iv) a minimum quantity of the carbohydrate chain susceptible to endo-beta-galactosidase ("polylactosaminoglycan"). In contrast, mature erythrocytes, whether of fetal, neonatal, or adult origin, are characterized by a high concentration of band 3 glycoprotein, polylactosaminoglycan, and glycophorins, but do not contain 105- and 95-kilodalton-glycoproteins. Thus, the process of erythroid maturation from erythroblasts to erythrocytes is accompanied by the appearance of band 3, the disappearance of 105- and 95-kilodalton glycoproteins, and a great increase in the quantity of polylactosaminoglycan. The structure of polylactosaminoglycan may not be different between mature erythrocytes and erythroblasts from the same ontogenic stage, but it is distinctively different from one stage to the other. The profiles of oligosaccharides released by endo-beta-galactosidase and immunofluorescence studies with anti-Ii antibodies indicated that a linear polylactosaminoglycan structure was present in erythroblasts as well as in erythrocytes of the fetal and newborn stage, whereas a branched polylactosaminoglycan structure was present in erythroblasts as well as erythrocytes of adult blood. Thus, two membrane characteristics are closely associated with the process of erythroid cell development; one-the membrane proteins band 3, band 4.5, and 95- and 105-kilodalton glycoproteins-determines the degree of maturation, and the other-polylactosaminoglycan-may determine the ontogenic stage of the erythroblast progenitors.

Lymphocyte blast transformation and granulocyte motility were studied in 20 clinically stable hemodialysis patients, 10 of whom were receiving 50 mg of zinc (as zinc acetate) per day and 10 of whom were not. Plasma zinc concentration was significantly higher in zinc-treated than in untreated patients (108 +/- 5 vs. 82 +/- 2 micrograms/dl). Mononuclear cell subpopulation analysis showed equivalent proportions of T lymphocytes and monocytes in both groups, but B lymphocytes were reduced in untreated patients (10 +/- 0.7 vs 14 +/- 0.5%). Lymphocyte blast transformations in response to nonspecific mitogens, soluble antigen and mixed lymphocyte culture were not significantly different in the two groups, nor was lymphocyte zinc concentration. Zinc-treated patients showed significantly greater granulocyte responsiveness to zymosan-activated serum (21 +/- 1 vs. 14 +/- 2 mean), greater chemokinetic activity (50 +/- 4 vs. 27 +/- 3 mean) and higher granulocyte zinc concentration (114 +/- 6 vs. 47 +/- 2 micrograms/10(6) cells) than untreated patients. Granulocyte zinc correlated significantly with plasma zinc (r = 0.81, P less than 0.001) and with granulocyte motility (r = 0.63, P = 0.001). Moderate zinc deficiency in hemodialysis patients does not result in abnormal lymphocyte blast transformation in vitro, but it does result in granulocyte zinc depletion and impaired granulocyte motility.

The cellular sediments of 42 malignant and 16 benign effusions (58 cases) were studied using the immunoperoxidase technique. Serial sections of formalin-fixed, paraffin-embedded residual sediments of effusions, sent for routine cytologic examination, were studied by commercially available polyclonal antisera against lysozyme, alpha 1-anti-trypsin, alpha 1-anti-chymotrypsin, tissue polypeptide antigen (TPA), a wide-spectrum anti-keratin, carcinoembryonic antigen (CEA) and, in single cases, thyroglobulin and prostate-specific antigen. A final definite diagnosis from histologic study of biopsy or autopsy specimens was known in all cases. All carcinomas, the mesotheliomas and the reactive mesothelial cells showed a positive reaction for TPA and, partly, the wide-spectrum keratin. Lysozyme could be demonstrated in the cells of the one proven malignant fibrous histiocytoma; all malignant epithelial cells were negative. Alpha 1-anti-chymotrypsin and alpha 1-anti-trypsin showed similar reactions: they were often positive in carcinoma cells of the breast, the bronchial system and the pancreas, in contrast to a mostly negative reaction in carcinomas of the stomach and ovary. CEA showed considerable differences; it was always negative in benign and malignant mesothelial proliferations but mostly positive in carcinomas of the stomach, pancreas and bronchial system. It was only positive in less than 20% of the carcinomas of the breast and always negative in the proven malignant effusions of primary carcinomas of the ovary and prostate. Studying a combination of several tumor markers is possible in serial paraffin-embedded sections and may be a valuable criterion in the cytologic diagnosis of effusions.

In order to isolate an endogenous inhibitor (chalone) of granulopoiesis, a source of chalone has been employed which consists of pure granulocytes. Optimal conditions for the isolation of these cells from bovine blood using EDTA are described. After incubation of the cells, the conditioned medium was lyophilised and extracted in two separate procedures with water or acetone. Sephadex G-10 chromatography yielded a fraction having a molecular weight under 700 which was capable of inhibiting the colony formation of myelopoietic stem cells in a nontoxic, cell-specific and reversible manner. THE INHIBITORy activity of the fraction was found to be dependent on the addition of 2-mercaptoethanol. Even then, aqueous solutions of the inhibitor are at present not indefinitely stable.

We have obtained cells in various stages of granulocytic development by a combination of isopycnic separation and electronic cell sorting. Not only were immature cells (blast cells, promyelocytes and myelocytes) separated from mature cells (bands and polys), but the immature cells were separated into proliferating (S + G2 + M) and resting (Go/G1) compartments of the cell cycle. This permits the study of the morphological and biochemical changes associated with development apart from those changes associated with proliferation.

A solid-phase radioimmunoassay has been developed for the detection of cell specific chromosomal proteins which require the presence of DNA for their immunoreactivity. The assay involves the binding of chromatin particles sonicated under highly controlled conditions to PVC plates followed by first and second antibody with extensive detergent washing. The sensitivity and specificity of the assay system compares favourably with the complement fixation technique.

1. A study was undertaken to investigate the distribution of ascorbic acid between various cellular components of blood, in normal individuals, and its relation to the plasma concentration. Forty-one unsupplemented individuals and sixteen supplemented (2 g/d for 5 d) individuals were studied. 2. Granulocytes, mononuclear leucocytes, platelets and erythrocytes were separated by differential sedimentation and centrifugation. Ascorbic acid contents were measured by the dinitrophenylhydrazine method. 3. Ascorbic acid content per cell was higher in mononuclear leucocytes and granulocytes than in platelets and erythrocytes. Intracellular ascorbic acid concentrations, calculated from published values for cell volumes, when compared with the plasma concentration showed a marked ability to concentrate ascorbic acid in mononuclear leucocytes (80 times), platelets (40 times) and granulocytes (25 times). 4. Erythrocytes showed little ability to concentrate ascorbic acid over the normal range of plasma concentration but because of their relative numbers they and the plasma fraction accounted for most of the blood-borne ascorbic acid (greater than 70%). 5. The ascorbic acid content of granulocytes, platelets and erythrocytes showed a significant positive correlation with the plasma concentration and supplementation with ascorbic acid significantly increased the content of these cell types. Mononuclear leucocytes in contrast did not show any such relationship. 6. The ability of the mononuclear leucocytes to maintain the highest levels of ascorbic acid in the cell types studied, despite variation in plasma availability, warrants further study, particularly in view of the importance of these cells in immunocompetence.

Structures of glycolipids isolated from human granulocytes were elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and exo- and endoglycosidase treatment. All neutral glycolipids, with saccharide residues ranging from 2 to 10, were found to have linear N-acetyllactosaminyl backbones. The majority of neutral glycolipids contain one or two fucosyl residues attached to N-acetylglucosamine residues through the Fuc alpha 1----3 linkage and were reactive with the monoclonal antibody specific to Gal beta 1----4(Fuc alpha 1----3)GlcNAc, the Lex structure. Their general structure can be expressed as follows: (formula; see text) where n = 0-3. Glycolipids containing sialic acid (gangliosides) were also found to have linear N-acetyllactosaminyl backbones with sialic acid joined to this backbone by either alpha 2----3 or alpha 2----6 linkage. The gangliosides have the following general structure: (formula; see text) where n = 0-3. The ceramide was composed of sphingosine with d18:1 as the long-chain base and C16:0 (as a major component) or C24:1 (as a minor component) fatty acid. Analysis of glycolipids isolated from granulocytes, erythrocytes, and whole blood cells revealed that, among the glycolipids prepared from the whole blood cells, dihexaosylceramide, lactoneotetraosylceramide, and the above described linear lactoneo series neutral glycolipids are present in granulocytes but barely present in erythrocytes.

Polylactosaminoglycans were isolated from human chronic myelogenous leukemia cells and their structures were elucidated. The lactosaminoglycan saccharides were isolated by hydrazinolysis and fractionated by QAE-Sephadex. The structures of fractionated oligosaccharides were analyzed by fast atom bombardment-mass spectrometry and methylation before and after treatment with specific exoglycosidases, such as alpha 2----3 specific neuraminidase. Based on these experiments, the structures of sialyl polylactosaminoglycans of chronic myelogenous leukemia cells were found to contain the following unique structure which is absent in normal mature granulocytes: (formula; see text) In addition to this, chronic myelogenous leukemia polylactosaminoglycans can be distinguished from normal granulocyte polylactosaminoglycans by the following characteristics. Leukemic polylactosaminoglycans are (a) shorter, (b) more highly sialylated and contain fully sialylated, tetrasialosyl polylactosaminoglycans, (c) are less fucosylated at C-3 of N-acetylglucosamine of polylactosaminyl side chains, and (d) contain a significant amount of sialyl Lex, NeuNAc alpha 2----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----3, structure. These results indicate that chronic myelogenous leukemia cells express unique polylactosaminoglycan structures which are distinct from normal mature granulocytes.

The human promyelocytic cell lines HL-60 can be induced to undergo differentiation to either granulocyte- or macrophage-like cells. We followed the changes in the synthesis and content of ferritin in this and other cell lines during differentiation. Ferritin content of HL-60 cells ranged from 11 to 81 fg/cell, depending on the clone tested. Following exposure to dimethylsulfoxide (DMSO) or retinoic acid (RA) an increase in ferritin and a decrease in total protein synthesis was observed, resulting in increased ferritin content, reaching a peak after 2 days. This increase occurred prior to the appearance of the typical morphological and functional characteristics of mature granulocytes. A correlation was found between concentrations of DMSO effective in inducing differentiation and the increase in ferritin content. Other inducers of granulocyte differentiation had a similar effect, while 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of macrophage differentiation, had not. Another human cell line (U-937), which was induced into monocyte-like cells by RA, showed a twofold increase in ferritin content following differentiation. Addition of iron to the culture medium increased ferritin content of both differentiating and non-differentiating cells, but the former responded to lower concentrations of iron. The increase in ferritin during differentiation, however, was not related to an accelerated iron uptake. The present results suggest that changes in the intracellular ferritin of the developing myeloid cells may play a regulating role in the process of maturation of these cells.

92 patients with acute myeloid leukemia were classified according to the FAB classification (M1 n = 20, M2 n = 43, M3 n = 1, M4 n = 19, M5a n = 2, M5b n = 2, and M6 n = 5 patients). Serum measurements of lactoferrin (LF), myeloperoxidase (MPO) and lysozyme (LYS) were performed before the start of treatment. LF was significantly lower in M1 when compared with M2 but not as compared to M4, MPO was significantly higher in M2 and M4 than in M1, but comparable MPO levels were found in M2 and M4. LYS was significantly elevated in M2 in comparison with M1, and in M4 when compared to both M1 and M2. Polymorphonuclear granulocytes (PMNs) in M1 were significantly reduced when compared with M2 and M4, whereas mononuclear cells were significantly increased in M4 in comparison with both M1 and M2. FAB classification did not generate any prognostic information. When the patients were, instead, subdivided according to LF levels were found prognostically significant differences. Of patients below 100 micrograms/l, 44% went into remission as compared to 77% with LF from 101 to 400 micrograms/l. In patients with LF levels above 400 micrograms/l the remission frequency was only 14%. Multivariate statistical analysis on the data further suggested that lactoferrin may be used as an independent prognostic indicator. We conclude that although determination of the serum-levels of lactoferrin, lysozyme and myeloperoxidase in certain cases may be valuable as a supplement to the morphological examination of acute myeloid leukemia, it is evident that none of the three determinations can be used alone to distinguish between the FAB groups.

Granulocyte chalone is a cell line-specific, species non-specific regulator substance, which inhibits cell proliferation in the granulocyte system in a reversible manner. The existence and specificity of action of this substance has been shown both in vitro and in vivo in widely different assay conditions and using a broad spectrum of assay techniques. Granulocyte chalone inhibits cell proliferation in the transit populations of the granulocyte system; it seems to act at the level of cell membrane and divert cells from the G1 phase to the "G0" phase with some direct or indirect effect on S phase cells as well. The substance is non-cytotoxic and it has no detectable effect on any other tissue than normal and leukaemic granulocytes even after long-lasting in vivo treatment. Granulocyte chalone offers an exciting potential for the treatment of myeloid leukaemia and, therefore, the substance is of more than academic interest.

An in vivo system has been developed for estimating the inhibitory activities of granulocyte chalone which overcomes the objection to the use of mixed proliferating target cells. It depends on the identification of labelled mature granulocytes in the peripheral blood of mice that received a radioactive precursor of DNA synthesis, with and without granulocyte extracts, several days previously. Two extracts of bovine granulocytes were tested. The first demonstrated that maximum inhibition was achived when it was given 2 h before the isotope, suggesting that its activity was short lived. It also failed to depress the labelling index of peripheral lymphocytes, thereby demonstrating tissue specificity. The second was used to show that the dose-response relationship was exponential.

A 39-year-old woman presented with mild anemia, glossitis, an increased MCV, a low serum cobalamin (Cbl) (vitamin B12), mild tissue deficiency of Cbl, but with neither malabsorption of Cbl, impaired intake, nor deficiency of or inactivity of transcobalamin II (TC II). Because of a persistently low holo-TC II (TC II carrying Cbl as the circulating complex of TC II-Cbl), much of the evaluation was focused on the patient's TC II. Her TC II promoted the uptake of Cbl, reacted with anti-TC II, and bound Cbl in vitro. A test dose of 200 micrograms of cyanocobalamin (CN-Cbl) i.m. increased her holo TC II to levels higher than those in healthy persons, but with a much more abrupt fall to a subnormal level. Two milligrams of CN-Cbl i.m. followed by 100 micrograms i.m. monthly failed to maintain normal amounts of circulating TC II-Cbl or to overcome the tissue deficiency of Cbl. One milligram i.m. weekly or daily p.o. corrected both. The low holo TC II was considered to be responsible for the clinical expression and may have been primary to the reduced amounts of total and holo R binder of Cbl in the circulation. This study of a newly recognized defect points out the need for circulating holo TC II, a rational use of pharmacologic amounts of Cbl, and a possible interrelationship between TC II and the R binder of Cbl.

L1, a major granulocyte protein, was purified and analysed by use of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Three subunits were visualized, and they were found to have molecular weights of 12.5 kDa, 13.3 kDa and 8.3 kDa. They were extracted from 2D-gels, and different combinations of subunits and two L1 antisera were analysed by immunodiffusion in agarose gel. The 8.3 kDa polypeptide in combination with one or both of the other polypeptides, gave immunoprecipitation with one of the L1 antisera, while no precipitation occurred when the three polypeptides were tested separately. Neither was there any precipitation when the two heavier polypeptides were tested in combination. By use of another L1 antiserum, all the L1 polypeptides were found to be antigenic and give immunoprecipitations. The L1 protein has a great affinity for calcium, and calcium was necessary for immunoprecipitation with the L1 subunits to occur. Autoradiographs of 2D-PAGE gels with labelled leucocytes visualized the L1 subunits in the same position as the subunits from purified, cold L1, indicating no significant alteration of the L1 protein during the purification procedure.

A series of maxillary sinus mucosal specimens and a series of smears of retained maxillary sinus secretions were studied with regard to the relationship between bacteria and inflammatory cells in order to illustrate aspects of the inflammatory process and to investigate the cause of mucosal damage in maxillary sinusitis. The results show that the granulocytes in the retained secretions are actively phagocytizing organisms if conditions are favourable for the granulocytes. Such situations are present in mucopurulent secretions but mostly not in strictly purulent secretions. A correlation was found between the presence of large numbers of inflammatory cells and tissue destruction, whereas bacterial invasion of the mucosa was a rare phenomenon and seen only in areas where the epithelial lining was destroyed. This indicates strongly that the mucosal damage in sinusitis is caused by inflammatory cells and not primarily by bacteria.

In this work granulocytes from normal human donors and patients suffering from chronic myeloid leukemia (CML) were externally labeled with 125Iodine, using the Iodogen method. 125Iodine labeled Concanavalin A binding proteins (CBP) and detergent-resistant proteins (DRP) were isolated from the cell lysates and characterized by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D- and 2D-SDS-PAGE). Autoradiographs of the 2D-gels of DRP show seven proteins with Mr 118,000 (spot 1 a), Mr 112,000 (spot 1b), Mr 78,000-85,000 (spot 2), Mr 85,000 (spot 4), Mr 52,000 (spot 3, 3 a and 3 b). Of this set, spot 1 b, 2 and 4 are also present in the autoradiographs of 2D-gels of CBP and, hence, may be considered to be transmembrane components. Spot 4 is expressed more intensely in the normal granulocytes while spots 3 a and 3 b are mainly expressed on the leukemic granulocytes.

Human leukocyte proteins from more than 150 patients with rheumatoid arthritis, together with age- and sex-matched controls, were analyzed by use of the ISO-DALT technique in two-dimensional polyacrylamide gel electrophoresis. Patients with ankylosing spondylitis, polymyalgia rheumatica, psoriatic arthritis, calcium tendinitis, post-infectious arthritis, and asymmetrical seronegative arthritis were also included as positive controls. Synthesis of several proteins, referred to by number as members of the "Rheuma" set, is shown to increase in the leukocyte preparations from patients with classical rheumatoid arthritis. Several of these proteins are specific to monocytes or granulocytes; others are of unknown cellular origin, but appear to be unique to rheumatoid arthritis. The Rheuma proteins appear to be indicators of disease activity, because their increased synthesis can be correlated with sedimentation rate and other clinical indices of rheumatoid disease activity.

Zinc status and the effect of zinc supplementation were assessed in groups of patients with non-Hodgkin's lymphoma and Hodgkin's disease; patients were either untreated or in remission. In the patients in remission, plasma zinc was normal; and whereas 30% of untreated patients had low plasma zinc, the group as a whole did not differ from normal. For mononuclear cell zinc, the range of values in the disease group was far wider than in controls, but there was no significant difference between the means of the groups. Granulocyte zinc was significantly lower in both the groups of patients in remission from non-Hodgkin's lymphoma and Hodgkin's disease compared with the control group. Significant increases were found in the plasma copper, ceruloplasmin, and the copper-to-zinc ratio in several of the patient groups. Plasma zinc increased by 23% with zinc supplementation (50 mg elemental Zn/day), but there was no effect on mononuclear cell or granulocyte zinc. Apart from granulocyte zinc, there is little evidence of zinc deficiency in non-Hodgkin's lymphoma or Hodgkin's disease. However, the presence of depleted granulocyte zinc levels could modify the immune function of this cell population.

Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) patients consistently show a rearrangement in a 5.8-kilobase length of chromosome 22, referred to as the breakpoint cluster region (bcr). In Ph1-positive acute lymphoblastic leukemia (ALL), the breakpoint in chromosome 22 is more heterogeneous and, in some instances, does not occur within this region. In such cases the cell of origin of the neoplastic clone and the relationship of the disease to CML has remained obscure. We have analyzed the bcr rearrangement in the malignant cells from three patients who presented with Ph1-positive ALL and who in cytogenetic studies had shown evidence of variable involvement of myeloid cells in the Ph1-positive clone. Rearrangements in bcr typical of most cases of CML were detected in purified granulocyte preparations from two of the ALL patients (nos. 1 and 2) and in the blasts from patient 3 at the time of her terminal relapse. In the same analysis the simultaneously obtained granulocytes from patient 3, however, did not show any evidence of bcr rearrangement. Patient 3 was also heterozygous for the BamHI polymorphism in the X-linked hypoxanthine phosphoribosyltransferase (HPRT) gene, thus permitting a different method of clonal analysis based on methylation differences in active and inactive alleles. When DNA from her granulocytes that had shown no bcr rearrangement was hybridized to an HPRT probe, a pattern typical of a polyclonal population was seen. A similar pattern was exhibited by her marrow fibroblasts. In marked contrast, her simultaneously isolated blasts showed an unambiguous monoclonal pattern. These findings demonstrate the origin of the disease in the first two patients in a cell with myelopoietic as well as lymphopoietic potential and confirm the restricted lymphoid cell origin of the neoplastic clone in the third Ph1-positive ALL patient. Furthermore, they indicate that different target cells for transformation within the hematopoietic system may be affected by very similar bcr rearrangements.

In the last period of time there has been stated that in human lysosomes of neutrophilic leukocytes an in ones of some experimental animals occur, which stimulate the growth of lymphocytes and fibroblasts in the study in vitro. With regard to this it was assumed that similar stimulating processes may take place in the surgical wounds. The experiments were carried out on guinea pigs which were wounded and afterwards the wounds were sutured and the allogenic proteins preparations were introduced into its in dose of 100 microgram as follows: lysosomal proteins of granulocytes, mixed and separated on the the DEAE-cellulose columns; lysosomal proteins of macrophages, the proteins isolated from plasma and nuclei both the granulocytes and macrophages. The evaluation of healing was based on the measurement of mechanical resistance of wounds adhesions and was completed with the pathomorphological examinations. It has been shown that the mixture of lysosomal proteins of granulocytes, intruduced into the wounds, caused the acceleration of the healing processes. After giving into the wounds some fractions of proteins which were separated on the colums with DEAE-cellulose, the stimulating processes of healing were seen, while after adding the other ones-the inhibiting processes. The protein with similar evidences were not show inside other structures studied of granulocytes and macrophages. The results obtained seem to support the supposition that in lysosomes of neutrophilic granulocytes there are the factors which regulate the repair processes in wounds.

Cytological examination of cerebrospinal fluid needs special techniques of cell collecting and staining. On condition of realizing the methodical requirements cytology of the cerebrospinal fluid brings more advancement in diagnostics than other laboratory examinations. The advantages of this procedure are: 1. Meningitis, meningoencephalitis, and subarachnoidal hemorrhage can be immediately diagnosed by cytological examination of the cerebrospinal fluid. In cases of encephalitis cytological findings depend on the extend of the inflammatory process and its distance from the cerebrospinal fluid compartement. Absence of tumor cells in preparations of cerebrospinal fluid cannot exclude primary or secundary brain tumors. 2. Cytological examination enables a better insight into the dynamics of cellular reactions proceeding in the cerebrospinal fluid as well as in its enlining tissues.

Cationic lysosomal proteins from human polymorphonuclears (PMN) were isolated by column chromatography and divided into five fractions. On acrylamide gel electrophoresis, fraction I had four bands slower than lysozyme (LZM) mobility; fraction II had five or six bands slower than LZM; fraction III had at least seven bands slower and two bands faster than LZM; fraction IV contained LZM, two bands faster and a few faint bands slower than LZM; fraction V was composed of almost pure LZM. Partial characterization of the fractions showed presence of neutral protease in fractions I-IV, chymotrypsin in fraction III, lysozyme in fractions IV and V, and phospholipase A2 mainly in fractions II and III. Modulatory activity of fractions I-V were tested at concentrations up to 50 micrograms/ml. Enhancement of phagocytosis of Staphylococcus aureus was observed by fractions I, IV, and V, whereas phagocytic index was enhanced by all but the fraction II. Intracellular bactericidal activity (ICBA) was markedly enhanced by fractions I, II, and V. Addition of DNA or cytochalasine B inhibited or abolished phagocytosis-enhancing activity of cationic fractions. Their influence on ICBA was much less pronounced. Fraction III enhanced phagocytic index and phagocytosis of E. coli, whereas fractions I and II enhanced intracellular bactericidal activity against this bacteria. Enhancement of phagocytic activity of monocytes has also been observed. The data suggest that some cationic lysosomal fractions from human PMNs enhance phagocytosis and phagocytic index by human PMNs and monocytes and intracellular bactericidal activity of human PMNs. This alternative pathway of phagocytic enhancement is unrelated to the previously described enhancers of phagocytosis and may play a role in defense mechanisms against infection.

The mass fraction of calcium (Ca), magnesium (Mg), manganese (Mn), iron (Fe). zinc (Zn), strontium (Sr) and copper (Cu) in isolated granulocytes, erythrocytes and platelets was measured using the nuclear microprobe technique. The cellular profile of metal variation was conspiciously altered in patients (n = 29) with ankylosing spondylitis compared with the profile found in age and sex matched healthy controls. Ca was accumulated in erythrocytes and granulocytes but decreased in platelets (p less than 0.001). Mg was increased in granulocytes but appeared in reduced concentrations in platelets and erythrocytes (p less than 0.001). The cellular amounts of Mn was increased in granulocytes (p less than 0.001), normal in platelets (p greater than 0.05) and subnormal in erythrocytes (p less than 0.001). Zn was reduced in all 3 cell types (p less than 0.001). Fe accumulation was evident in granulocytes and platelets (p less than 0.001). Sr was only measurable in granulocytes from patients. Cu was below the detection limit in the different cell types isolated from patients, but appeared in measurable amounts in erythrocytes and platelets from controls. The granulocyte amounts of Ca, Mg, Mn and Sr were strongly related to the acute phase reaction. Negative correlations were found between erythrocyte Mg and Zn and the inflammatory activity. The patients had increased serum levels of Cu, normal levels of Ca, Mg and Sr and decreased levels of Zn, Fe and Mn. No relationship was found between the serum concentrations of these elements and their respective cellular stores, except for a weak negative correlation between granulocyte and serum Fe.(ABSTRACT TRUNCATED AT 250 WORDS)

A new method of assessing disease activity in ulcerative colitis has been developed utilizing indium-111-labelled granulocytes. Faecal 111In excretion after intravenous administration of labelled granulocytes ranged from 1.1% to 45% of the injected dose in patients with ulcerative colitis, showing significant differences between mild, moderate, and severely active groups. There was a significant correlation between faecal 111In excretion and a clinical index based on the Crohn's disease activity index (r = 0.79, p less than 0.001) and the erythrocyte sedimentation rate (r = 0.73, p less than 0.001). Quantitative faecal 111In-labelled granulocyte excretion is an objective and specific method of assessing disease activity in ulcerative colitis.