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Magnesium and potassium were analyzed in plasma, erythrocytes, and urine collected during 24 h and in muscle biopsies from 25 subjects with insulin-dependent, type I diabetes mellitus (IDDM). Magnesium was also measured in mononuclear cells. The results were compared with those of 28 healthy controls, and were also correlated with the degree of diabetic control as estimated by analysis of the level of glycosylated hemoglobin (HbA1c). Subjects with IDDM had significantly lower muscle (P less than 0.01) and plasma (P less than 0.001) concentrations of magnesium compared with those of healthy controls. The HbA1c levels correlated significantly with the concentrations of magnesium in muscle (r = -0.62, P less than 0.001), plasma (r = -0.62, P less than 0.001), and mononuclear cells (r = -0.47, P less than 0.05). The results indicate that some patients with IDDM have lowered contents of magnesium in striated muscular and/or plasma, and that those parameters are dependent on the degree of diabetic control.

Intra- and extracellular phase transitions in human peripheral blood monocyte suspensions with and without the cryoprotectant 1 M dimethylsulfoxide were measured using differential scanning calorimetry. Using an fluorescence diacetate/ethidium bromide assay for membrane integrity and a phagocytosis assay for cell function, it was found that mortality was correlated with several phase transitions under a variety of cooling and warming regimens. As a result of these studies we concluded that: intracellular freezing is lethal, but avoidance of freezing during fast cooling is not sufficient to provide complete protection; a subtle freezing injury in the cryoprotected monocytes can be correlated with a measurable increase in devitrification on warming; and the cell contents form more stable glasses than the Hanks' balanced salt solution with fetal calf serum used as the extracellular medium.

Four monoclonal antibodies, termed AMH-1, AMH-2, AMH-3, and AMH-4, raised against human lung macrophages in bronchoalveolar lavaged fluid, alveolar spaces, and interstitia of lung tissue are described. The antibodies were produced according to hybridoma technique by immunizing mice with bronchoalveolar lavaged cells. All four monoclonal antibodies reacted with macrophages in bronchoalveolar lavaged fluid and alveolar spaces by immunohistochemical staining and flow cytometric analysis, but they gave different reactivity patterns with the monocyte-macrophage lineage. AMH-1 did not react with peripheral blood monocytes, peritoneal macrophages, or pulmonary interstitial macrophages. Although AMH-2 reacted weakly with blood monocytes and with some of the pulmonary interstitial macrophages, it did not react with peritoneal macrophages. AMH-3 did not show reactivities with either blood monocytes or peritoneal macrophages but was positive for most of the pulmonary interstitial macrophages. AMH-4 was reactive with cells from the monocyte-macrophage lineage. There was a correlation between the reactivity patterns of all four antibodies to macrophages in bronchoalveolar lavaged fluid and the patients' smoking habits. Most significantly, epithelioid cells of lung granulomas obtained from patients with sarcoidosis and hypersensitivity pneumonitis were negative for AMH-1 but were strongly stained by AMH-2, AMH-3, and AMH-4. Differences among the four antibodies in their reactivities with macrophages and granulomas in lungs indicate that lung macrophages contain heterogeneous populations which are in various states of differentiation and maturation and that the epithelioid cells and lung macrophages share the same membrane antigens. Therefore, these antibodies would be useful reagents for investigating the subpopulations and functions of macrophages in lungs and for clarifying the pathogenesis of granulomatous lung diseases.

Three trace elements, manganese, copper and zinc, selected as normal constituents were measured in the chromatin fraction of peripheral blood mononuclear cells from four pemphigous patients and compared with values form 4 matching control cases treated with corticosteroid. Manganese and copper were decreased in pemphigous patients significantly against controls whereas zinc was decreased slightly without statistical significance. Decreased trace element levels in chromatin revealed an unique pattern characteristic only for pemphigus as compared with earlier data of this group and of others in different pathological states. Decrease in chromatin zinc content might reflect the fact of corticosteroid treatment rather than a pathognomic feature which has to be taken into consideration while working with samples from patients on long-term steroid therapy. The theory of trace element depletion in pemphigus is confronted with known reports of penicillamine--a chelating agent--induced cases of the disease.

A radioimmunoassay (RIA) was used to quantitate biologically active gamma interferon (INF-gamma) in sera and in supernatants of cultured mononuclear cells obtained from 50 patients with aplastic anemia. Only five of the 50 serum samples had INF-gamma levels above background (greater than 0 less than 0.5 units per mL). Detectable levels of spontaneous INF-gamma (0.3 to 868 U/mL) were found in 18 of the 50 mononuclear cell supernatants tested. The addition of patient sera or INF-gamma positive supernatants to cultures of normal hematopoietic colonies did not result in reduced colony growth. Flow cytofluorimetric analysis of mononuclear cells failed to establish a correlation between the presence of INF-gamma in supernatants and the number of activated T cells or natural killer (NK) cells in the mononuclear cell population. However, a significant correlation did exist between the presence of monocytes and the production of INF-gamma. Contrary to previous reports, our data suggest that patients with aplastic anemia do not have high circulating levels of INF-gamma. Unstimulated mononuclear cells from some patients will produce significant levels of INF-gamma, but this does not result in decreased in vitro hematopoiesis.

Five Persian Jews were detected with the polyglandular deficiency syndrome (PDS). Primary hypoparathyroidism and hypogonadism were present in each, adrenal insufficiency in two, and insulin-dependent diabetes mellitus and latent hypothyroidism in single subjects. The percentage of T and B cells, and the mononuclear cell response to phytohemagglutinin and Concanavalin A were normal in all five. IgG and IgA levels and the OKT4+/OKT8+ cell ratio were low in one subject. Antinuclear and antithyroid antibodies were present in one subject. HLA-DR5 was present in 4/4, HLA-24 and B5 (B51) in 3/4 subjects. A single case of isolated hypoparathyroidism (IHP) was detected among 12 first degree relatives. HLA antigens B8, DR3, were absent in all of these subjects. Seven non-Iranian Jews with IHP were also examined. HLA A26 or A25 were present in all seven. Persian Jews appear to have a unique variant of PDS.

A biotin-avidin horseradish peroxidase system is described for the localisation of membrane-bound monoclonal antibodies by low power transmission electron microscopy. An indirect bridge technique was used, with a biotin-labelled goat anti-mouse immunoglobulin antibody and horseradish peroxidase-conjugated avidin D. The technique provided preparations of high morphological and ultrastructural quality which facilitated accurate cellular identification. Positive immunostaining was specific and precisely defined.

Surface proteins radiolabeled by lactoperoxidase catalyzed radioiodination and/or sialoglycoproteins tritium-labeled by sodium metaperiodate/NaB3H4 technique, obtained from neoplastic cells of more than 50 patients with malignant hemopoietic disease have been analyzed with acrylamide gradient gel electrophoresis under denaturing conditions (SDS-PAGE). Electrophoretic protein and glycoprotein patterns were essentially similar within the group of examined chronic lymphocytic leukemia (CLL) patients with major surface glycoproteins gp44 and gp29,35. Glycoprotein gp29,35 corresponds by its electrophoretic mobility to Ia-like (HLA-DR antigen), which was identified on some CLL cells also by immunoprecipitation with a conventional anti-Ia antigen serum and subsequent electrophoretic analysis of immuno-precipitated antigen. Electrophoretic patterns of cell surface proteins and glycoproteins of CLL cells were markedly different from those of acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML) cells which possessed a characteristic major cell surface glycoprotein gp95 (with relative molecular weight of 95-105k). This glycoprotein was markedly decreased or absent on examined CLL cells. Electrophoretic glycoprotein patterns revealed variations in the expression of several glycoproteins, as well as in the electrophoretic mobility of major glycoprotein gp95 also within the examined groups, particularly in acute myeloblastic leukemia.

Human articular chondrocytes in culture produced large amounts of specific mammalian collagenase, gelatinase and proteoglycanase when exposed to dialysed supernatant medium derived from cultured human blood mononuclear cells (mononuclear cell factor) or to conditioned medium, partially purified by fractionation with ammonium sulphate (60-90% fraction), from cultures of human synovial tissue (synovial factor). Human chondrocytes and synovial cells also released into culture medium an inhibitor of collagenase of apparent molecular weight about 30 000, which appeared to be similar to the tissue inhibitor of metalloproteinases synthesised by tissues in culture. The amounts of free collagenase inhibitor were reduced in culture media from chondrocytes or synovial cells exposed to mononuclear cell factor or synovial factor. While retinol inhibited the production of collagenase brought about by mononuclear cell factor or synovial factor, it restored the levels of inhibitor, which were reduced in the presence of mononuclear cell factor or synovial factor. Dexamethasone markedly reduced the production of collagenase by synovial cells, while only partially inhibiting factor-stimulated collagenase production by chondrocytes. Addition of puromycin as an inhibitor of protein synthesis reduced the amounts of both collagenase and inhibitor to control or undetectable levels.

Monocytic features can be induced in the myeloid cell line HL60 in order to provide a suitable in vitro model for the investigation of in vitro activity in mononuclear phagocytes. 1,25-Dihydroxyvitamin D3 (calcitriol) induced the HL60 cell line to express the monocytic differentiation antigen Leu M3 in about 30-50% of the cells along with an increase (up to 20%) in the expression of HLA-DR but not HLA-DQ class II antigen. Functional investigation showed that calcitriol-treated cells formed rosettes with sheep erythrocytes coated with an anti-sheep erythrocyte-specific IgG2a mouse MoAb and readily ingested them. In addition, these same sensitized erythrocytes were lysed in an 18-hr antibody-dependent cellular cytotoxicity (ADCC) assay. All together these data indicate the presence of functionally active Fc-IgG receptors (FcR). Sorting experiments demonstrated that only Leu M3+ HLA-DR+ cells contained the effector cell population; such was also the case for blood monocytes. This phenotypic profile was, however, not predictive per se of FcR presence and function, as 12,O-tetradecanoylphorbol-13-acetate (TPA)-induced HL60 cells neither formed rosettes nor phagocytosed nor exhibited ADCC activity, although they express Leu M3 and HLA-DR (as well as HLA-DQ) antigens. These results suggest that calcitriol and TPA cause the differentiation of HL60 cells along distinct pathways. On the other hand, different subpopulations with given predetermined differentiation capabilities may coexist in HL60 cell line. This hypothesis gains support by the observation that when TPA and calcitriol were added together to the undifferentiated cells, the resulting phenotypic pattern was representative of the different activities of both of the inducers as they were used separately.

Normal, unstimulated alveolar macrophages (AM) and monocytes (Mo) from both mice and rats have been shown to exhibit an angiotensin-I-forming enzyme. This protease has a similar or identical molecular weight to plasma renin as judged by HPLC analysis. In addition, the enzyme activity could be almost completely inhibited by a specific antibody to pure mouse submaxillary gland renin. This indicates that the angiotensin-I-generating protease in AM and Mo is identical to true renin. These results are in accordance with those of immunocytochemical studies, since the cells sharing immunoreactive renin are macrophages and monocytes. The low percentage of renin positive cells may indicate a functionally different and probably distinct subpopulation of AM and Mo. The presence of renin and angiotensins and angiotensin-I-converting enzyme in normal macrophages and monocytes seems to be common. Hence, the data presented raise the possibility that an intracellular renin-angiotensin system may infact exist in a certain subpopulation(s) of normal macrophages and monocytes.

A simple, sensitive, rapid and reproducible method is described for the measurement of HLA-DR expression on monocytes. The assay is based on an enzyme-linked immunostaining of the cells in suspension form followed by colour development. The non-specific binding of anti-HLA-DR to Fc receptors was taken into account by incubation of cells with an irrelevant monoclonal antibody (antirubella-G7H9). The binding of the anti-rubella monoclonal with the cells was subtracted from the binding of anti-HLA-DR to cells. Using three different cell lines (K562, CTLL and Daudi), the specificity of the assay proved to be acceptable and the assay remained sensitive even when only 10(4) cells/well were used. The assay has a number of potential applications.

Previous investigations have suggested that beta-adrenoceptor-mediated responses were decreased in uremia. To evaluate this phenomenon further, beta 2-receptor density in mononuclear cells, plasma catecholamines and plasma parathyroid hormone were studied in two groups of normotensive patients: group U, twenty-five chronic uremic patients with end-stage renal failure; group C, twenty-eight control subjects. Each group was divided into three age and sex-matched subgroups. Beta 2-receptor density was determined using (-)125 iodocyanopindolol. Despite a significant increase in plasma epinephrine in the group of uremic patients, there was a significant increase in beta 2-adrenoceptor density. On the other hand the uremic state did not influence (-)125 iodocyanopindolol binding affinity and plasma norepinephrine. Parathyroid hormone, as expected, was significantly elevated in all the uremic subgroups. It can be concluded that the uremic state is associated with an unexpected upregulation of beta 2-receptor density in mononuclear cells. The role of an endogenous beta-blocking substance is suggested.

Using a method based on the differential affinity of mononuclear cells to insolubilized histamine, we were able to distinguish several differences in the composition of T cell subpopulations between healthy control subjects and sarcoidosis patients. In 5 of 10 patients with sarcoidosis, the ability of T cells to respond to three mitogens was severely depressed, while in the remaining 5 it was similar to the response of normal subjects. After the elimination of a subpopulation of peripheral blood mononuclear cells (PBMC) that adhered to the histamine column, the response to mitogens of the unbound cells of normal control subjects was significantly lower than values obtained before separation. The histamine-unbound lymphocytes of the five patients with active sarcoidosis, whose unseparated PBMC responded poorly to mitogens, reacted significantly better to the mitogens than before the separation. This pattern of responsiveness has been previously only in PBMC of patients with autoimmune diseases. It seems that some patients with sarcoidosis possess a subpopulation of T-lymphocytes with suppressor activity and a specific affinity to insolubilized histamine, the elimination of which restores the level of response to almost normal.

We have previously shown that the human monocyte-like cell line U937 synthesizes C1-INA and expresses cell surface C1-INA. In this report we provide evidence that this surface-expressed C1-INA is functionally active. Intact U937 cells demonstrated functional C1-INA activity in a hemolytic assay. This activity was blocked when the cells were incubated with monospecific antibody to C1-INA, and was not detectable in cell-free supernatants of U937 cells. SDS-PAGE analysis of radiolabeled U937 cell surface proteins purified by anti-C1-INA affinity chromatography revealed two distinct bands. One protein had a Mr of 105 kDa identical to plasma C1-INA, and the second had a Mr of 200 kDa. We were unable to determine the identity of the 200 kDa protein by Western blotting with anti-C1-INA. However, the possibility exists that this 200 kDa molecule may represent a C1-INA receptor, a dimeric form of C1-INA, or an unrelated cell surface protein with affinity for C1-INA. Furthermore, we show that treatment of U937 cells with phorbol ester resulted in an increase in the percentage of cells expressing surface C1-INA. These results suggest that U937 cells express functional cell surface C1-INA, which could function in vivo to protect these human tumor cells from lysis by host complement.

Cytokines released from monocytes upon stimulation by lipopolysaccharide cause a number of cells to undergo proliferative and synthetic changes. At least one of these cytokines affects hepatocytes in vivo causing increased synthesis of a series of acute-phase proteins. We have established an in vitro micro-assay for hepatocyte stimulating factor (HSF) using primary cultures of normal rat hepatocytes. Measurement of increased synthesis of alpha 2-macroglobulin and decreased synthesis of albumin caused by exogenously added factor constitute a sensitive parameter for quantifying HSF. For comparing various cytokines preparations, we have defined a unit of HSF activity in terms of a stimulation index. We have used this assay to follow some preliminary attempts to isolate the factors responsible for stimulation of synthesis of acute-phase reactant by the liver.

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.

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.

Seven patients with Hodgkin's disease were studied for the presence of lysozyme and alpha-1-antitrypsin activity by immunoelectron microscopy. As a result, Reed-Sternberg cells, Hodgkin's cells, and atypical cells were distinctly positive for lysozyme in four cases and weakly positive in the remaining three cases. These cells were also positive for alpha-1-antitrypsin in all cases. Because the cells of the monocyte-macrophage lineage also bore lysozyme and alpha-1-antitrypsin, it is suggested that Reed-Sternberg cells, Hodgkin's cells, and the atypical cells are derived from the monocyte-macrophage lineage.

The thromboplastin synthesis of the human monocytoid cell line U-937 and its two subclones designated U-937-3 and U-937-4 has been studied. U-937-4 seems by several functional criteria to represent a more advanced stage of monocyte differentiation than the original U-937. U-937-3 appears to be arrested at an even more immature stage than the original population. The basal thromboplastin activity was higher in U-937-4 than in U-937-3 or U-937 cells (7.0 +/- 1.9 (SEM), 1.0 +/- 0.2 and 1.6 +/- 0.6 units/mg protein, respectively) although not as high as in human normal monocytes (14.1 +/- 2.4). The thromboplastic expression of the two clones was maximal when cells were in logarithmic growth. Both clones responded with a weak to moderate thromboplastin synthesis upon addition of stimulants like phytohaemagglutinin (PHA), immune complexes or endotoxin. Thromboplastin production was also potentiated in the presence of lymphocytes. The supporting effect of lymphocytes was strong in the case of U-937-3 as well as in U-937 cells, but less pronounced in U-937-4 cells as it also is in human monocytes. The thromboplastin response after PHA stimulation was more rapid in U-937-4 cells (maximal after 4-8 h) than in U-937 or U-937-3 cells (12-16 h). Human monocytes also responds quickly to PHA (maximally 4 h). Total phospholipid content and the relative distribution of individual phospholipids were essentially similar in U-937-3, U-937-4 and U-937. With regard to thromboplastin production, U-937-4 cells seem to be more monocyte-like than the more immature cells U-937-3 and U-937. It is concluded that thromboplastin seems to be a useful marker for monocyte differentiation.

Plasma and buffy layer vitamin C concentrations have been measured in 19 patients before and following major surgery, and falls of 36% and 43% respectively were recorded on the first post-operative day. However, the change in the buffy layer could not be accounted for by a change in the concentration of vitamin C in platelets, mononuclear cells or polymorphonuclear leucocytes, the cell types which form the buffy layer. The buffy layer change could be explained by a post-surgical increase in the number of polymorphonuclear leucocytes which, when the buffy layer vitamin C is expressed per 10(8) white cells, dilutes the platelet and mononuclear cell contribution to the buffy layer vitamin C concentration. The fall in buffy layer vitamin C does not, therefore, reflect any increased demand for white cell vitamin C in the immediate post-operative period, although the decrease in plasma vitamin C could imply some increased utilisation or redistribution of the non-cellular compartment.

The distribution of some cytoskeletal structures (microtubules, microfilaments, intermediate filaments) has been studied by indirect immunofluorescence microscopy and affinity purified antibodies in osteoclasts isolated from medullary bone of laying hens and in hen blood monocytes cultured in vitro. Both cell types show similar patterns of distribution of cytoskeletal structures and this further supports the concept that these cells are closely related. Osteoclasts and monocytes are also similar in their adhesion patterns, because they adhere to fibronectin-free areas and show closely comparable cell-to-substrate interactions when observed with interference reflection microscopy.

We describe a procedure for lipopolysaccharide (LPS) biotinylation using N-biotinyl-L-lysine and application of the biotinylated LPS (Bi-LPS) to localization of LPS binding sites and subcellular distribution. Biotinylation of LPS was confirmed by enzyme-linked immunosorbent assay (ELISA), gel immunodiffusion, and immunodot techniques. The biological and toxicological activity of the Bi-LPS was tested by Limulus amoebocyte lysate (LAL) assays and histopathological examinations, respectively. Results showed that biotin was conjugated to LPS without disrupting the biological/toxicological activity of the molecule, which indicates that the biotin is directly linked to the polysaccharide portion of LPS. Localization of binding sites and subcellular distribution of Bi-LPS in human platelets and monocytes were studied by electron microscopy using an avidin-biotin-horseradish peroxidase (HRP) or streptavidin-gold method. Platelet surfaces were intensely stained by the reaction product of horseradish peroxidase (HPR) 5 min after incubation, and Bi-LPS was localized in small vesicles and vacuoles of platelets and in the phagocytic vacuoles of monocytes 60 min post incubation. Bi-LPS provides a reliable, stable, and sensitive tool for determination of LPS binding sites and subcellular distribution.

We determined the activity of IL 1, obtained from various human monocyte subcellular compartments, when associated with liposomes. Soluble IL 1, bound to the outer surface of lyophilized liposomes, stimulated responsive target cells. However, this activity was not preserved when soluble IL 1 was incorporated into the inner chambers of classical liposomes. In contrast, monocyte plasma membranes that exhibited IL 1 activity had the same level of activity when presented on lyophilized liposomes and when incorporated inside the classical liposomes. However, monocyte plasma membranes bound to the outer surface of the liposomes exhibited greater activity than the monocyte membrane IL 1 itself in its soluble form. This suggests that membrane IL 1 is an integral membrane protein, readily integrated into the lipid bilayers. Like soluble IL 1, the expression of IL 1 activity present in the cytosol of activated monocytes was decreased by incorporation into liposomes, but was high and active when presented on lyophilized liposomes. The best artificial cell reconstitution was obtained with lyophilized liposomes in association with monocyte cytosol and plasma membranes. When an unactivated monocyte compartment was mixed with one from an activated monocyte, the signal was equal to that of the activated cell compartment alone. The IL 1 activity of activated cell fractions associated with lyophilized liposomes was determined, and an increase of IL 1 activity for both plasma membranes and cytosol was observed, whereas a decrease of the signal was obtained for the lysosomal compartment. Endoplasmic reticulum showed no IL 1 activity, even after trypsin treatment. The highest activity after trypsin treatment was recovered in the cytosol associated with lyophilized liposomes, suggesting that molecules obtained after this treatment were able to bind tightly to the lipid bilayers.

Mononuclear leucocytes (MNL) were isolated from blood of 11 healthy blood donors. Lymphocyte subsets were sorted in a Cytofluorograf after direct (B cells) or indirect immunofluorescence labeling with monoclonal antibodies directed against the phenotypes of T-, T helper (Th)- and T suppressor (Ts) cells. The sorted cells were incubated with (+/-)125iodocyanopindolol ([125I]CYP) for the determination of beta-adrenergic receptors. Beta-adrenergic receptors on B cells were increased two-fold (3700 sites/cell, p less than 0.004) and had a lower affinity (dissociation constant KD 40pM, p less than 0.03) when compared with T cells (1400 sites/cell, KD 17pM). Receptors on Th- and Ts cells showed a similar binding capacity, but Ts cells had a slightly higher [125I]CYP binding affinity (KD 13 pM) than Th cells (KD 27 pM, p less than 0.02). The different densities and affinities of beta-adrenergic receptors in human lymphocyte subsets, as assessed by antagonist binding should be considered in the interpretation of receptor alterations of unfractionated MNL, which may occur simultaneously to changes of the blood leucocyte distribution.

Repopulation and maturation of rat mesenteric and peritoneal mast cells were studied after mast cell depletion by intraperitoneal injection of distilled water. Immature mast cells were first identified in the mesentery and peritoneal fluid 5 and 6 days, respectively, after water injection. The most immature mast cells that could be identified contained a few orthochromatic granules. Upon maturation, the granules became metachromatic and increased in size and number. Heparin, revealed by toluidine blue staining and berberine sulfate fluorescence, appeared simultaneously with orthophthaldialdehyde (OPT)-induced histamine fluorescence. Paraformaldehyde-induced serotonin fluorescence appeared somewhat later. Repopulation of mesentery and peritoneal fluid by mast cells seemed to be independent of each other and to occur from undifferentiated precursor cells.

Measurement of intracellular magnesium (Mg2+) may have advantages over serum Mg2+ measurements in the assessment of Mg2+ homeostasis in patients. An accurate and reproducible method of measuring the Mg2+ content of mononuclear cells (lymphocytes and monocytes) from the venous blood of human subjects utilizing commonly available reagents and equipment is described. A well-defined clinical population of 88 subjects (48 males and 40 females) was used as the normal population. The mean mononuclear cell Mg2+ content was 67.8 +/- 13.8 (SD) fg/cell giving a 95% confidence interval normal range of 40-95 fg/cell which compared favorably to the empirical 95% limits of 44-94 fg/cell. The serum Mg2+ measured at the same time was 0.90 +/- 0.06 (SD) mmol/l. No significant correlation was found when comparing serum and mononuclear cell Mg2+ concentrations. Additionally, no significant differences were found when serum and intracellular Mg2+ were analyzed for sex and age with the exception that intracellular Mg2+ tended to be slightly higher in the younger age groups. The routine performance of this assay may require as little as 7 ml of venous blood and was done with a coefficient of variation of 3.0-3.6%.

Samples from 49 cases of myeloproliferative diseases were tested by an immunocytochemical technique for leucocyte lysozyme and lactoferrin. The presence of these constituents in myeloid precursors from cases of acute and chronic myeloid leukaemia reflected the degree of cellular maturation, lysozyme appearing (as it does in normal myeloid cells) at the stage of primary granule production (in promyelocytes), while lactoferrin wad detectable only in more mature, secondary granule-containing myeloid cells. Auer rods stained positively for lysozyme, in keeping with their relationship to primary granules. Monocytes from five cases of leukaemia showing predominantly monocytic differentiation were indistinguishable from normal monocytes in their staining reactions for lysozyme despite the presence of raised serum and urinary lysozyme levels. In four cases of acute myeloid leukaemia circulating polymorphs deficient in lactoferrin were detected: in one of these cases a similar percentage of polymorphs was lysozyme negative.

In these studies CR 1 polymorphism previously demonstrated on erythrocytes (E) was also found on CR 1-bearing peripheral blood leukocytes including polymorphonuclear (PMN), eosinophils, monocytes, and B lymphocytes. However several cell-specific differences in CR1 were found: (a) an approximately 5,000-dalton increase in CR 1 on PMN and eosinophils, (b) unequal band intensity among heterozygotes suggests that there is preferential expression of 220,000- or 225,000-dalton receptors on leukocytes compared to E, and (c) "minor" bands, approximately 15,000 daltons larger than the major receptor molecule, were found on E but not on leukocytes. These observations constitute a unique example of heterogeneity of an integral membrane receptor.

Frozen sections of kidney tissue from 12 patients with systemic lupus erythematosus were examined for the expression of oncogene proteins. Polyclonal rabbit antibody to c-myc peptide or to whole 65,000 D c-myc protein, was used to identify c-myc protein within tissue sections. Patterns of nuclear staining in the Hep-2 cell line were similar to those seen with monospecific human SLE sera showing anti-Sm reactivity. In addition c-myc staining was not abolished by prior incubation of tissue sections with human serum containing anti-Sm antibodies. Five of 12 kidney biopsy tissues from patients with systemic lupus erythematosus (SLE) showed positive speckled c-myc protein staining within nuclei of monocyte/macrophage cells of glomerular tufts. Positive staining was in all instances completely abolished by prior absorption of anti-c-myc antibody with c-myc protein. No c-myc protein was identified within SLE immune complex deposits.

We investigated the effects on immune function after progressive hypobaric hypoxia simulating an ascent to 25,000 ft (7620 m) over 4 weeks. Multiple simultaneous in vitro and in vivo immunologic variables were obtained from subjects at sea level, 7500 ft (2286 m), and 25,000 ft during a decompression chamber exposure. Phytohemagglutinin-stimulated thymidine uptake and protein synthesis in mononuclear cells were reduced at extreme altitudes. Mononuclear-cell subset analysis by flow cytometry disclosed an increase in monocytes without changes in B cells or T-cell subsets. Plasma IgM and IgA but not IgG levels were increased at altitudes, whereas pokeweed mitogen-stimulated in vitro IgG, IgA, and IgM secretion was unchanged. During exposure to 25,000 ft, in vitro phytohemagglutinin-stimulated interferon production and natural killer-cell cytotoxicity did not change statistically, but larger intersubject differences occurred. IgA and lysozyme levels (nasal wash) and serum antibodies to nuclear antigens were not influenced by altitude exposure. These results suggest that T-cell activation is blunted during exposure to severe hypoxemia, whereas B-cell function and mucosal immunity are not. Although the mechanism of altered in vitro immune responsiveness after exposure to various environmental stressors has not been elucidated in humans, hypoxia may induce alterations in immune regulation as suggested by in vitro immune assays of effector-cell function.

Several techniques were compared for collection of peritoneal mononuclear phagocytes from cynomolgus monkeys. A multiple-holed cannula proved superior to a hypodermic needle for the collection of peritoneal washings because it was not occluded by the omentum. With the cannula technique 55--70% of the harvest medium could be recovered from the peritoneal cavity with a yield of 2.5--5 x 10(5) macrophages per ml. A closed system for injecting and collecting harvest medium was better than a syringe in preventing bacterial contamination of peritoneal washings. After collection, cells were resuspended in growth medium to a concentration of 5 x 10(5) macrophages per ml, distributed into Leighton tubes, and incubated at 36 degrees C. After 12 hours, cultures were washed to remove non-adherent cells and refed with growth medium. This technique provided a sterile, reliable method for the collection and cultivation of macrophages from monkeys.

A 1,25-dihydroxyvitamin D3 receptor macromolecule was detected in peripheral mononuclear leukocytes from normal humans. This macromolecule was found to be present in monocytes but absent from normal resting peripheral B and T lymphocytes. However, it was present in established lines of malignant B, T, and non-B, non-T human lymphocytes, as well as in T and B lymphocytes obtained from normal humans and activated in vitro.

This paper describes information-handling aspects of the TYCHO I analysis system (Clin, Chem. 27: 1807--1820, 1981), which analyzes two-dimensional electrophoresis gels, matches the individual protein spots with those in a reference pattern, and stores various information--including spot measurements, identifications, treatment profiles, set memberships, and comments--in a computerized database. This and additional information such as amino acid composition and cellular localization is then accessible from an interactive program that includes a pictorial user interface and presents much of the data in graphical form. Use of the TYCHO I system is illustrated by examples drawn from analyses of gel patterns from human leukocytes.

Prussian blue has been widely used to localize iron in a variety of tissues at the light and electron microscopic level. In the present study, thin sections of human marrow and blood cells and rat duodenal cells were exposed to silver proteinate (SP) after staining en bloc with acid ferrocyanide (AF), with and without prior iron saturation using iron nitrilotriacetate (FeNTA). Silver deposition was observed over Prussian blue-reactive sites and significantly enhanced sites of minimal AF and FeNTA-AF staining. AF-SP stain deposits were present in the cytoplasmic matrix, granules, and occasionally on the surfaces of macrophages, monocytes, and erythroblasts. FeNTA-AF-SP stained additional cytoplasmic and surface sites in erythroblasts and stained neutrophil granules intensely. Duodenal epithelium from iron-loaded rats demonstrated strong AF-SP staining of ferric iron in microvilli, apical cytoplasmic matrix, and lateral membranes. Similar preparations from iron-replete rats stained sparsely; however, intense AF-SP staining was observed after iron saturation with FeNTA. SP similarly enhanced luminal ferrous iron deposits stained with acid ferricyanide in rats given intraluminal ferrous iron. AF-SP stain deposits were removed by exposure of thin sections to NH4OH, KCN, or HNO3 but were not affected by prior exposure to HIO4 or NaBH4, consistent with a silver cyanide or complex stain precipitate rather than reduced silver or silver ferriferrocyanide. SP enhancement of Prussian blue allows identification of reactive sites not readily visualized with AF or FeNTA-AF alone, and offers the potential for differentiating AF staining from other deposits or organelles of comparable density.

Density centrifugation and counterflow centrifugal elutriation were utilized to prepare enriched fractions of megakaryocytes from human bone marrow aspirates. This separation method enriched megakaryocytes in initial marrow aspirates by 116- to 463-fold. Approximately 63% of megakaryocytes were recovered from the initial samples, composing 18.7% of the nucleated cells in the final preparations. Mean megakaryocyte diameters of 51.6 micron and 33.8 micron were obtained from fixed and unfixed cellular specimens, respectively. Smaller platelet glycoprotein-positive mononuclear cells with a mean diameter of 20.5 micron were found in the highest concentrations in this final fraction. These cells presumably represent immature megakaryocytic forms. Counterflow centrifugal elutriation provides a means of isolating enriched populations of marrow megakaryocytes. This accessibility to viable populations of human megakaryocytes will allow additional investigation of the terminal events of megakaryocyte development.

We investigated the modifications that diet-induced hypercholesterolemia, in rabbit, can produce in the cell surface charge and chemistry of arterial endothelium (E) and blood monocytes (M). Weekly, up to 8 weeks, after blood samples were taken for lipid analysis and blood cell preparation, the vasculature was washed free of blood and the endothelial luminal surface (ES) exposed to cytochemical probes for detecting charged groups, sialoconjugates and oligosaccharides. After fixation in situ, specimens collected from lesion-prone regions (aortic arch and coronary artery) and vena cava, were processed for electron microscopy. Morphometric analysis of tracer distribution on endothelium of nonlesional and lesional areas occurring in various stages of structural alterations, showed a remarkable resistance of the cell coat to very high level of serum cholesterol. In nonlesional zones the E surface charge and glycoconjugates were not significantly changed. In lesional areas, including those with forming fatty streaks, while cationic sites, galactosyl-, and N-acetyl-galactosaminyl residues were not altered whereas mannosyl moieties increased in density. A reduction in anionic groups and sialoconjugates appeared only after advanced extracellular and intracellular accumulation of lipoprotein-derived material and stromal proliferation developed in the intima. Moreover, these ES changes were usually restricted to the relatively rare E cells heavily loaded with lipid inclusions. The modulations were generally paralleled by comparable variations in the M surface. Regardless the extent of surface charge reduction, monocytes continued to migrate and foam cells to egress from the vessel wall. The results suggest that the onset and progression of early intimal lesions are not preceded but followed by significant restricted alterations in cell surface charge and glycoconjugates of arterial endothelium and monocytes.

Ferritin levels in blood serum and mononuclear cells of the peripheral blood were determined in 60 patients with chronic lymphatic leukemia (group I), and 31 patients with other lymphomas of the low and medium degree of malignancy (group II). Significantly higher blood serum and mononuclear cells ferritin was found in the examined patients than in 54 healthy individuals. Particularly high ferritin levels were seen in blood serum of patients of group II in whom clinical stage of the disease was high. Moreover, moderate correlation of ferritin content in mononuclear cells and absolute leucocytosis was found in group I. This parameter correlated well with the percentage of poorly differentiated cells (group II).

A protein with interleukin 1 (IL-1) activity was obtained from the human acute monocytic leukemia cell line (THP-1) and purified by ultrafiltration, Ultrogel AcA54 chromatography, isoelectric focusing, and discontinuous polyacrylamide gel electrophoresis. Like the pI 6.8 species of IL-1 from human peripheral blood monocytes (PBM), the cell line IL-1 has a molecular weight (MW) of 14,000, a pI of 6.8, is heat labile, and does not bind to concanavalin A-Sepharose. Chemical modification of arginine residues by phenylglyoxal or sulfhydryl groups by N-ethyl maleimide and iodoacetamide completely destroys the activity of IL-1 from THP-1 cells as well as that of the pI 6.8 component from PBM. In contrast, the pI 5.1 component of PBM IL-1 is resistant to heat denaturation and sulfhydryl reagents, although it is totally inactivated by phenylglyoxal. IL-1 from THP-1 cells enhanced the proliferative response of the same subpopulations of PNA- thymocytes as IL-1 from PBM. These observations suggest that IL-1 derived from this cell line is similar to the IL-1 pI 6.8 species produced by human monocytes, and distinct from the pI 5.1 species.

The tumor-promoting ester 4 beta-phorbol 12-myristate 13-acetate (PMA) has been shown to induce the differentiation of the immature monocytelike cell line U-937 c in vitro into a heterogeneous population of cells, including small "dense" cells, large vacuolized or "foamy" cells, spindle-shaped cells, and cells with multiple filopodia ("stellate" cells). The effect of PMA was dose- and time-dependent, the optimal conditions being 40-162 nM PMA for 48 hours. The minimum time of exposure to PMA to ensure further differentiation of U-937 cells was about 5 hours. The PMA-stimulated cells acquired morphologic, ultrastructural, and functional characteristics typical of cells of the monocyte/macrophage lineage. The PMA-treated U-937 cells became adherent, ceased to proliferate, and exhibited increased expression of monocyte-specific antigens (Leu-M2, - M3, HLADr), surface receptors (FcR, C3bR), enzymes (nonspecific esterase, transglutaminase), and ability to mediate chemotaxis, phagocytosis, superoxide anion production, and antibody-dependent cytotoxicity reactions. The induced cells lost their morphologic differentiation and ability to attach to surfaces and regained proliferative capacity upon repeated subculture in PMA-free media.

We have used IL-1 alpha and IL-1 beta specific RNA standards in an RNA dot blot assay to rapidly and quantitatively assess the differential expression of the two human interleukin-1 mRNAs. This approach enabled us to minimize inaccuracies due to differences in specific activities of the probes, transcript size, and transfer and/or hybridization efficiencies of the two transcripts. We were thus able to accurately determine the steady state levels and molar ratio of the two species of IL-1 mRNA in human peripheral blood monocytes (PBM) and alveolar macrophages (AM). PBM, upon stimulation by lipopolysaccharide (LPS), expressed approximately ten-fold more IL-1 beta mRNA than IL-1 alpha. AM, however, expressed only two-fold more IL-1 beta then IL-1 alpha. PBM aged in vitro for 7 days prior to LPS stimulation, expressed at least 20-fold more IL-1 beta than IL-1 alpha RNA and at levels significantly lower than either PBM or AM.