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Biopsy specimens were taken at gingivectomy from 18 adult patients undergoing treatment for chronic marginal periodontitis. They were embedded so that the cut surface of the gingiva was parallel to the top of the block to obtain a comprehensive view in a transversal plane of the inflammatory cell infiltrate near the bottom of the pocket. Sections were stained with HES or with toluidine blue for histological description, and acid alpha-naphthyl acetate esterase (ANAE) was used to differentially stain T lymphocytes, plasma cells and monocytes/macrophages. Sections stained with HES showed that the density and size of the cell infiltrates varied along the circumference of a tooth over very short distances and on various surfaces on neighbouring teeth. Differential counts of cells stained for ANAE demonstrated great variation in the composition of the cell infiltrates, particularly along the pocket epithelium. The predominating ANAE positive cell type in this area was T lymphocytes, while in the central connective tissue, plasma cells predominated. There was no systematic covariation between the localization of the gingiva (i.e. mesial, facial, etc.) and the composition of the cell infiltrates. The local variation in the composition of the cellular infiltrate most likely reflects local variability in the noxious substances (i.e. plaque composition) within the periodontal pocket, and in the resulting local inflammatory response.

We examined peripheral blood mononuclear cells (PBMC) from 16 patients with Sjögren's syndrome (SS) and 7 normal control subjects for the expression of the c-myc proto-oncogene. Patients with SS were found to have a significantly increased expression of c-myc messenger RNA compared with normal individuals. No abnormal forms of c-myc RNA were detected in the SS patients. DNA analysis did not show deletion, rearrangement, or amplification in the c-myc proto-oncogene. The methylation status of the c-myc gene in patients with SS was found to be comparable with that of the control subjects. Nuclear run-off assays showed increased transcription of the c-myc gene in some patients but normal transcription in others, suggesting that posttranscriptional mechanisms are also involved in the increased c-myc messenger RNA observed in these patients. Two patients with primary SS and B cell lymphomas were found to have normal c-myc expression in their PBMC. These results demonstrate the presence of activated PBMC in patients with primary SS and delineate some of the mechanisms that are involved at the molecular level. We speculate that increased c-myc expression may represent an early permissive event in the progression toward neoplasia in these patients.

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.

The present study was performed to analyse glucocorticoid receptor (GR) binding in peripheral blood mononuclear leukocytes (MNL) from 39 myasthenia gravis (MG) patients (unoperated patients (n = 13), thymectomized patients (n = 14) and patients receiving glucocorticoids: thymectomized (n = 11) and unoperated (n = 6]. A whole cell binding assay with 3(H) dexamethasone was used. GR mean values were significantly higher in the MNL of MG patients (thymectomized or not) not receiving glucocorticoid than in the MNL of healthy donors. Affinity was within the normal range. Sex, age or clinical forms of illness did not influence the results. In patients receiving prednisone (Pd) the GR values were significantly lower than in MG patients without Pd therapy, independent of Pd dose or time of administration. No differences in receptor binding between normal subjects and MG patients receiving Pd have been found.

Selected populations of human peripheral blood T lymphocytes, B lymphocytes, monocytes and polymorphonuclear cells were examined for their intracellular content of lactoferrin transferrin and ferritin by an indirect immunofluorescence technique. Lactoferrin was found in polymorphs but not in lymphoid cells. Two different lactoferrin staining patterns were observed which we designated 'perinuclear', characterized by a ring of positive material round the nucleus, and cytoplasmic, in which most positive material was distributed in the cytoplasm. The former staining pattern was found in high density polymorphs the latter was associated with low density polymorphs occasionally found contaminating the peripheral blood mononuclear cell suspension. After incubation in vitro, the perinuclear pattern changed to cytoplasmic staining. Transferrin was found in T cells and polymorphs. In contrast, only a few transferrin containing cells were detected in the B cell and monocyte fractions. Following overnight incubation, a halo of positive material was found surrounding T cells stained for transferrin, suggesting that T cells released transferrin during incubation. Ferritin was also found in T cells and adherent cells. Following latex particle ingestion, the intensity of ferritin staining was markedly increased. Only a small proportion of lymphoid cells and monocytes stained for transferrin or ferritin in the total peripheral blood mononuclear cell suspension. The results indicate, therefore, that in response to manipulation procedures used routinely for the selection of human peripheral blood lymphoid cells, detectable amounts of transferrin and ferritin are present in T but not B cells. The fact that T cells are equipped with proteins known to participate in binding and storage of iron may constitute, at least in part, the basis for the contribution of these cells to the regulation of other major biological systems.

Complex carbohydrate in granules of monocytes was compared with that in granules of neutrophils by ultrastructural cytochemical methods. The acid mucosubstance in granules of both cell types stained with dialysed iron after brief fixation with dilute glutaraldehyde, but that in monocyte granules differed in failing to stain after stronger fixation. Approximately 10% granules in normal blood monocytes stained with this method, whereas more than 90% of granules in leukaemic monocytes from two of seven patients with acute myelomonocytic leukaemia stained intensely. This difference presumably results from unmasking of acid groups in immature granules or increased synthesis of granule mucosubstance in some leukaemic monocytes. Granules of monocytes differed further from those of neutrophils in failing after either type of fixation to stain with a high iron diamine technique for for demonstration of sulphated mucosubstance. The absence of high iron diamine staining could reflect a lack of sulphate esters in monocyte granule mucosubstance, masking of the sulphate groups by other components, or extraction of the sulphated mucosubstance during specimen processing.

Spleen and light density peripheral blood leukocytes of 10 hairy cell leukemia (HCL) patients and total leukocytes of one patient and blood donor cells were stained quantitatively for cellular DNA. The DNA content of single cells was measured by flow cytometry (FC) and compared to the DNA content of sheep cells admixed as an internal control. Eight of eleven patients (72 per cent) showed deviations from blood donor DNA content. Two female patients showed increased cellular DNA content, the six male patients had hypodiploid cells. Chromosomal aberrations are therefore likely to exist in the majority of HCL patients. Separation of HCL cells into sheep erythrocyte rosette and non rosette forming cells revealed similar DNA abnormalities in both cell populations, suggesting that the leukemia encompasses cells with B and with T markers.

Monocytes, lymphocytes and polymorphs were separated from the peripheral blood of normal human subjects. Ferritin concentrations were determined with antibodies to both human spleen and heart ferritins. The heart type ferritin concentration in monocytes was 38.4 +/- 21.6 fg/cell (mean +/- SD), in lymphocytes 8.6 +/- 6.6 fg/cell and in polymorphs 3.2 +/- 2.4 fg/cell. Spleen type ferritin concentrations (fg/cell) were 15.6 +/- 7.0 in monocytes, 6.6 +/- 5.7 in lymphocytes and 7.0 +/- 4.6 in polymorphs. The mean heart/spleen ferritin ratios were 2.8/1 for monocytes, 2.0/1 for lymphocytes and 0.6/1 for polymorphs. The cell extracts were also subjected to anion exchange chromatography. Heart type ferritin eluted at a higher chloride ion concentration than spleen type ferritin. Both H and L subunits were synthesized by mononuclear cells when incubated with 3H-leucine. Human leucocytes contain a wide range of isoferritins and ferritin concentrations may be considerably underestimated in conventional assays for serum ferritin which employ antibodies to liver or spleen ferritin.

Previous studies have shown that human platelets, megakaryocytes and peripheral blood monocytes contain the subunit a of plasma factor XIII (FXIII) which plays a key role in fibrin stabilization. To study the expression of FXIII subunit a during differentiation of the mono- and megakaryocytic cell lines, bone marrow smears were examined by immunomorphological methods. In addition to megakaryocytes, FXIII was detected in a large number of cells by a highly sensitive immunoperoxidase staining. Characterization of these cells was carried out by double immunofluorescent labelling in which the detection of FXIII subunit a was combined by the labelling of either Leu M3 monocyte/macrophage surface antigen or platelet factor 4 (PF4) a marker for megakaryocytic cells. On the course of differentiation from early precursors to mature megakaryocytes all phenotypic form of the megakaryocytic cell line expressed FXIII subunit a though there were considerable changes in its subcellular distribution pattern. Leu M3 positive cells of monocytopoiesis, i.e. monocytes and promonocytes and in all probability monoblasts as well, were also labelled for FXIII. On this basis FXIII subunit a could be considered as a common marker of megakaryo- and monocytopoiesis and its immunomorphological detection might provide a useful diagnostic tool for identifying normal and perhaps also malignant differentiation forms of these cell lines.

A sensitive thymocyte co-stimulator assay of IL-1 using a beta-D-galactoside specific lectin (allo A) obtained from the beetle (Allomyrina dichotoma) is reported here. Allo A stimulated [3H]thymidine uptake of mouse thymocytes in the presence of IL-1. The allo A assay was more sensitive than the PHA or PNA- thymocyte assay, especially at low doses of IL-1. Optimal conditions for the allo A assay were as follows: allo A, 2.5-5.0 micrograms/ml; whole thymocytes, 0.5-1.0 x 10(6) cells/well; incubation time, 72-96 hr. The assay is sensitive and convenient and can easily be performed in any laboratory.

We studied three patients with cyclic thrombocytopenia which occurred in phase with the menstrual cycle. The platelet count in each patient reached a nadir of 5-20 x 10(9)/l at the onset of menses. Thrombocytopenia was associated with bruising, epistaxis and menorrhagia and was followed 5-14 d later by normal or elevated platelet counts (up to 900 x 10(9)/l). Repeat bone marrow examinations performed at the time of reduced platelet counts showed megakaryocytic hyperplasia. 111In-platelet-disappearance from the circulation was measured in one patient; T50, time to half activity, was shortened to 0.7 d during the period of thrombocytopenia and was prolonged to 3.2 d when the platelet count increased (normal platelet T50 is about 4.8 d). In two of three patients platelet-associated anti-glycoprotein Ib autoantibodies were present and remained elevated despite normalization of the platelet count. In both of two patients the decrease in platelet count at the onset of menses was associated with an increase in the expression of monocyte Fc gamma receptors. Based on the reported capacity of oestrogenic hormones to modulate macrophage Fc gamma receptor expression, we propose that hormonal changes during the menstrual cycle may alter the Fc gamma receptor-mediated clearance of antibody-coated platelets by macrophages, modulate platelet survival, and cause cyclic thrombocytopenia.

Supernatants of mononuclear cells (MNC-SN) were shown to increase synthesis of glycosaminoglycan (GAG) by cultured normal dermal fibroblasts. Fibroblasts from the skin of patients with progressive systemic sclerosis (PSS, scleroderma) were hyporesponsive. We exposed fibroblasts outgrowing from explants of normal adult skin to MNC-SN for up to 30 generations in culture. MNC-SN were obtained by incubating normal MNC with concanavalin A. Four experimental, 4 normal control, and 3 PSS control lines were passaged by trypsinizing and splitting the cultures 1:2 every 7 days. At the third and fifth passages, portions of the experimental fibroblasts were removed from MNC-SN, then passaged in medium alone. Cell counts, assays for GAG, and electron microscopy were performed and increases in GAG after brief reexposure to MNC-SN were determined at the third, fifth, and eighth passages. In normal dermal fibroblasts, baseline GAG production, measured by 3H-glucosamine uptake, was low and increased as much as 15 times after reexposure to MNC-SN. In contrast, production was high in both experimental and PSS lines, and increases after reexposure to MNC-SN were consistently small. This PSS-like behavior persisted in experimental fibroblasts removed from MNC-SN at the third and fifth passages. Growth of experimental and scleroderma fibroblasts was slower than that of control fibroblasts. Ultrastructurally, both scleroderma and experimental dermal fibroblasts differed from normal fibroblasts by their oval cellular shape, indentations in nuclear membrane, numerous organelles and bundles of microfilaments, prominent Golgi, and intranuclear inclusions. These experiments indicate that normal adult dermal fibroblasts subjected to MNC-SN in vitro acquire a scleroderma-like phenotype that persists for many generations.