Cytochemical observations in preleukemic states and remission of akute leukemia

In preleukemia and in remission of acute leukemia abnormal mononuclear cells ("string of bead" cells) have been observed and characterized with cytochemical methods. These cells may be resistant to cytostatic therapy and may be characterized as a source of a newly developing leukemic blast cells. Preleukemia and leukemic remissions seem to be identical states. Based on the morphological and cytochemical parallelity of cells in CFU-c-enriched fractions of the bone marrow and the so called string of bead cells we consider these cells to be closely related to the human commited stem cell.     

Prospective study on the influence of gastroduodenal ulcer hemorrhage on the diagnostic methods in Helicobacter pylori infection]

Peripheral giant cell granuloma consists of mononuclear cells and osteoclast-like giant cells. The proliferative ability of peripheral giant cell granuloma is restricted to the mononuclear cell compartment, whereas multinucleated giant cells lack mitotic activity. Although the proliferative compartment of peripheral giant cell granuloma has been investigated in detail, the expression and distribution of proteins regulating apoptosis is unknown. The present study demonstrates strong expression of bak and bax in the majority of giant cells. In contrast, giant cells show only weak positivity for bcl-2 and moderate positivity for bcl-x. Mononuclear cells were negative to weakly positive for bcl-x. Only scattered mononuclear cells were positive for bak, bax and bcl-2. The frequency of apoptotic nuclei detected by TUNEL-staining compared to regular nuclei was 18 times higher in giant cells than in mononuclear cells. In summary, our findings support the presumption that giant cells of bone and soft tissue tumors are reactive cell forms and not of neoplastic origin.     

Accidental persistent infection of cell lines by Newcastle disease virus, showing three unusual features--defective neuraminidase, temperature sensitivity and intranuclear inclusions

A persistent, defective infection by an unknown strain of Newcastle disease virus (NDV) appeared accidentally in established lines of pig, ox and sheep kidney cells. Virus particles released from the persistently infected cells were not infectious and were deficient in neuraminidase activity. Synthesis of some of the virus-specified proteins in the persistently infected cells was temperature-sensitive. Co-cultivation of mixed populations of carrier cells and healthy chick embryo cells induced cell fusion with the formation of multinucleate heterokaryons and intra-nuclear inclusions. The development of inclusions in the chicken nuclei was not accompanied by 'rescue' of infectious NDV.     

Morphology of bone remodeling

In this review, first the basic structure of bone and the histological and cytological characteristics of bone remodeling are reviewed. Then, topics on the ultrastructural and cytochemical characteristics of the biological mineralization, such as matrix vesicle and advanced collagen mineralization, are discussed. The recent advances in the understanding of the fine structural and cytochemical characteristics of bone cells in bone remodeling and their role in the regulation of bone metabolism, with special focus on the ultrastructural and cytochemical evidences of cell to cell, cell to metrix interaction of bone cells are also reviewed.     

Cytochemical demonstration of extraperoxisomal catalase. I. Sheep liver

In sheep hepatocytes catalase activity was demonstrated both within peroxisomes and within the cytosol. In the cytosol the catalase reaction product is contiguous to the plasma membrane and surrounds the nuclei, rough endoplasmic reticulum, cisternae, mitochondria and Golgi apparatus. This is the first cytochemical demonstration of guine extraperoxisomal catalase. No catalase reaction product was seen in the cytosol of nonparenchymal cells. To demonstrate catalase, both glutaraldehyde and formaldehyde fixation were used, followed by a diaminobenzidine technique modified from Novikoff and Goldfischer. Control reactions were performed to distinguish catalase reaction product from adsorption of oxidized diaminobenzidine and from precipitate due to oxidase-, peroxidase- or heat-stable peroxidatic activities. The results were evaluated in the light and electron microscopes.     

Identification of monocytes in suspensions of mononuclear cells

A new histochemical technique for morphological studies of mononuclear cells and granulocytes, based on fluorescent staining with methyl green, pyronine Y, and stilbene-isothiocyanato disulfonic acid (MPS stain) is described. The method was applied to mononuclear cells isolated from the blood of normal human subjects by Ficoll-Isopaque density centrifugation. Three cell populations were distinguished, mainly on the basis of differences in morphology and cytochemistry, utilizing the MPS stain. One of the cell types had many of the morphological characteristics of the monocyte. This technique, augmented by lysosomal content and endocytosis capacity studies, revealed contimination of the cell suspension with a larger percentage of monocytes than has usually been reported in the literature.     

Fetal cells in maternal blood: the use of primed in situ (PRINS) labelling technique for fetal cell detection and sex assessment

Prenatal diagnosis is presently performed following invasive procedures with variable risks of fetal loss; non-invasive procedures using fetal cells in maternal blood would be welcome for the early detection of fetal sex or aneuploidy. We describe a simple and rapid protocol to detect fetal cells and thus to assess fetal sex. In a first step, nucleated blood cells were separated into mononuclear and polynuclear cells using a double density gradient centrifugation. In a second step primed in situ (PRINS) labelling technique was performed to label Y-chromosomes. 15 samples were studied and correct gender assignment was made in 13/15. The number of labelled nuclei was higher in polynuclear cell phases than in mononuclear cell phases. Moreover, the polylobular aspect of labelled nuclei from polynuclear cell phases strongly suggested that they could belong to fetal polynuclear cells. The PRINS technique combines some advantages of FISH, such as visual assessment of in situ chromosome labelling and the powerful specificity and sensitivity of PCR. In association with a simple enrichment procedure it constitutes a rapid protocol for fetal cell detection, non-invasive early prenatal sex assessment, and could further be applied to detect the main viable aneuploidies.     

Bovine leukemia virus-induced lymphocytosis and increased cell survival mainly involve the CD11b+ B-lymphocyte subset in sheep

In this study, we show that bovine leukemia virus (BLV)-induced persistent lymphocytosis (PL) results from the in vivo expansion of the CD11b+ B-lymphocyte population. This subset shares phenotypic characteristics with murine and human B-1 cells. BLV interactions with the sheep B-1-like subset were explored. We found that B-1- and B-2-like cells are initially infected to similar extents. However, in long-term-infected sheep, the viral load is higher in B-1-like cells and only B-1- and not B-2-like cells show increased ex vivo survival compared to that in uninfected sheep. Ex vivo viral expression was found in both B-1- and B-2-like cells, indicating that both cell types support viral replication. Finally, cycloheximide and a protein kinase C inhibitor (H7) that blocks the ex vivo activation of viral expression did not affect the increased survival in B-1-like cells, suggesting that resistance to apoptosis is acquired in vivo. Collectively, these results indicate a peculiar susceptibility of sheep B-1-like cells to BLV transforming effects and further support the involvement of increased survival in BLV pathogenesis.     

Immunological reactivity of the lung. I. A guinea pig model for the study of pulmonary mononuclear cell subpopulations

The effects of various regimens of cyclophosphamide administration on guinea pig peripheral blood leukocytes were studied. Cyclophosphamide-induced immunosuppression was assessed by the effect of drug administration on the proportions and absolute numbers of leukocyte populations, and by the effect on functional capabilities of unfractionated and adherent cell-depleted mononuclear cell suspensions as measured by the PHA-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity assays against chicken erythrocyte targets. Intraperitoneal administration of five daily doses of cyclophosphamide (5 mg/kg) caused a modest absolute leukopenia but no change in cytotoxic effector function of the mononuclear cells remaining in the circulation. As the dosage of cyclophosphamide was increased to 20 mg/kg/day to produce a pronounced leukopenia, a profound neutropenia (less than 300 polymorphonuclear leukocytes/mm3) together with a marked decrease in mononuclear cell effector function was noted. A single i.p. injection of cyclophosphamide (100 mg/kg), which produced identical degrees of leukopenia of each leukocyte class as did daily administration of cyclophosphamide (20 mg/kg/day), caused no change in mononuclear cell effector function when compared to saline controls. Complement receptor-bearing and Fc-receptor bearing mononuclear cells were decreased to the same degree by both regimens of cyclophosphamide administration. Removal of adherent cells from mononuclear cell suspensions by column purification resulted in a marked decrease in cytotoxic effector function at low effector to target ratios. At higher effector to target ratios there was no difference in cytotoxic effector function between unfractionated and column-purified cells. In contrast, the functional defect in mononuclear cell suspensions from animals that received five daily doses of cyclophosphamide (20 mg/kg) could not be compensated for at higher effector to target ratios, indicating that this functional defect was not an artifact of relative depletion of monocytes by cyclophosphamide, but was due to an actual suppression of the effector functional capabilities of the killer cells. This study indicates that, dependent on the particular regimen of drug administration, the quantitative depletion of mononuclear cell populations by cyclophosphamide administration can be clearly distinguished from the qualitative effect on certain functional capabilities of surviving cells.     

Size distribution, electronic recognition, and counting of human blood monocytes

During a study on the separation of human blood monocytes from lymphocytes, a method was developed to recognize and count monocytes by electronic means. Lightscattering (Cytograf, Bio/Physics), and changes in electrical resistance (Channelyzer, Coulter) were used to size mononuclear leukocytes directly in cell suspensions. Both methods revealed a size distribution profile in which two populations of mononuclear leukocytes could be distinguished. The largest cells were virtually eliminated after phagocytosis of iron particles. We confirmed that these cells were monocytes by three different criteria: the intracellular lysozyme activity, the number of phagocytes, and the percentage of cells with kidney-shaped nuclei. The highly significant correlations we found showed that monocytes could be recognized and counted by electronic sizing. For this method, purified mononuclear leukocyte preparations had to be used, since the presence of erythrocytes, platelets, and polymorphonuclear cells interfered. Statistical analysis revealed that electronic sizing permitted discrimination of differences in monocyte content of 4.5%, with a probability of 95%. It was calculated that this sensitivity of electronic monocyte counting was about three times higher than the sensitivity of microscopic methods. Since 100,000 cells can be sized within a few seconds, not only the efficiency of the preparation but also minor changes in the size of monocytes and lymphocytes introduced during the isolation can be followed.     

Ultramicrochemical determination of nucleic acids in individual cells using the Zeiss UMSP-I microspectrophotometer. Application to isolated rat hepatocytes of different ploidy classes

Edstr?m's method for the ultramicrochemical determination of RNA and DNA in individual cells was modified for the measurement of extinction in u.v. light with the aid of the Zeiss scanning microspectrophotometer UMSP-I. With this new procedure, nucleic acids down to about 3 pg RNA or about 4 pg DNA can be measured with a very high accuracy. The method was applied to enzymatically isolated rat liver parenchymal cells. A mean DNA content of 6.52 pg was found for diploid cells. The DNA content of mononuclear cells of different ploidy levels and of binuclear cells showed a close proportionality with the nuclear ploidy and the number of nuclei per cell. The RNA content of mononuclear diploid cells amounted to 33.4 pg, yielding an RNA/DNA ratio of 5.12. The RNA/DNA ratio was similar for binuclear and mononuclear cells of the same ploidy level but decreased considerable with increasing nuclear ploidy.     

Growth inhibition of malignant hypernephroma cells by autologous lysophospholipid incubated macrophages obtained by a new method

In order to obtain pure human macrophages, mononuclear cells from peripheral blood were cultured on teflon membranes and the non-adherent lymphocytes removed. After 24 hours, all remaining adherent cells were detached from the membranes with 100% viability. They showed all the morphological and cytochemical characteristics of human monocytes. Within 10 days of cultivation they differentiated into monolayers of pure macrophages. Untreated macrophages of this origin showed only limited cytostatic effects on autologous hypernephroma cells in vitro. After preincubation with different alkyl-lysophospholipids they revealed a tumor growth inhibition capability of up to 90%.     

The Notch ligand, Jagged-1, influences the development of primitive hematopoietic precursor cells

We examined the expression of two members of the Notch family, Notch-1 and Notch-2, and one Notch ligand, Jagged-1, in hematopoietic cells. Both Notch-1 and Notch-2 were detected in murine marrow precursors (Lin-Sca-1+c-kit+). The Notch ligand, Jagged-1, was not detected in whole marrow or in precursors. However, Jagged-1 was seen in cultured primary murine fetal liver stroma, cultured primary murine bone marrow stroma, and in stromal cell lines. These results indicate a potential role for Notch-Notch ligand interactions in hematopoiesis. To further test this possibility, the effect of Jagged-1 on murine marrow precursor cells was assessed by coculturing sorted precursor cells (Lin-Sca-1+c-kit+) with a 3T3 cell layer that expressed human Jagged-1 or by incubating sorted precursors with beads coated with the purified extracellular domain of human Jagged-1 (Jagged-1(ext)). We found that Jagged-1, presented both on the cell surface and on beads, promoted a twofold to threefold increase in the formation of primitive precursor cell populations. These results suggest a potential use for Notch ligands in expanding precursor cell populations in vitro.     

Physical and cytochemical properties of neutrophils activated in situ in the lung during ZAP infusion in sheep

Increased retention of activated neutrophils in the lungs contributes to endothelial cell injury. However, characterization of the morphological changes that occur in neutrophils during activation in the pulmonary microcirculation has not been fully determined in vivo. Therefore, the present study was designed to determine structural and cytochemical properties of neutrophils in situ in pulmonary arterioles and alveolar capillaries during the infusion of zymosan-activated plasma (ZAP) or plasma (control) in anesthetized sheep. Quantitative morphological methods showed that ZAP infusion caused significant retention of neutrophils in alveolar capillaries [2.19 +/- 0.40 (SD) x 10(8) neutrophils/ml of capillary blood volume] and pulmonary arterioles (1.02 +/- 0.46 x 10(8) neutrophils/ml of arterial blood volume) compared with plasma infusion (1.03 +/- 0.15 and 0.30 +/- 0.10 x 10(8) neutrophils/ml, respectively; P < 0.05). Harmonic mean diameter of ZAP-activated neutrophils in situ (7.19 +/- 0.44 microns) was significantly greater than the diameter of neutrophils in plasma-treated sheep (6.29 +/- 0.17 microns; P < 0.05). Neutrophil cross-sectional area (54 +/- 3 microns2) and volume (248 +/- 27 microns3) in situ in alveolar capillaries were also significantly greater in ZAP-treated sheep than in control sheep (41 +/- 4 microns2 and 184 +/- 9 microns3, respectively; P < 0.05). Similarly, microvascular neutrophils in ZAP-treated sheep were vacuolated and elongated, filamentous actin was redistributed peripherally, and the cells were degranulated. We conclude that during ZAP infusion, neutrophils become enlarged and degranulated in pulmonary microvessels, especially in alveolar capillaries. The structural and cytochemical changes that occur are consistent with the hypothesis that neutrophil activation is accompanied by alterations in neutrophil physical properties, alterations that may facilitate retention and contribute to endothelial cell injury.     

Determination of distances from tyrosine D to QA and chlorophyllZ in photosystem II studied by ''2+1'' pulsed EPR

The immunomodulating effect of a new polysaccharide-peptide complex from culture mycelia of Lentinus edodes (LE) was studied for elucidation of the mechanism of augmentation of cell-mediated immunity. RNA samples were isolated from the untreated and treated murine splenocytes and human peripheral blood mononuclear cells. RT-PCR was used to analyze the cytokine gene expression and bioassay was used to analyze the cytokine production. By administration of LE, the expression levels of IL-2 and TNF-alpha genes were augmented in the treated murine spleen mononuclear cells and human peripheral blood mononuclear cells. The production of IL-2 were augmented in the treated murine spleen mononuclear cells, and the production of TNF-alpha were augmented in the treated murine peritoneal exudate macrophages. The production of IL-2 and TNF-alpha were augmented in the treated human peripheral blood mononuclear cells. These results suggest that LE may induce Th immune responses.     

G-CSF-mobilized CD34 peripheral blood stem cells are significantly less apoptotic than unstimulated peripheral blood CD34 cells: role of G-CSF as survival factor

Vi bacterial polysaccharide is a homopolymer of alpha 1-4 N-acetyl polygalacturonic acid with variable O-acetylation at position C-3 and forms a capsule around many bacteria. It has been referred to as the virulence factor of Salmonella typhi and is also a candidate vaccine against typhoid fever. The present study reports the interaction of this polysaccharide with murine mononuclear phagocytes and lymphocytes, and with human monocytes. Vi showed a dose-dependent binding to the murine monocyte cell lines WEHI-274.1 and J774. This binding was abrogated if the polysaccharide was deacetylated, suggesting involvement of acetyl groups in this interaction. Vi also bound to the murine B-cell lymphoma line A20, to peritoneal exudate cells and to a lesser degree to spleen cells and thymocytes from BALB/c mice. The polysaccharide also interacted with the human histiocytic lymphoma line U937 but not with the human monocyte cell line THP-1. Stimulation with Vi led to up-regulation of surface major histocompatibility complex (MHC) class II expression on A20 cells. Immunoprecipitation of Vi-bound molecules from cell surface biotinylated A20 and WEHI-274.1 revealed two bands with MW of about 32,000 and 36,000. The study demonstrates that Vi capsular polysaccharide can interact with mononuclear phagocytes and lymphocytes through specific cell surface molecules and modulate MHC class II expression.     

Screening for bacterial vaginosis and cervicitis aimed at preventing premature delivery

BACKGROUND/AIMS: We previously reported that the populations of lymphocytes and the expression of activated antigens in human sinusoidal mononuclear cells were different from those in peripheral blood mononuclear cells. Attempts to culture these cells for further study failed because they died rapidly under standard culture conditions in vitro after isolation from the liver. In this study, we evaluated the characteristics of cell death and the effects of various culture conditions on the viability of these cells. METHODS: Sinusoidal mononuclear cells were isolated from University of Wisconsin solution that had been perfused through the portal veins of normal healthy human livers harvested for transplantation into living related recipients. RESULTS: 70% of sinusoidal mononuclear cells cultured in vitro were nonviable within 48 h after isolation, while only 10% of peripheral blood mononuclear cells died under the same conditions. Sinusoidal mononuclear cells showed DNA ladder formation of DNA on electrophoresis and characteristic morphological pattern on electron microscopic examination that suggested they had died in an apoptotic manner. The addition of human liver extracts or 2-mercaptoethanol and reduced glutathione to the cultures rescued the sinusoidal mononuclear cells from apoptosis. Furthermore, diamide, a sulfhydryl group specific oxidant, negated the effect of the liver extract. CONCLUSION: In comparison with peripheral blood mononuclear cells, human sinusoidal mononuclear cells were more subject to death by apoptosis ex vivo, which was reversed by exogenous agents producing reducing conditions. These results suggested that hepatic sinusoidal mononuclear cells might express a different sensitivity to redox environment than peripheral blood mononuclear cells.     

Interaction of lactoferrin, monocytes, and T lymphocyte subsets in the regulation of steady-state granulopoiesis in vitro

Colony-stimulating activities (CSA) are potent granulopoietic stimulators in vitro. Using clonogenic assay techniques, we analyzed the degree to which mononuclear phagocytes and T lymphocytes cooperate in the positive (production/release of CSA) and feedback (inhibition of CSA production/release) regulation of granulopoiesis. We measured the effect of lactoferrin (a putative feedback regulator of CSA production) on CSA provision in three separate assay systems wherein granulocyte colony growth of marrow cells from 22 normal volunteers was stimulated by (a) endogenous CSA-producing cells in the marrow cells suspension, (b) autologous peripheral blood leukocytes in feeder layers, and (c) medium conditioned by peripheral blood leukocytes. The CSA-producing cell populations in each assay were varied by using cell separation techniques and exposure of isolated T lymphocytes to methylprednisolone or to monoclonal antibodies to surface antigens and complement. We noted that net CSA production increased more than twofold when a small number of unstimulated T lymphocytes were added to monocyte cultures. Lactoferrin's inhibitory effect was also T lymphocyte dependent. The T lymphocytes that interact with monocytes and lactoferrin to inhibit CSA production are similar to those that augment CSA production because their activities are neither genetically restricted not glucocorticoid sensitive, and both populations express HLA-DR (Ia-like) and T3 antigens but not T4 or T8 antigens. These findings are consistent with results of our studies on the mechanism of lactoferrin's inhibitory effect with indicate that mononuclear phagocytes produce both CSA and soluble factors that stimulate T lymphocytes to produce CSA, and that lactoferrin does not suppress monocyte CSA production, but does completely suppress production or release by monocytes of those factors that stimulate T lymphocytes to produce CSA. We conclude that mononuclear phagocytes and a subset of T lymphocytes exhibit important complex interactions in the regulation of granulopoiesis.