Rapid detection of malaria and other bloodstream parasites by fluorescence microscopy with 4'6 diamidino-2-phenylindole (DAPI).

DAPI is a fluorescent dye which appears to complex specifically with DNA. We have used this probe to detect and identify malarial infections by fluorescence microscopy. Experiments were conducted using Plasmodium berghei yoeli--infected mouse blood, P. lophurae--infected duck blood, and P. vivax--infected human blood. Infected avian blood was used to detect parasites within nucleated erythrocytes. Control blood smears from uninfected hosts revealed fluorescence only in the leukocytes of mammalian blood or in nuclei of leukocytes and erythrocytes of avian blood. Cytoplasmic staining of red blood cells was absent in all controls. In contrast, the cytoplasm of infected red blood cells was stippled with fluorescence centers. Ring forms, trophozoites, segmenters, and merozoites frequently were observed. This simple procedure can be applied directly to routine clinical analysis, as well as experimental procedures, DAPI can also be used to stain other parasites, including nuclei in microfilariae.     

Synthesis and assembly of membrane skeletal proteins in mammalian red cell precursors

The synthesis of membrane skeletal proteins in avian nucleated red cells has been the subject of extensive investigation, whereas little is known about skeletal protein synthesis in bone marrow erythroblasts and peripheral blood reticulocytes in mammals. To address this question, we have isolated nucleated red cell precursors and reticulocytes from spleens and from the peripheral blood, respectively, of rats with phenylhydrazine-induced hemolytic anemia and pulse-labeled them with [35S]methionine. Pulse-labeling of nucleated red cell precursors shows that the newly synthesized alpha- and beta-spectrins are present in the cytosol, with a severalfold excess of alpha-spectrin over beta-spectrin. However, in the membrane-skeletal fraction, newly synthesized alpha- and beta-spectrins are assembled in stoichiometric amounts, suggesting that the association of alpha-spectrin with the membrane skeleton may be rate-limited by the amount of beta-spectrin synthesized, as has been shown recently in avian erythroid cells (Blikstad, I., W. J. Nelson, R. T. Moon, and E. Lazarides, 1983. Cell, 32:1081-1091). Pulse-chase experiments in the rat nucleated red cell precursors show that the newly synthesized alpha- and beta-spectrin of the cytosol turn over coordinately and extremely rapidly. In contrast, in the membrane-skeletal fraction, the newly synthesized polypeptides of spectrin are stable. In contrast to nucleated erythroid cells, in reticulocytes the synthesis of alpha- and beta-spectrins is markedly diminished compared with the synthesis and assembly of proteins comigrating with bands 2.1 and 4.1 on SDS gels. Thus, in nucleated red cell precursors, the newly synthesized spectrin may be attached to the plasma membrane before proteins 2.1 and 4.1 are completely synthesized and incorporated in the membrane.     

The effects of repeated bleedings on bone marrow and blood morphology in adult laboratory rats

There were slight and rapidly disappearing changes in the bone marrow and blood morphology of laboratory rats after repeated bleedings on consecutive days, except marked increases in the counts of circulating reticulocytes. However, the significantly elevated reticulocyte count was not accompanied by the similar increase in the count of marrow erythroid nucleated cells. Marked increments could be found in the counts of splenic erythroid nucleated cells and, therefore, it is possible for rat circulating reticulocytes to originate following repeated bleedings from the spleen. The validity of investigations of bone marrow and blood morphology for detecting haemorrhage is discussed with a view to preclinical safety drug evaluations performed on adult laboratory rats.     

Nectary structure and nectar secretion in Maxillaria coccinea (Jacq.) L.O. Williams ex Hodge (Orchidaceae)

BACKGROUND AND AIMS: It had previously been assumed that Maxillaria spp. produce no nectar. However, nectar has recently been observed in Maxillaria coccinea (Jacq.) L.O. Williams ex Hodge amongst other species. Furthermore, it is speculated that M. coccinea may be pollinated by hummingbirds. The aim of this paper is to investigate these claims further. METHODS: Light microscopy, histochemistry, scanning and transmission electron microscopy. KEY RESULTS: This is the first detailed account of nectar secretion in Maxillaria Ruiz & Pav. A 'faucet and sink' arrangement occurs in M. coccinea. Here, the nectary is represented by a small protuberance upon the ventral surface of the column and nectar collects in a semi-saccate reservoir formed by the fusion of the labellum and the base of the column-foot. The nectary comprises a single-layered epidermis and three or four layers of small subepidermal cells. Beneath these occur several layers of larger parenchyma cells. Epidermal cells lack ectodesmata and have a thin, permeable, reticulate cuticle with associated swellings that coincide with the middle lamella between adjoining epidermal cells. Nectar is thought to pass both along the apoplast and symplast and eventually through the stretched and distended cuticle. The secretory cells are collenchymatous, nucleated and have numerous pits with plasmodesmata, mitochondria, rough ER and plastids with many plastoglobuli but few lamellae. Subsecretory cells have fewer plastids than secretory cells. Nectary cells also contain large intravacuolar protein bodies. The floral morphology of M. coccinea is considered in relation to ornithophily and its nectary compared with a similar protuberance found in the entomophilous species M. parviflora (Poepp. & Endl.) Garay. CONCLUSIONS: Flowers of M. coccinea produce copious amounts of nectar and, despite the absence of field data, their morphology and the exact configuration of their parts argue strongly in favour of ornithophily.     

Smad3基因剔除对小鼠造血功能的影响

研究Smad3基因剔除对小鼠造血功能的影响。实验小鼠分为 5组 ,每组有Smad3基因剔除小鼠(Smad3 - - )和其同窝孪生的野生型小鼠 (Smad3 + + )各 1只。小鼠的造血功能用 14天形成的脾结节 (CFU S1 4 )、多系祖细胞 (CFU GEMM)、粒 单系祖细胞 (CFU GM)、红系祖细胞 (BFU E)测定及外周血象、骨髓象等实验血液学指标来确定。每组小鼠取尾血作白细胞、红细胞和血小板计数 ,涂片作白细胞分类计数。将一侧股骨的骨髓冲出 ,制成单细胞悬液 ,计数其中有核细胞数 ,测定CFU GM、BFU E、CFU GEMM值。将每只小鼠的 4× 10 4个骨髓有核细胞 ,经尾静脉注入 3只 8～ 10周经致死量射线照射的同系雌性小鼠体内 ,测定 14天的CFU S。取一部分胸骨、肝脏、脾脏固定做病理切片 ,其余胸骨冲出骨髓 ,涂片作分类计数。结果Smad3 - - 小鼠外周血白细胞和血小板计数明显高于Smad3 + + 小鼠 ,红细胞数无显著差异。外周血白细胞分类结果也表明粒细胞显著增高。骨髓有核细胞数无显著差异 ,CFU GM显著增高 ,BFU E无显著差异 ,CFU GEMM明显减少 ,CFU S显著减少。病理形态学观察发现骨髓增生极度活跃 ,以粒系为主 ,肝脾无显著差别。骨髓涂片分类表明粒系增多 ,粒系 :红系比例增高。因此得出结论Smad3基因剔除使小鼠造血干祖细胞数目     

Chicken thrombocytes in culture: lymphocyte-conditioned medium delays apoptosis.

Chicken thrombocytes are nucleated cells, analogs to mammalian platelets. These cells are involved in hemostasis, phagocytosis and secretion of specific products. Most of the properties of avian thrombocytes have been established in experiments that employed recently isolated blood cells. Attempts to cultivate these cells for a long period of time under optimal culture conditions for peripheral blood cells were unsuccessful; thrombocytes died after 24 h of cultivation unlike macrophages cocultured with them. Here we investigate the reasons and type of thrombocyte death in culture. Thrombocytes were separated from peripheral blood of roosters and cultured for 48 h. The influence of different culture conditions on thrombocyte viability was studied. Cells were cultured as adherent cell monolayers or under agitation (preventing adherence), in the presence or lack of lymphocytes or their soluble factors, and various concentrations of fetal bovine serum. After 24 h in standard culture thrombocytes displayed cytoplasm and chromatin condensation, DNA cleaved into oligonucleosomal fragments and unaltered mitochondria. These results strongly suggest that thrombocytes suffer an apoptotic cell death in culture. Apoptosis could be delayed by culturing thrombocytes in the presence of lymphocytes or their soluble factors.     

QSAR modeling and transmission electron microscopy stereology of altered mitochondrial ultrastructure of white blood cells in patients diagnosed as schizophrenic and treated with antipsychotic drugs.

Treatment of patients diagnosed as schizophrenic with antipsychotic drugs (neuroleptics) is known to cause occasional unexplained depletion of white blood cells, especially neutrophil granulocytes. It has been known for many years that neuroleptics can interfere with the mitochondrial respiratory chain in vitro. Because there has been a growing interest recently in mitochondrial targeting of drugs, and since a quantitative structure-activity relationship (QSAR) model that predicts mitochondrial accumulation of neuroleptics has been published, we investigated the effects of neuroleptics on white blood cell mitochondria. Venous blood samples were collected from both patients undergoing treatment with neuroleptics and healthy volunteers. The samples were processed for transmission electron microscopy. The resulting images of white blood cells were analyzed using stereology to compare quantitatively mitochondrial morphology in the patient and control groups. We found that in patients, but not in controls, there was swelling of mitochondria and fragmentation of the mitochondrial cristae. There also were fewer mitochondria in patients than in controls, although due to the swelling of the organelles, the volume density of mitochondria in the two groups was not significantly different. Such changes are typical of a toxic insult. Consequently, it seems plausible that, since schizophrenia is not a disease considered to affect white blood cells per se, these changes probably are due to the medication.     

QSAR modeling and transmission electron microscopy stereology of altered mitochondrial ultrastructure of white blood cells in patients diagnosed as schizophrenic and treated with antipsychotic drugs.

Treatment of patients diagnosed as schizophrenic with antipsychotic drugs (neuroleptics) is known to cause occasional unexplained depletion of white blood cells, especially neutrophil granulocytes. It has been known for many years that neuroleptics can interfere with the mitochondrial respiratory chain in vitro. Because there has been a growing interest recently in mitochondrial targeting of drugs, and since a quantitative structure-activity relationship (QSAR) model that predicts mitochondrial accumulation of neuroleptics has been published, we investigated the effects of neuroleptics on white blood cell mitochondria. Venous blood samples were collected from both patients undergoing treatment with neuroleptics and healthy volunteers. The samples were processed for transmission electron microscopy. The resulting images of white blood cells were analyzed using stereology to compare quantitatively mitochondrial morphology in the patient and control groups. We found that in patients, but not in controls, there was swelling of mitochondria and fragmentation of the mitochondrial cristae. There also were fewer mitochondria in patients than in controls, although due to the swelling of the organelles, the volume density of mitochondria in the two groups was not significantly different. Such changes are typical of a toxic insult. Consequently, it seems plausible that, since schizophrenia is not a disease considered to affect white blood cells per se, these changes probably are due to the medication.     

The spleen ofmustelus schmitti (chondrichthyes, triakidae): A light and electron microscopic study

The spleen ofMustelus schmitti is described, being an elongated organ running dorsally along the stomach and surrounded by a thin capsule without muscular tissue. The classical division between red and white pulps was evident, with a marginal sinus surrounding the latter. Numerous ellipsoids were located in the red pulp, inside nodular-like structures, or in the marginal sinus. Two types of reticular cells were apparent as well as macrophages and melanomacrophages. Hemopoiesis was present through immature and mature cells of the erythroid and thrombocytoid lineages, but no evidence of granulopoiesis was found. Comparison amongMustelus species and between chondrichthyan and mammalian spleens are made.     

Enucleation of mammalian cells by cytochalasin B. II. Formation of hybrid cells and heterokaryons by fusion of anucleate and nucleated cells

The development of methods for the formation of hybrid cells and heterokaryons by virus-induced fusion of chemically-enucleated cells and nucleated cells has been described. Heterokaryons and hybrid cells formed by fusion of anucleate mouse peritoneal macrophages (MPM) and nucleated mouse L and human HEp-2 cells were identified by mixed haemadsorption, by their sensitivity to trypsin and by their capacity to ingest antibody-coated sheep red blood cells. The expression of macrophage markers in these cells declined rapidly after fusion. Hybrid cell and heterokaryon formation was identified in mixed cultures of anucleate L cells and nucleated MPM, and was accompanied by the reactivation of DNA synthesis in the macrophage nuclei. Other hybrids and heterokaryons were formed by virus-induced fusion of anucleate MPM and nucleated chick embryo erythrocytes and anucleate L cells and nucleated HEp-2 cells. The value of anucleate-nucleate cell hybrids in the study of metabolic and genetic regulation in mammalian cells is discussed.     

Expression of an onco-developmental antegen among avian species.

Rabbit antisera directed against an onco-developmental antigen on chicken red blood cells have been serologically dissected through specific adsorptions. It is now possible to detect 13 antigenic determinants with the fractionated antisera. The onco-developmental antigen referred to as chicken fetal-leukemic antigen (CFA) is fetal-specific in the white Leghorn chicken, being present on the embryonic but not adult peripheral red blood cells of non-being present on the embryonic but not adult peripheral red blood cells of non-leukemic birds. However, one or more of the onco-developmental antigenic determinants have been detected on adult peripheral red blood cells of non-Gallus avian species, as well as on red blood cells from two adult chicken varieties. For phylogenetic purposes, red blood cells from avian species were characterized for their combinations of CFA determinants. Comparisons among species revealed specific patterns of antigenic expression within phylogenetic groups. Several CFA determinants were restricted in their occurrence to species within a single family, and one determinant was found in all cases where CFA was expressed. The distribution of CFA determinants was used to determine immunological distances among four Galliform species. These distances agreed with the immunological relationships established using different serological markers.     

Morphology and Longevity of Cells Cultured from Primate, Carnivore and Rodent Tissues

Most workers agree that cells derived from avian and from human sources, and propagated rapidly in monolayer culture, usually come to be dominated by diploid cells of fibroblastic morphology, and are usually also limited in their ability to divide in vitro. The experiments reported here, as well as those reported in some other laboratories, suggest that this phenomenon may occur in primates generally. It does not seem to apply, however, to the cells of some other forms, such as some amphibian groups, the rat, and the ferret: cells from these sources can sometimes be propagated in euploid form for much longer than expected on the basis of experience with primate and avian cells. In addition, such long-term euploid lines are sometimes epithelioid in morphology. This is a feature of special interest in the context of much observation which is consistent with the proposal that fibroblastic (i.e. stroma) cells could indeed be limited in in vivo division potential, and with the observation that epithelial (i.e. most organ parenchymal) cells in many cases cannot be so limited, or must have limits on division potential at very high levels in vivo. Much recent work in other laboratories suggests, however, that many differences between these groups may be explained or circumvented with appropriate medium composition.     

Morphological Changes Induced By Extremely Low‐Frequency Electric Fields

In this paper, morphological effects of electric fields on avian erythrocytes (nucleated red blood cells) have been studied in detail. Morphological changes include rounding and cytoplasm transparency. It has been shown that the effect is non‐thermal. Careful imaging and image analyses have been carried out to show that the degree of this effect is frequency‐dependent, and has a higher conversion rate at higher temperatures. Furthermore, to better understand the mechanisms behind the morphological changes, we investigated the dedifferentiation hypothesis and performed a series of tests on avian erythrocytes including fluorescence spectroscopy for hemoglobin, and tests on human umbilical cord blood, mesenchymal stem cells, and bone marrow mesenchymal stem cells including flow‐cytometry analysis for expression of certain markers and calcium staining. Bioelectromagnetics. 2019;40:375–390. © 2019 Bioelectromagnetics Society.     

Multidimensional flow cytometric blood cell differentiation without erythrocyte lysis.

Forward light scattering, orthogonal light scattering, and the fluorescence intensities of unlysed peripheral blood cells, labeled with CD45-phycoerythrin and the nucleic acid dyes LDS-751 and thiazole orange, were measured simultaneously, utilizing a flow cytometer. Erythrocytes, reticulocytes, platelets, neutrophils, eosinophils, basophils, monocytes, lymphocytes, nucleated erythrocytes, and immature nucleated cells occupied unique positions in the five-dimensional space created by the listmode storage of the five independent parameters. A software program was developed which identified and enumerated each of these cell populations. Platelets in this study were identified by LDS-751 staining, in addition to their forward and orthogonal light-scattering characteristics. Validation of this approach was obtained by demonstrating that all CD41- or CD42-expressing platelets also stained with LDS-751. Furthermore, the staining by LDS-751 did not change following platelet activation with ADP. The quantification of erythrocytes, platelets, neutrophils, eosinophils, monocytes, and lymphocytes correlated well with data obtained with a commercial hematology whole blood analyzer (H-1). Reproducibility of the identification of these populations was shown by repeated measurement of the same sample and by staining and analysis of multiple aliquots of identical blood samples. Stability studies demonstrated that 8 hours after blood collection, the number of damaged cells increased. This could be measured by a greater thiazole orange uptake by the damaged cells. This investigation demonstrates the feasibility of multidimensional flow cytometric blood cell differentiation for an automated whole blood cell analysis without the necessity of erythrocyte lysis. The ability to simultaneously identify reticulocytes, nucleated erythrocytes, and immature nucleated cells in one measurement is unique and promises to be a powerful tool for the assessment of abnormal blood samples.     

Brief report: a new profile of terminal <Emphasis Type="Italic">N</Emphasis>-acetyllactosamines glycans on pig red blood cells and different expression of α-galactose on Sika deer red blood cells and nucleated cells

It has been reported that: (1) large variations were found in the number of sialic acid (SA) capped with N-acetyllactosamines (SA-Galβ1-4GlcNAc-R) and α-Gal epitopes (Galα1-3Galβ1-4GlcNAc-R) or uncapped N-acetyllactosamines (Galβ1-4GlcNAc-R) on different mammalian red blood cells, and on nucleated cells originating from a given tissue in various species; (2) goat, sheep, horse and mouse red blood cells lack α-Gal epitopes, despite the expression of this epitope on a variety of nucleated cells in these species, including lymphocytes differentiated from the same hematopoietic origin. In this study, flow cytometry and Western blot analyses of pig red blood cells showed that α-Gal epitopes on pig red cells developed concomitantly after treatment with neuraminidase, suggesting that the terminal N-acetyllactosaminide glycans were capped with SA-α-Gal epitopes. Whereas, the expression of the α-Gal epitopes on red blood cells from Sika deer (Cevus nippon hortulorum) were found to be absent even though the epitopes were present on their white blood cells. Thus, these results add new data not only for the terminal carbohydrate structures on cell surface glycans of various mammalian cells, but also for wide variety of epitope expression on the cells from different tissues, which might be useful for understanding their unique states resulting from differentiation and evolution.     

A new method for high speed, sensitive detection of minimal residual disease

Investigations of rare cell types in peripheral blood samples, such as tumor, fetal, and endothelial cells, represent an emerging field with several potentially valuable medical applications. Peripheral blood is a particularly attractive body fluid for the detection of rare cells as its collection is minimally invasive and can be repeated throughout the course of the disease. Because the number of rare cells in mononuclear cells can be very low (1 in 10 million), a large number of cells must be quickly screened, which places demanding requirements on the screening technology. While enrichment technology has shown promise in managing metastatic disease, enrichment can cause distortions of cell morphology that limit pathological identification, and the enrichment targeting adds additional constraints that can affect sensitivity. Here, we describe a new approach for detecting rare leukemia cells that does not require prior enrichment. We have developed an immunocytochemical assay for identification of leukemia cells spiked in peripheral blood samples, and a high-speed scanning instrument with high numerical aperture and wide field of view to efficiently locate these cells in large sample sizes. A multiplex immunoassay with four biomarkers was used to uniquely identify the rare cells from leukocytes and labeling artifacts. The cytometer preserves the cell morphology and accurately locates labeled rare cells for subsequent high resolution imaging. The sensitivity and specificity of the approach show promise for detection of a low number of leukemia cells in blood (1 in 10 million nucleated cells). The method enables rapid location of rare circulating cells (25 M cells/min), no specific enrichment step, and excellent imaging of cellular morphology with multiple immunofluorescent markers. The cell imaging is comparable to other imaging approaches such as laser scan cytometry and image flow cytometry, but the cell analysis rate is many orders of magnitude faster making this approach practical for detection of rare cells.     

Separation of human and animal cells by steric field-flow fractionation

In this work, the feasibility of separating and characterizing cell populations by steric field-flow fractionation (steric FFF) is demonstrated by application to fixed human and avian red cells, fresh blood from several species, and viable HeLa cells. The basis for this work is established by means of a discussion of the role of steric FFF in the broad family of field-flow fractionation techniques. The behavior of steric FFF is then characterized by application to standard polystyrene latex beads and to fixed red blood cells. Studies of these standards and of the other cells noted under various conditions of field strength and flow velocity are used to improve the separation conditions and approach optimization. It is shown that the fixed human and avian red cells can be separated in a time of less than 15 min. In addition, it is shown that HeLa cells maintain their viability after passage through the separation channel.     

Partial oxidative-stress perturbs membrane permeability and fluidity of fish nucleated red blood cells

We investigated the influence of partial oxidative stress on permeability and fluidity of nucleated fish red blood cells for simulating nucleated somatic cells. Peroxide value indicating lipid hydroperoxide level in nucleated red blood cells of common carp (Cyprinus carpio) increased with increasing body size. We detected that oxidation of nucleated red blood cells led to the degraded PUFA compositions and accelerated the permeability of calcein and ATP in the nucleated red blood cells restrictedly oxidized with 1 mM AAPH treatment for 30 min at 21 degrees C in the dark. Using fluorescence probes, PC3P, we found that oxidative stress reduced the membrane fluidity of nucleated red blood cells. It was also observed that AAPH had no significant influence on the osmotic fragility and electrophoretic profiles of red blood cell proteins. These results suggest that partial oxidative-stress, even if failure to fragment the membrane, may affect membrane permeability of fish nucleated red blood cells for an important energy molecule, ATP.     

Separation of human and animal cells by steric field-flow fractionation

In this work, the feasibility of separating and characterizing cell populations by steric field-flow fractionation (steric FFF) is demonstrated by application to fixed human and avian red cells, fresh blood from several species, and viable HeLa cells. The basis for this work is established by means of a discussion of the role of steric FFF in the broad family of field-flow fractionation techniques. The behavior of steric FFF is then characterized by application to standard polystyrene latex beads and to fixed red blood cells. Studies of these standards and of the other cells noted under various conditions of field strength and flow velocity are used to improve the separation conditions and approach optimization. It is shown that the fixed human and avian red cells can be separated in a time of less than 15 min. In addition, it is shown that HeLa cells maintain their viability after passage through the separation channel.