An automated method of differential red blood cell classification with application to the diagnosis of anemia.

A method of automated red cell analysis suitable for the rapid classification of large numbers of red cells from individual blood specimens has been developed, and preliminarily tested on normal bloods and clinically proven cases of anemias and red cell disorders. According to this method digital image processing techniques provide several features relating to shape and internal central pallor configurations of red cells. These features are used with a fully automated decision logic to rapidly provide a quantitative "red cell differential" analysis, a report of the percentage subpopulations of recognized categories of red cells. For each subpopulation, measurements of mean cell area, mean cell hemoglobin content and mean cell hemoglobin density are provided. The nine types of red cell disorders studied with this method were: (a) iron deficiency anemia, (b) the anemia of chronic disease, (c) beta-thalassemia trait, (d) sickle cell anemia, (e) hemoglobin C disease, (f) intravascular hemolysis, (g) hereditary elliptocytosis, (h) hereditary spherocytosis, and (i) megaloblastic anemia due to folic acid deficiency. Preliminary indications are that the red cell differential is useful in distinguishing between these conditions.     

Studies on the properties and tissue distribution of the isozymes of guanylate kinase in man.

The isozyme patterns of guanylate kinase were examined in fetal and adult tissues, in cultured cells and also in red cells separated by density gradient fractionation. Results from fetal and cultured cells inidcated that there are three primary isozymes a, c, and e among the seven isozymes of guanylate kinase in man. Serial secondary isozyme production in red cells in vivo showed that isozyme a produces b, c produces d, and e produces f and g. The three sets of isozymes were found to differ in the following properties: activation/inhibition by EDTA; thermostability, and molecular weights. Isoelectric points of several of the isozymes were estimated by isoelectric focusing. It was concluded that the isozymes of guanylate kinase are determined by three separate gene loci.     

Flow conditions of red cells and plasma in microvascular bifurcations

The flow conditions of red cells and plasma in microvascular ramifications were investigated in a biological model of the frog's retrolingual membrane. Upon the controllable reduction of blood flow from the arterioles into the microvascular bed, with an appropriate decrease of red cell: plasma ratio in the blood, a tendency of the red cells to be drawn along the parent main capillaries without entering the branching capillaries was in evidence. These latter thus transformed into the plasmatic capillaries deprived of red cells. The factors being responsible for this process were found to be as follows: (a) the diameter of branching capillaries, (b) the angles of off-shoots, (c) the degree of slow-down of blood flow velocity in the branches, and (d) the reduction of red cell: plasma ratio in parent vessels. The direct relationship was found between these factors and the transformations of the off-shoots into the plasmatic capillaries.     

Knockdown of phosphotyrosyl phosphatase activator induces apoptosis via mitochondrial pathway and the attenuation by simultaneous tau hyperphosphorylation

Phosphotyrosyl phosphatase activator (PTPA) is decreased in the brains of Alzheimer's disease (AD) and the AD transgenic mouse models. Here, we investigated whether down‐regulation of PTPA affects cell viability and the underlying mechanisms. We found that PTPA was located in the integral membrane of mitochondria, and knockdown of PTPA induced cell apoptosis in HEK293 and N2a cell lines. PTPA knockdown decreased mitochondrial membrane potential and induced Bax translocation into the mitochondria with a simultaneous release of Cyt C, activation of caspase‐3, cleavage of poly (DNA ribose) polymerase (PARP), and decrease in Bcl‐xl and Bcl‐2 protein levels. Over‐expression of Protein phosphatase 2A (PP2A) catalytic subunit (PP2A_C) did not rescue the apoptosis induced by PTPA knockdown, and PTPA knockdown did not affect the level of and their phosphorylation of mitogen‐activated protein kinases (MAPKs), indicating that PP2A and MAPKs were not involved in the apoptosis induced by PTPA knockdown. In the cells with over‐expression of tau, PTPA knockdown induced PP2A inhibition and tau hyperphosphorylation but did not cause significant cell death. These data suggest that PTPA deficit causes apoptotic cell death through mitochondrial pathway and simultaneous tau hyperphosphorylation attenuates the PTPA‐induced cell death.

     

The characterization of human uterine smooth muscle cells in culture

Primary cultures initiated from normal human uterine endometrium after total enzymatic dissociation contained epithelioid cells and smooth muscle cells. The smooth muscle cells were subsequently isolated by differential trypsinization and grown in culture for 36 +/- 4 generations. Ultrastructural examination of log and post-confluent cultures of cells at low and high population doubling levels revealed characteristics similar to those of published reports on other smooth muscle cells studied in vivo and in vitro. Among the common features present were: (a) abundant bundles of 60--70 A myofilaments; (b) branched mitochondria; (c) stacks of cisternae of rough endoplasmic reticulum; (d) caveolae intracellulares; (e) nexuses. Other features included ovoid nuclei, a well developed Golgi apparatus and abundant free ribosomes. The subcultured cells exhibited features of dedifferentiation in the log phase of growth and at post-confluency. However, the post-confluent cells showed characteristics indicating redifferentiation back towards their in vivo morphology. Smooth muscle cells isolated from endometrial curettings may provide a useful model for biochemical and pharmacological studies of a cell type derived from a hormonal target tissue as the cells "age" in culture.     

Assessing the efficacy of coherent anti‐Stokes Raman scattering microscopy for the detection of infiltrating glioblastoma in fresh brain samples

Coherent anti‐Stokes Raman scattering (CARS) microscopy is an emerging technique for identification of brain tumors. However, tumor identification by CARS microscopy on bulk samples and in vivo has been so far verified retrospectively on histological sections, which only provide a gross reference for the interpretation of CARS images without matching at cellular level. Therefore, fluorescent labels were exploited for direct assessment of the interpretation of CARS images of solid and infiltrative tumors. Glioblastoma cells expressing green fluorescent protein (GFP) were used for induction of tumors in mice (n = 7). The neoplastic nature of cells imaged by CARS microscopy was unequivocally verified by addressing two‐photon fluorescence of GFP on fresh brain slices and in vivo. In fresh unfixed biopsies of human glioblastoma (n = 10), the fluorescence of 5‐aminolevulinic acid‐induced protoporphyrin IX was used for identification of tumorous tissue. Distinctive morphological features of glioblastoma cells, i.e. larger nuclei, evident nuclear membrane and nucleolus, were identified in the CARS images of both mouse and human brain tumors. This approach demonstrates that the chemical contrast provided by CARS allows the localization of infiltrating tumor cells in fresh tissue and that the cell morphology in CARS images is useful for tumor recognition.

Experimental glioblastoma expressing green fluorescent protein.     

T细胞亚群CD4测定方法的研究

为比较外周血T淋巴细胞亚群CD4不同测定方法的差别,以流式细胞术为定量手段,测定了猴外周血中三种不同方法处理后CD4的表达.结果表明：先标后溶法——先用异硫氰荧光素标记的单克隆抗体（FITC-CD4 McAb）标记后,再加入红细胞溶解液溶掉红细胞的处理方法,结果基本等同于传统的淋巴细胞分离法,但样本用量仅为传统方法的1/5,且操作简单.激光共焦显微术的形态学研究也证实：先标后溶法与淋巴细胞分离法相似,其细胞膜表面荧光标记清晰,优于先溶后标法.     

Label‐free and non‐invasive monitoring of porcine trophoblast derived cells: differentiation in serum and serum‐free media

Traditional approaches to characterize stem cell differentiation are time‐consuming, lengthy and invasive. Here, Raman microspectroscopy (RM) and atomic force microscopy (AFM) – both considered as non‐invasive techniques – are applied to detect the biochemical and biophysical properties of trophoblast derived stem‐like cells incubated up to 10 days under conditions designed to induce differentiation. Significant biochemical and biophysical differences between control cells and differentiated cells were observed. Quantitative real time PCR was also applied to analyze gene expression. The relationship between cell differentiation and associated cellular biochemical and biomechanical changes were discussed.

Monitoring trophoblast cells differentiation     

Pyrogallol inhibits the growth of human pulmonary adenocarcinoma A549 cells by arresting cell cycle and triggering apoptosis

Pyrogallol (PG) is a polyphenol compound and has been known to be an O generator. We evaluated the effects of PG on the growth of human pulmonary A549 cells in relation to the cell cycle and apoptosis. Treatment with 50 or 100 μM PG significantly inhibited the cell growth of A549 for 72 h. DNA flow cytometric analysis indicated that PG slightly induced a G1 phase arrest of the cell cycle at 24 or 48 h, but did not induce the specific cell cycle arrest at 72 h. Intracellular GSH depletion was observed in PG‐treated cells. PG induced apoptosis in A549 cells, as evidenced by sub‐G1 cells, annexin V staining cells, and the loss of mitochondrial membrane potential (Δ Ψ_m). The intracellular ROS (reactive oxygen species) level including O increased in PG‐treated A549 cells at 24 and 48 h, and persisted at 72 h. The changes in GSH as well as ROS levels by PG affected the cell viability in A549 cells. In conclusion, PG inhibited the growth of human pulmonary A549 cells by inducing cell cycle arrest as well as triggering apoptosis. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:36–42, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20263     

Unidirectional block between isolated rabbit ventricular cells coupled by a variable resistance.

We have used pairs of electrically coupled cardiac cells to investigate the dependence of successful conduction of an action potential on three components of the conduction process: (a) the amount of depolarization required to be produced in the nonstimulated cell (the "sink" for current flow) to initiate an action potential in the nonstimulated cell, (b) the intercellular resistance as the path for intercellular current flow, and (c) the ability of the stimulated cell to maintain a high membrane potential to serve as the "source" of current during the conduction process. We present data from eight pairs of simultaneously recorded rabbit ventricular cells, with the two cells of each pair physically separated from each other. We used an electronic circuit to pass currents into and out of each cell such that these currents produced the effects of any desired level of intercellular resistance. The cells of equal size (as assessed by their current threshold and their input resistance for small depolarizations) show bidirectional failure of conduction at very high values of intercellular resistance which then converts to successful bidirectional conduction at lower values of intercellular resistance. For cell pairs with asymmetrical cell sizes, there is a large range of values of intercellular resistance over which unidirectional block occurs with conduction successful from the larger cell to the smaller cell but with conduction block from the smaller cell to the larger cell. We then further show that one important component which limits the conduction process is the large early repolarization which occurs in the stimulated cell during the process of conduction, a process that we term "source loading."     

Cathodal proteins from primitive (embryonic) red cells of amphibia. Isolation and characterization.

A group of abundant (15% of the soluble protein) nonhemoglobin proteins was isolated from the primitive (embryonic) red cells found in tadpoles, using the cationic properties of the proteins at pH 8.6 to separate them from hemoglobin and other red cell proteins. The cathodal proteins (CP) were resolved into five components, and the two most predominant proteins were separated and characterized. Purified CP-1b and CP-2 had an amino acid composition similar to that of unfractionated cathodal proteins and to each other, except for small variations in the lysine and half-cystine content. The molecular weight of the purified CP-1b and CP-2 was 13 to 14,000, determined by gel filtration chromatography and electrophoresis in the presence of sodium dodecyl sulfate. Cathodal proteins were immunologically related although there were quantitative differences in reactivity. The concentration of cathodal proteins in primitive (embryonic) red cells was 100 times that in definitive (adult) red cells coincided with the replacement of primitive red cells. The synthesis of the cathodal proteins appeared to continue throughout the life of the primitive red cells; when hemoglobin synthesis declined in primitive red cells, approximately half of the protein synthesized by the cells was cathodal protein. Although the function of the cathodal proteins is as yet unknown, the data suggest that the cathodal proteins are a unique characteristic of erythroid differentiation in early development.     

Prevention of cerebral malaria by adoptive transfer of malaria-specific cultured T cells into mice infected with Plasmodium berghei

Murine T cell populations specific for Plasmodium berghei parasites were generated in vitro from BALB/c immune lymph node cells. The malaria-specific T lymphocytes were shown: a) to proliferate specifically in vitro in response to stimulation with P. berghei-infected red blood cells; b) to exhibit the Thy-1+, Lyt-1+2- cell surface phenotype; c) to provide specific helper activity for an in vitro anti-hapten (TNP) plaque-forming cell antibody response; and d) to protect P. berghei-infected mice from early mortality due to cerebral malaria.     

In vivo immune response suppression by the supernatant from concanavalin A-activated spleen cells.

Supernatants from concanavalin A- (Con A) activated murine spleen cells have been shown to suppress the in vitro plaque-forming cell (PFC) response to sheep red blood cells (SRBC). The present study examined the effect of such Con A-activated spleen cell supernatants (herein termed CONS) on the in vivo immune response to SRBC in C57BL/6, BALB/c and CDF1 mice. CONS derived from BALB/c spleen cells suppressed direct PFC 4 and 8 days after immunization with 2 X 10(8) SRBC. CONS also suppressed indirect PFC 8 days after immunization, as well as serum hemagglutinins to SRBC. The PFC response of C57BL/6 (H-2b) mice was suppressed as much as that of BALB/c (H-2d) by CONS derived from BALB/c mice, indicating a lack of H-2 specificity of the CONS. In addition to suppression of the antibody response to SRBC, in vivo CONS administration resulted in reduction in spleen cell number. This reduction was not sufficient to explain the decreased PFC response. When the CONS was separated into less than 10,000 m.w. and greater than or equal to 10,000 m.w. fractions, the immunosuppressive activity was found in the less than 10,000 m.w. fraction. This observation suggests that intact interferon, SIRS, and MIF were not responsible for the results obtained.     

Folate status of the population in the Canadian Health Measures Survey

Background

Low folate concentrations are inversely associated with birth defects, including neural tube defects, congenital heart disease and oral clefts. Conversely, high folate concentrations may be associated with adverse outcomes, including increased risk of colorectal cancer among those with pre-existing neoplasms. The purpose of our study was to investigate the folate status of a nationally representative sample of Canadians, including a subset of women of childbearing age.

Methods

We examined red blood cell folate concentrations among members of the general population aged 6–79 years (n = 5248) and separately among women of childbearing age (15–45 yr, n = 1162), as recorded by the Canadian Health Measures Survey and measured by immunologic assay. We assessed the data for significant differences by age, sex and socioeconomic status.

Results

Less than 1% of Canadians showed folate deficiency (red blood cell folate < 305 nmol/L) and 40% showed high folate concentrations (> 1360 nmol/L). Among women of childbearing age, 22% showed concentrations below those considered optimal for maximal neural tube defect-risk reduction (< 906 nmol/L). Significant differences by age and socio-economic status, but not sex, were evident in median red blood cell folate concentrations, although concentrations in all groups exceeded recommended levels. No differences by age or income were found among women of child-bearing age.

Interpretation

Folate deficiency is virtually nonexistent in the Canadian population, although high folate concentrations are evident. Additional research is needed to better understand the determinants of red blood cell folate among women of childbearing age who have concentrations below levels that are maximally protective against neural tube defects. Ongoing monitoring of the folate status of Canadians and the relationship between red blood cell folate and health outcomes is warranted.Folic acid represents both a public health success and a controversial debate over associated health risks. Fortification with folic acid of Canadian white wheat flour (150 μg/100 g) and other selected grains in 1998 has been linked to a 46% reduction in the prevalence of neural tube defects.¹ Declines in rates of neural tube defects have also been documented in the United States and Chile after fortification of grains with folic acid.^2,3 To further reduce the risk of folate-dependent neural tube defects, women of childbearing age are encouraged to eat folate-rich foods and take a multivitamin supplement containing folic acid (0.4 mg/d).⁴ Higher-dose supplements (4–5 mg/d) are recommended for women at increased risk of giving birth to a baby with a neural tube defect (e.g., those who regularly use folic acid antagonist medications or have a family history of neural tube defects).⁵ Although biochemical assessment of the folate status of select subgroups of Canadians has been done, it has not been studied on a nationally representative sample in over 30 years.^6–9 The recent Canadian Health Measures Survey provides data to fill this gap.⁷ The purpose of our study was to describe the current folate status of Canadians, including a subgroup of Canadian women of childbearing age, and to assess whether folate concentrations vary by age, sex and socio-economic status.     

Photon emission from chemically perturbed yeast cells

Photon emission (PE) from yeast cells Saccharomyces cerevisiae strain SP-4 in normal conditions and in conditions perturbed by the addition of formaldehyde was investigated using single-photon counting equipment. PE from yeast cells, growing in a standard nutrient medium (YPG) then centrifuged and resuspended in a phosphate buffer (pH = 6.5), was measured in the presence of oxygen or argon. The solution of formaldehyde (2%) was injected into the sample. The intensity of PE increased and reached a maximum, then slowly decreased to a level which was higher than the PE level without the perturbing factor. The kinetics of PE was found to be strongly dependent upon the presence of oxygen. The model of formation and recombination of free radicals was tested. The results indicate that PE can arise during the recombination reactions of free radicals like R^? + R^?, RO^? + RO^?, RO^?₂ + RO which are formed in the enzymatic oxidative reactions.     

Subgroups in Group III of RNA Phages

In our previous studies, RNA phage strains were separated into 3 major groups on the basis of filtration efficiency through Millipore filters. In the present study, the strains of group III were shown to be further divided into 3 subgroups: (a) Qβ, NH, SG; (b) VK, SO; (c) ST. This subgrouping was found to be compatible with the serological grouping and pronase sensitivity with the exception of strain NM. Strain NM was classified in subgroup (a) by the Millipore filtration and in subgroup (b) by the other two methods.     

Primary in vitro immunogenicity of liposomal model membranes in mouse spleen cell cultures.

Neither 2,4-dinitrophenyl-6-N-aminocaproylphosphatidylethanolamine (DNP-Cap-PE) nor fluoresceinthiocarbamylphosphatidylethanolamine (F1-PE) induces hapten-specific plaque-forming cells (PFC) when incubated with suspensions of spleen cells from unimmunized C57BL/6J mice. However, PFC are produced after incorporation of these synthetic lipid antigens into liposomal model membranes. The in vitro response is characterized by the following: a) it is time and dose dependent; b) the frequency of IgM PFC exceeds IgG PFC; c) both nonadherent and adherent cells are required (2-mercaptoethanol can replace the requirement for adherent cells in some experiments); d) depletion of thymus-derived cells by treatment with anti-theta antiserum plus complement does not diminish the response; e) spleen cells from nude BALB/c mice also produce PFC. Thus, the essential features of the in vivo immunogenicity of DNP-Cap-PE and F1-PE sensitized liposomes, which have been previously described, can be replicated in an in vitro cell culture system.     

Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased 45Ca2+ uptake into the cell monolayer, and (f) increased 86Rb+ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca2+ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca2+-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca2+ gating.     

Optical and photoacoustic radiofrequency spectroscopic analysis for detecting red blood cell death

Under stress, red blood cells (RBCs) undergo programmed cell death (eryptosis). One of the signaling molecules for eryptosis, sphingomyelinase (SMase), plays an important role in monitoring the efficacy of vascular targeted cancer therapy. The high optical absorption of erythrocytes coupled with the changes of eryptotic RBCs makes RBCs ideal targets for the photoacoustic (PA) detection and characterization of vascular treatments. In this work, experiments characterizing eryptosis were performed: PA detection of high frequencies (>100 MHz) that enabled analysis at the single‐cell level and of low frequencies (21 MHz) that enabled analysis at the RBC ensemble level. Ultrasound spectral analysis was performed on control and SMase‐treated RBCs. Spectral unmixing was applied to quantify methemoglobin production as a by‐product of RBC death. Validation was performed using a blood gas analyzer and optical spectrometry. Our results indicate that PA radiofrequency spectra could be used to differentiate the biochemically induced morphological changes as RBCs lose their native biconcave shape, and release hemoglobin into the surroundings. Spectral unmixing revealed a 7% increase in methemoglobin content for SMase‐treated samples due to the oxidative stress on the RBCs. These findings suggest that PA spectral analysis of RBC death can potentially serve as a biomarker of the efficacy of vascular targeted cancer therapies.