The distribution of chlorine e6 and its derivatives in HeLa cells studied using luminescence microscopy

The influence of the chemical structure of porphyrin pigments on their accumulation and localization in HeLa cells has been examined by the scanning fluorescence microphotometry. It has been found that the replacement of carboxyl groups of chlorine e6 for methyl and amino groups has no influence on the pigment distributions in cells. All the pigments are bound by cell membrane structures. The chemical modification of chlorine e6 structure is essential for the ability of pigment to be accumulated by cells that can be used to increase the efficiency of cancer phototherapy. The charge and hydrophobic properties of pigment molecules are of great importance for accumulating porphyrin sensitizers by cells.     

Distribution of porphyrin sensitizers among protein and cellular blood elements

The distribution of porphyrin pigments between plasma proteins and blood cells was studied. It was shown that the relative fraction of sensitizer bound by blood cells changed significantly depending on the physicochemical features of pigment molecules. This parameter strongly correlates with porphyrin polarity. Polar watersoluble tetraphenylporphin derivatives, chlorine e6 and hematoporphyrin are bound by plasma proteins only. The decrease in pigment polarity by substitution of polar side groups results in a drastic increase of pigment affinity to blood cells. The binding of extremely apolar pigments by cells in blood occurs for a long period of time, probably as a result of a low rate of pigment redistribution between serum proteins and cellular membrane. The data obtained show that blood cells may be involved into the control of pigment transport and distribution in organism during photodynamic therapy. The parameters of porphyrin distribution between plasma proteins and cells in blood are of certain importance when the pharmacokinetic behavior of various sensitizers is compared.     

Interaction of the photosensitizer 13,15-<Emphasis Type="Italic">N</Emphasis>-(3′-hydroxypropyl)cycloimide chlorin p<Subscript>6</Subscript> with normal and cancerous blood cells

The interaction of 13,15-N-(3′-hydroxypropyl)cycloimide chlorin p₆ (CIC) with normal blood cells and human myeloid leukemia K562 and HL60 cells was studied. CIC was found to be bound by the erythrocyte membrane but did not penetrate into the cytoplasm. It is characterized by a diffuse distribution in the cytoplasm of normal leukocytes, whereas its diffuse distribution in K562 and HL60 cells is accompanied by perinuclear accumulation and binding to the plasma membrane. The average cytoplasmic concentration corresponding to the CIC accumulation in leukemic cells at saturation is 2.2 to 2.6 times higher than that in normal leukocytes. CIC is more intensely accumulated in granulocytes than in lymphocytes. The kinetics of the cellular uptake and efflux was characterized. The normal leukocytes and erythrocytes were found to be 1.5 times and 3 to 4 times less sensitive, respectively, to the photodynamic action of CIC than the K562 and HL60 cells.     

Photosensitization by chlorine and porphyrin causing structural damage of erythrocytes

Kinetics of hemolysis of human erythrocytes photosensitized by porphyrin and chlorin derivatives was investigated by small angle light scattering method. The compounds used were arranged in the following order of photosensitizing activity decrease: ethylendiamide of chlorine e6, chlorine e6 dimethylester chlorine e6 haematoporphyrin diacetate tetracarboxyphenylporphyn. The substances having a greater number of binding sites with liposomes (chlorine e6 dimethylester) or with albumin (ethylendiamide of chlorine e6) showed the greatest photohemolytic activity. The photohemolytic activity of porphyrins was suggested to depend on the number of the pigment molecules bound by erythrocytes membranes.     

Rubidium uptake in single cells

Summary Rubidium uptake was measured in single erythroid and myeloid cells of rabbit by means of X-ray microanalysis. It was found in the nucleated bone marrow cells that after incubation in rubidium the sums of potassium and rubidium concentrations were similar to the original potassium concentrations, indicating that there was one-to-one replacement of potassium by rubidium. Although the nuclear potassium and rubidium concentrations were higher than those in the cytoplasm, the nuclear and cytoplasmic ratios of K/Rb were similar. This implies that the potassium in both compartments exchanged freely with rubidium. In the erythroid line of cells there was a continuous reduction of potassium transport activity during the maturation process as indicated by the decrease in rubidium uptake rates. The uptake was measured in seven groups of cell types that could be distinguished on the basis of morphology and chemical composition. The order of the groups from high to low rubidium uptake were: esosinophilic myelocyte > early erythroblast and thinrimmed erythroblast > late erythroblast > early bone marrow red cell > late bone marrow red cell > peripheral blood red cell. Thus, there is a continuous decrease in rubidium transport as the erythroid cells mature.     

Damage photosensitized by chlorine e6 in bacteria with various defects of the DNA repair system

The goal of the work was to study the sensitivity of isogenic Escherichia coli cells differing in their ability to mediate DNA repair steps to the action of visible light sensitized by chlorine e6. Cells incapable of excision repair as well as those deficient in post-replicative recombination DNA repair were found to be much more sensitive to the combined action of visible light and chlorine e6 as compared to cells whose genes responsible for DNA repair were not damaged. The results indicate that visible light damages bacterial DNA in the presence of chlorine e6.     

Validation of SYTO 9/Propidium Iodide Uptake for Rapid Detection of Viable but Noncultivable <Emphasis Type="Italic">Legionella pneumophila</Emphasis>

Legionella pneumophila is an ubiquitous environmental microorganism that can cause Legionnaires’ disease or Pontiac fever. As a waterborne pathogen, it has been found to be resistant to chlorine disinfection and survive in drinking water systems, leading to potential outbreaks of waterborne disease. In this work, the effect of different concentrations of free chlorine was studied (0.2, 0.7, and 1.2 mg l^?1), the cultivability of cells assessed by standard culture techniques (buffered charcoal yeast extract agar plates) and viability using the SYTO 9/propidium iodide fluorochrome uptake assay (LIVE/DEAD® BacLight?). Results demonstrate that L. pneumophila loses cultivability after exposure for 30 min to 0.7 mg l^?1 of free chlorine and in 10 min when the concentration is increased to 1.2 mg l^?1. However, the viability of the cells was only slightly affected even after 30 min exposure to the highest concentration of chlorine; good correlation was obtained between the rapid SYTO 9/propidium iodide fluorochrome uptake assay and a longer cocultivation with Acanthamoeba polyphaga assay, confirming that these cells could still recover their cultivability. These results raise new concerns about the assessment of drinking water disinfection efficiency and indicate the necessity of further developing new validated rapid methods, such as the SYTO 9/propidium iodide uptake assay, to assess viable but noncultivable L. pneumophila cells in the environment.     

The effects of the adrenoreceptor agonists phenylephrine and isoproterenol on the intracellular ion concentrations of branchial epithelial cells of brown trout (Salmo trutta L.)

Brown trout were fitted with indwelling, intraperitoneal catheters and injected with 4–6 mol · kg^-1 of the -receptor agonist phenylephrine or the -receptor agonist isoproterenol. The intracellular concentrations of sodium, chlorine, potassium and phosphorus in the pavement epithelial cells and the mitochondria-rich cells of the branchial epithelium were measured by X-ray microanalysis 1 h after the injection of the adrenoreceptor agonists. Injection with phenylephrine resulted in a significant increase in intracellular chlorine and potassium in mitochondria-rich cells and a significant but relatively smaller increase in chlorine in pavement epithelial cells. Injection with isoproterenol resulted in a significant increase in sodium and chlorine concentration in pavement epithelial cells and a significant decrease in potassium concentration. The only significant effect of isoproterenol injection on mitochondria-rich cells was a decrease in intracellular chlorine concentration. The results suggest that these adrenoreceptor agonists have a direct effect on the influx of Na⁺ and Cl^- across the branchial epithelium. These effects may be a mechanism for acid-base regulation during the severe stress conditions that elicit catecholamine release in vivo. These results corroborate previous studies using X-ray microanalysis which suggested that pavement epithelial cells are the sites of Na⁺ uptake in freshwater fish whilst Cl^- uptake occurs via mitochondria-rich cells.Abbreviations LTSEM low-temperature scanning electron microscope - MR cells mitochondria-rich cells - PE cells pavement epithelial cells - XRMA X-ray microanalysis     

The role of tetrapyrrol photosensitizers in photoinduced variation of free radical characteristics of rat blood in endotoxic shock

The main goal of the present study was to evaluate the comparative effectiveness of tetrapyrrol photosensitizers (protoporphyrine IX and chlorine e6) in red (632.8 nm) and green (532.5) spectrum bands on rat blood free radical status, using the experimental model of endotoxic shock. Endotoxic shock was produced by intraperitoneal injection of lipopolysaccharide B. Irradiation effectiveness was estimated by leukocyte activation (measured with luminol-dependent chemiluminescence), superoxide dismutase activity of blood plasma (nitro blue tetrasolium assay) and lipid peroxidation (assay with cis-parinaric acid). It was found that laser irradiation has multidirectional effects on leukocyte activation, membrane lipid peroxidation and plasma SOD activity and all these effects were more pronounced in the case of endotoxic shock. Protoporphyrin was more effective in leukocyte activation and chlorine e6 demonstrated maximal effects on blood SOD activity.     

大草蛉成虫复眼的外部形态及其显微结构

用扫描电镜和光学显微镜观察了大草蛉Chrysopa pallens Ramber成虫复眼的外部形态及明、暗适应和性别对其显微结构的影响。结果发现：（1）其复眼呈半球形,位于头部两侧,略成“八”字形排列,单个复眼约由3 600个小眼组成,最前和最后小眼之间的夹角约为180°,最上和最下小眼之间的夹角约200°;（2）小眼主要由角膜、晶锥和6～8个小网膜细胞、基膜组成,外围环绕有2个初级虹膜色素细胞和6个次级虹膜色素细胞,基膜处有色素颗粒分布;（3）暗适应时,晶锥开裂程度较大,远端5～7个网膜细胞核向远端移动,与晶锥近端相接或接近,次级虹膜色素颗粒亦向远端移动包围晶锥;明适应时,晶锥开裂程度小或闭合,远端网膜细胞核向近端移动,透明带显现,大部分次级虹膜色素颗粒亦向近端移动分布在小网膜细胞柱周围,包被透明带;（4）在相同的明、暗适应下,雌、雄成虫复眼的显微结构无明显差异。结果表明大草蛉复眼为透明带明显的重叠象眼,其小眼不但具有次级虹膜色素颗粒纵向移动的常规调光机制,还存在晶锥开闭、远端网膜细胞核移动和基膜色素颗粒纵向扩散的调光新机制。     

Merocyanine 540 as a fluorescent probe of membranes: selective staining of leukemic and immature hemopoietic cells.

We have reported (Easton, Valinsky and Reich, 1978) that merocyanine 540 (MC 540) specifically stains a variety of living excitable cells, but not nonexcitable cells. This paper describes the exceptional permeability to MC 540 of leukemic leukocytes and immature hemopoietic precursor cells. We have used fluorescence microscopy and uptake of radioactive dye to study MC 540 staining of peripheral blood leukocytes from 80 leukemic and 34 normal individuals; leukemic leukocytes stain, whereas normal leukcytes do not. The leukocyte staining reaction differs from that previously described for excitable cells since it is independent of the ionic composition of the staining medium, kinetically complex, enhanced by light, enhanced by oxygen and essentially irreversible. Virtually all circulating nucleated cells from leukemic individuals are stained to approximately the same extent, and there is no qualitative or quantitative distinction between the various forms of leukemia. We have also found that MC 540 interacts with granulopoietic colony-forming cells (CFU-C) and with spleen colony-forming cells derived from mouse bone marrow (CFU-S). We cannot as yet identify a specific property of leukocyte plasma membranes that determines MC 540 permeability; since changes in MC 540 uptake appear to be correlated with cellular maturation during normal hemopoiesis, the retention of staining by leukemic cells, some of which appear morphologically normal, may indicate of failure in membrane maturation during leukemic blood cell development.     

The effect of colony stimulating factor on the synthesis of ribonucleic acid by mouse bone marrow cells in vitro.

The effect of granulocyte-macrophage colony stimulating factor (GM-CSF) on the synthesis of RNA in liquid cultures of mouse bone marrow, spleen, thymus, peritoneal, peripheral blood leukocytes and lymph node cells was investigated. GM-CSF appeared to stimulate RNA-synthesis in syngeneic bone marrow cells within ten minutes of adding it to the culture. In the presence of GM-CSF bone marrow cultures maintained their initial rate of RNA synthesis for approximately ten hours. GM-CSF had no apparent effect on the uptake of 3H-uridine into bone marrow cells. This stimulation was still observed in the presence of puromycin and cycloheximide, but was abrogated by actinomycin D. The magnitude of the stimulation was not affected by the density of cells between 1 and 20 x 10(6) cells/ml but was slightly smaller at 0.1 and 40 x 10(6) cells/ml. Increasing concentration of GM-CSF (up to 2 X 105 units per ml) led to increased stimulation of RNA synthesis in bone marrow cells, but a significant stimulation could be detected at concentrations as low as 800 units/ml. GM-CSF did not significantly stimulate RNA synthesis in spleen, thymus, mesenteric or subcutaneous lymph node cells. However a small stimulation was observed in peripheral blood leukocytes and peritoneal cells. Autoradiographic studies showed that GM-CSF stimulated RNA synthesis in blast cells, myelocytes, metamyelocytes and polymorphs. Nucleated erythroid cells showed no increased labeling with GM-CFS. Labeling in lymphoid-like cells was highly variable but the level of labeling did not appear to be influenced by GM-CSF.     

The decreasedin vitro chemotactic activity of polymorphonuclear leukocytes of newborns and infants

Using Boyden's technique, a statistically significant decrease in the chemotactic activity of polymorphonuclear (PMN) leukocytes was found during the early postnatal period, i.e. in the cord blood and in blood of newborns within the first 10-15 d of life after stimulation of cells with both zymosan-activated adult serum (ZAS) and with an abacterial filtrate of Escherichia coli broth culture (ECF). After this period, the responsiveness of leukocytes to both chemotactic agents increased and remained at the same level during the whole observation period, i.e. up to the age of 6 months. Nevertheless even then it did not reach fully the responsiveness of the leukocytes of mothers and pregnant women. Zymosan-activated serum was shown to be a more potent chemotactic stimulus to leukocytes of infants as compared to the E. coli filtrate.     

甲肝病毒在人血白细胞中体外连续传代和在分类白细胞中增殖的研究

1985年我们采用间接免疫荧光法(IF法)检测出甲肝患者外周血白细胞中有甲肝抗原(HAAg)存在,继而又将HAAg阳性白细胞直接种入PLC/PRF/5细胞,分离到两株甲肝病毒(HAV)NJ—3株和H—1株。为了弄清白细胞所携带的病毒究竟仅为吸附吞饮,抑或能在其中复制增殖,我们将分离到的HAV用正常人血白细胞进行体外增殖试验,现将结果报告如下。     

Histamine uptake by leukocytes in vitro

Human leukocytes at the concentration of 10(6) cells/ml were incubated for 1h at 37.5 degrees C in a tris-albumin buffer, containing 0, 6.25, 12.5, 25, 50, and 100 micrograms/ml of histamine. Histamine uptake was measured by a photofluorometric method. We found that the leukocytes take up exogenous histamine and attain respective intracellular concentration of 307%, 423%, 438%, 1000% and 645% above controls. It was found that neutrophils and eosinophils possess the greatest capacity to take up exogenous histamine whereas, contrary to the expectation, basophils display poor capacity.     

Preliminary observation of blood cells and hematopoietic organs in Lutjanus herenbergi and Lutjanus flaviflammus

The morphology of differentiated and differentiating cells of the red and white series in Lutjanus herenbergi and in Lutjanus flaviflammus is described. Early stages of red and white blood cells may be found only in smears of hemopoietic organs. Polychromatic erythroblasts, myelocytes and lymphoblasts may also occasionally be found in blood smears. Mature blood cells may be found both in blood smears and in hemopoietic organs. Differential white cell counts seem to demonstrate that the granulocytic series elements are the most common leukocytes in blood smears. Almost all granulocytes may be classified in the first three Arneth classes. An analysis of hemopoietic organs in these species was also performed. It was found that the only organs carrying on a hemopoietic function are the kidney and the spleen. The kidney is essentially a site of granulocytic differentiation while the spleen is a lymphopoietic organ. An erythropoietic activity may generally be observed in the kidney although weak erythropoietic activity may at times be found in the spleen.     

Expression of Toll-like Receptor 9 in nose, peripheral blood and bone marrow during symptomatic allergic rhinitis

Background

Allergic rhinitis is an inflammatory disease of the upper airway mucosa that also affects leukocytes in bone marrow and peripheral blood. Toll-like receptor 9 (TLR9) is a receptor for unmethylated CpG dinucleotides found in bacterial and viral DNA. The present study was designed to examine the expression of TLR9 in the nasal mucosa and in leukocytes derived from different cellular compartments during symptomatic allergic rhinitis.

Methods

The study was based on 32 patients with seasonal allergic rhinitis and 18 healthy subjects, serving as controls. Nasal biopsies were obtained before and after allergen challenge. Bone marrow, peripheral blood and nasal lavage fluid were sampled outside and during pollen season. The expression of TLR9 in tissues and cells was analyzed using immunohistochemistry and flow cytometry, respectively.

Results

TLR9 was found in several cell types in the nasal mucosa and in different leukocyte subpopulations derived from bone marrow, peripheral blood and nasal lavage fluid. The leukocyte expression was generally higher in bone marrow than in peripheral blood, and not affected by symptomatic allergic rhinitis.

Conclusion

The widespread expression of TLR9 in the nasal mucosa along with its rich representation in leukocytes in different compartments, demonstrate the possibility for cells involved in allergic airway inflammation to directly interact with bacterial and viral DNA.     

A Proton-Dependent Zinc Uptake in PC12 Cells

Intracellular pH in pheochromocytoma (PC12) cells was manipulated by acid loading the cells and the effect of such a change on radioactive zinc uptake was studied. It was found that zinc uptake was stimulated in cells loaded with protons without causing any measurable change in the intracellular pH. To confirm our assumption that the proton flux due to zinc entry is too small to be measured, we calculated the pH change that one would expect because of zinc influx. The intrinsic buffer capacity of PC12 cells was determined to be 8.03 mM/pH unit and was used in these calculations. It was found that at the five-minute incubation, zinc uptake occurring under our experimental conditions could cause a pH change of 0.000277 pH units per minute (assuming a 1:2 zinc:proton stoichiometry). This study adds a new dimension towards understanding the role played by intracellular pH in causing zinc entry into cells.     

Lack of the long pentraxin PTX3 promotes autoimmune lung disease but not glomerulonephritis in murine systemic lupus erythematosus

The long pentraxin PTX3 has multiple roles in innate immunity. For example, PTX3 regulates C1q binding to pathogens and dead cells and regulates their uptake by phagocytes. It also inhibits P-selectin-mediated recruitment of leukocytes. Both of these mechanisms are known to be involved in autoimmunity and autoimmune tissue injury, e.g. in systemic lupus erythematosus, but a contribution of PTX3 is hypothetical. To evaluate a potential immunoregulatory role of PTX3 in autoimmunity we crossed Ptx3-deficient mice with Fas-deficient (lpr) C57BL/6 (B6) mice with mild lupus-like autoimmunity. PTX3 was found to be increasingly expressed in kidneys and lungs of B6lpr along disease progression. Lack of PTX3 impaired the phagocytic uptake of apoptotic T cells into peritoneal macrophages and selectively expanded CD4/CD8 double negative T cells while other immune cell subsets and lupus autoantibody production remained unaffected. Lack of PTX3 also aggravated autoimmune lung disease, i.e. peribronchial and perivascular CD3+ T cell and macrophage infiltrates of B6lpr mice. In contrast, histomorphological and functional parameters of lupus nephritis remained unaffected by the Ptx3 genotype. Together, PTX3 specifically suppresses autoimmune lung disease that is associated with systemic lupus erythematosus. Vice versa, loss-of-function mutations in the Ptx3 gene might represent a genetic risk factor for pulmonary (but not renal) manifestations of systemic lupus or other autoimmune diseases.     

The effect of temperature on cellular reactions of Crassostrea virginica to the injection of avian erythrocytes

Intracardial injection of chicken red blood cells (RBC) into the oyster, Crassostrea virginica, at 15–19°C elicited ingestion, digestion, and removal of the cells by the leukocytes. A rapid response of fibroblastlike cells and characteristic cell aggregates were also detected as early as 1 hr post-injection. While the former was engaged in an unsuccessful attempt to encapsulate red blood cell laden leukocytes intravascularly, the function of the latter cell type was undetermined. The process of disposing the red blood cells in the oysters under running seawater conditions was not noticeably different from that observed in the oysters under standing water conditions.In oysters previously sensitized with red blood cells, response to a challenge injection was similar to that observed following the first injection, and there was no indication that the activities of the leukocytes were enhanced. However, it was demonstrated that oyster leukocytes remained functionally viable for at least 22 days. There is evidence indicating that the number of the characteristic cell aggregates increases considerably after a secondary injection of red blood cells, although the specificity of this response is yet to be determined.At 6°C, although phagocytosis of the blood cells occurred quite extensively, leukocytes exhibited considerably low digestive and migratory activity which resulted in far less effective disposal of the blood cells. Preliminary observations also revealed possible adhesion of oyster leukocytes to the wall of blood vessels following the inoculation at this temperature.