Technical considerations in evaluating the adhesion of leukocytes to aortic endothelium of the rat

Comments on techniques for characterizing leukocytes adhered to the aortic endothelium of the rat are given. Alpha-naphthyl acetate esterase positive leukocytes were studied by optical microscopy of en face intima-media preparations. Results indicate 1) 1% paraformaldehyde-2% glutaraldehyde is a better fixative than formalin-calcium or 4% paraformaldehyde with or without 1.5 mM CaCl2; the latter produces distortion of leukocytes, endothelial desquamation and enzymate inhibition, 2) washing the aorta with phosphate-buffered saline for 90 sec prior to fixation-perfusion produces a notable decrease in the number of leukocytes adhered, 3) diazotized parasaniline is better than fast blue RR salt as coupling agent in the esterase reaction, and 4) counterstaining with 1% methyl green for 1 min, before or after the esterase reaction, is not adequate because of limited contrast and the heavy staining of smooth muscle. Counterstaining with Gill's hematoxylin No. 3 for 90 sec is adequate only when done before the esterase reaction. Inhibition of endothelial esterase activity by hematoxylin decreases background, favors contrast of adhered leukocytes and makes it possible to observe nucleus-cytoplasm relations.     

Phagocytosis of Intravenously Administered Particles by Leukocytes Adhered to the Aortic Endothelium of the Rat

Phagocytosis has been used to characterize on a functional basis leukocytes adhered to the aortic endothelium of the rat. After intravenous administration of particles, phagocytosis was observed microscopically in esterase-positive leukocytes adhered to the endothelium in whole mounts of aorta. Polybead^R blue and red, 0.5 and 1 μm particle size, were inadequate because they were insufficiently colored to be identified individually at 400. Fluoresbrite^tm YG 0.25 and 0.50 μm at doses of 0.2 and 2 ± 0.3 m1/100 g, respectively, produced endothelial lesions. The same occurred with Monastral blue B^R (MbB) at 0.3 ml/100 g, red iron at 2 ± 16 mg/100 g and India ink at different concentrations depending on the supplier. At lower particle doses, lesions were not found. Deferoxamine mesylate 1.5 mg/100 g intravenous and allopurinol 5 mg/100 g intraperitoneal administered before the particles diminished the number and intensity of lesions. In none of the cases studied was the percentage of phagocytic cells greater than 50%. Clearance curves of MbB and Fluoresbrite^tm indicated rapid disappearance of particles from the blood. Results indicate that administration of particulate suspensions is not a good method for characterizing the phagocytic leukocytes adhering to the aortic endothelium because low doses produce rapid clearance of particles, thus impeding sufficient leukocyte loading, and higher doses produce endothelial lesions that often impair reliable counting of the adhering leukocytes.     

Quantitative assessment of the adherence of normal human blood mononuclear cells to vascular endothelial cell monolayers

We have developed a method to reliably quantitate the in vitro adherence of ⁵¹Cr-labeled blood mononuclear leukocytes to cultured monolayers of vascular endothelial cells from human umbilical veins. Normal mononuclear leukocytes adhered to endothelial cells more than to cover glass at all studied time periods over 4 hr with major differences seen at 2 hr (9.7 ± 1.2% vs 3.7 ± 1.1%; P < 0.01). Only a minority of cells adhering to endothelium were esterase positive. Similar patterns of binding were seen using varying concentrations of suspended mononuclear cells (1–4 × 10⁶/ml) simulating that occurring in vivo in different clinical states. This approach shows promise for in vitro approaches to lymphocyte-vascular endothelial interactions in human immune/inflammatory disorders.     

Alterations by leukocytes of erythrocyte flow in microchannels

The effects of leukocytes on blood rheology in the microcirculation may be predicted to result from the rheological characteristics of the individual white blood cell types and their behavior at entrances of small vessels; their influence on flow resistance of blood due to their disturbance of erythrocyte flow; and the obstruction caused by their adherence to endothelial cells or geometrical hindrance of their flow at irregular or narrow regions. Deformation of leukocytes in micropipettes indicates the relative rigidity of lymphocytes and blast cells from leukemias in contrast to granulocyte viscoelastic properties, and entry times for lymphocytes in 2.6-4.6-microns channels were 11-151 s but milliseconds in 9.1-micron tubes. Lymphocytes and erythrocytes rarely (less than 1%) adhered to cultured endothelium; however, granulocytes adhered avidly at shear stresses of 10-100 dyn/cm2, typical of microcirculation. In the 9.1-microns flow tube at Hct = 17.7 +/- 2.6%, increasing [WBC] caused a linear increase in flow resistance, but above [WBC] = 1000/mm3 resistance tended to plateau. These data support the interpretation that granulocyte adherence to endothelial cells with the potential for obstruction of flow may be a more significant rheological consequence of leukocytes in blood than their resistance to flow because of their relative rigidity or their influence on flow resistance by perturbation of the erythrocyte flow.     

Hematoxylin-Eosin Staining of OsO4-Fixed Epon- Embedded Tissue; Prestaining Oxidation by Acidified H2O2

Successful application of hematoxylin-eosin staining to 0.5-1 μ sections of OsO₄-fixed Epon-embedded mammalian tissue is made possible by first treating the sections for approximately 1 min at 25-30 C with 10% H₂O₂ acidified with 0.1 or 0.01 N H₂SO₄ to pH 3.2. Subsequent steps are: washing; drying; Hams hematoxylin at 50 C, 1-2 min; washing; drying; 0.2-0.3% NH₄OH in 70% ethanol, 3-5 sec, drying at 50 C; 5% aqueous eosin for 3 & 45 sec at 25-30 C, washing; drying; clearing in xylene and mounting in resin. The use of acidified H₂O₂ prevents the staining of Epon and permits the characteristic staining picture to be obtained. Sections were attached to glass slides without adhesive and processed horizontally on a rack. Slides should be well drained and blotted before each drying step, to prevent formation of precipitate on the section.     

Functional role of gap junctions in cytokine-induced leukocyte adhesion to endothelium in vivo

To assess the hypothesis that gap junctions (GJs) participate on leukocyte-endothelium interactions in the inflammatory response, we compared leukocyte adhesion and transmigration elicited by cytokine stimulation in the presence or absence of GJ blockers in the hamster cheek pouch and also in the cremaster muscle of wild-type (WT) and endothelium-specific connexin 43 (Cx43) null mice (Cx43e(-/-)). In the cheek pouch, topical tumor necrosis factor-alpha (TNF-alpha; 150 ng/ml, 15 min) caused a sustained increment in the number of leukocytes adhered to venular endothelium (LAV) and located at perivenular regions (LPV). Superfusion with the GJ blockers 18-alpha-glycyrrhetinic acid (AGA; 75 microM) or 18-beta-glycyrrhetinic acid (50 microM) abolished the TNF-alpha-induced increase in LAV and LPV; carbenoxolone (75 microM) or oleamide (100 microM) reduced LAV by 50 and 75%, respectively, and LPV to a lesser extent. None of these GJ blockers modified venular diameter, blood flow, or leukocyte rolling. In contrast, glycyrrhizin (75 microM), a non-GJ blocker analog of AGA, was devoid of effect. Interestingly, when AGA was removed 90 min after TNF-alpha stimulation, LAV started to rise at a similar rate as in control. Conversely, application of AGA 90 min after TNF-alpha reduced the number of previously adhered cells. In WT mice, intrascrotal injection of TNF-alpha (0.5 microg/0.3 ml) increased LAV (fourfold) and LPV (threefold) compared with saline-injected controls. In contrast to the observations in WT animals, TNF-alpha stimulation did not increase LAV or LPV in Cx43e(-/-) mice. These results demonstrate an important role for GJ communication in leukocyte adhesion and transmigration during acute inflammation in vivo and further suggest that endothelial Cx43 is key in these processes.     

Occurrence of ofloxacin ester-hydrolyzing esterase from Bacillus niacini EM001

A Bacillus niacini strain (EM001) producing an ofloxacin ester-enantioselective esterase was isolated from the soil samples collected near Taejon, Korea. The cloned gene showed that the esterase EM001 composed of 495 amino acids corresponding to a relative molecular weight (M_r) of 54,098 kDa. Based on the M_r and the protein sequence, the esterase EM001 was similar to p-nitrobenzyl esterase from Bacillus subtilis with an identity of 41.8%. The optimum temperature and pH of the purified His-tagged enzyme were 45 °C and 9.0, respectively. The purified esterase EM001 hydrolyzed preferably (R)-ofloxacin propyl ester than (S)-form ester at the initial reaction phase with an ee_P of 67% until the conversion rate become up to 35%.     

Iron-Hematoxylin and Safranin O As A Polychrome Stain for Epon Sections

A 3-step procedure performed at 85 C was as follows: Sections were treated with 4% aqueous ferric ammonium sulfate for 15-300 sec, washed, placed in ripened, 0.5% hematoxylin for 15-300 sec, washed, and counterstained in 1% Safranin O for 15-90 sec. This procedure produced a durable stain with clear cytoplasmic and nuclear differentiation.     

Differential Effects of Cold Atmospheric Plasma in the Treatment of Malignant Glioma

Objective

Cold atmospheric plasma (CAP) has recently been shown to selectively target cancer cells with minimal effects on normal cells. We systematically assessed the effects of CAP in the treatment of glioblastoma.

Methods

Three glioma cell lines, normal astrocytes, and endothelial cell lines were treated with CAP. The effects of CAP were then characterized for viability, cytotoxicity/apoptosis, and cell cycle effects. Statistical significance was determined with student''s t-test.

Results

CAP treatment decreases viability of glioma cells in a dose dependent manner, with the ID50 between 90-120 seconds for all glioma cell lines. Treatment with CAP for more than 120 seconds resulted in viability less than 35% at 24-hours posttreatment, with a steady decline to less than 20% at 72-hours. In contrast, the effect of CAP on the viability of NHA and HUVEC was minimal, and importantly not significant at 90 to 120 seconds, with up to 85% of the cells remained viable at 72-hours post-treatment. CAP treatment produces both cytotoxic and apoptotic effects with some variability between cell lines. CAP treatment resulted in a G2/M-phase cell cycle pause in all three cell lines.

Conclusions

This preliminary study determined a multi-focal effect of CAP on glioma cells in vitro, which was not observed in the non-tumor cell lines. The decreased viability depended on the treatment duration and cell line, but overall was explained by the induction of cytotoxicity, apoptosis, and G2/M pause. Future studies will aim at further characterization with more complex pre-clinical models.     

Double Staining of Plant Materials in Bulk

Ovaries and ovules of Oryza sativa and Zea mays were collected between 9-30 and 10-30 AM, fixed in formalin-acetic-alcohol, stained in Delafield's hematoxylin for 2-4 hr, dehydrated through graded ethanol, counterstained for 3-4 hr either in light green, orange G or fast green (0.05-0.1%) at the 1:1 alcohol-xylene stage and embedded. A few ovaries were hydrolysed in 1 N HCI for 25 min at 60 C, stained in leuco basic fuchsin for 60-90 min, rinsed 3 times with a mixture of: 10% Na₂S₂O₅, 1; N HC1, 1; and distilled water, 18; washed repeatedly in distilled water, dehydrated through graded ethanol, counter-stained for 3-4 hr either with light or fast green (0.05-0.1%) at the 1:1 alcohol-xylene stage and embedded. Microtome sections were cut, ribbons mounted, dried, paraffin removed with xylene, and mounted in balsam. Uniformly stained preparations resulted and the dilute stains gave vivid color contrasts. Large numbers of ovules and ovaries can be processed in a short time, and reliable percentages of viable embryo sacs in normal, sterile and semisterile plants obtained.     

Forces Required to Initiate Membrane Tether Extrusion from Cell Surface Depend on Cell Type But Not on the Surface Molecule

When a cell adhered to another cell or substratum via surface proteins is forced to detach, lipid membrane tethers are often extruded from the cell surface before the protein bond dissociates. For example, during the inflammatory reaction leukocytes roll on the surface of activated endothelial cells. The rolling adhesion is mediated by interactions of selectins with their ligands, e.g., P-selectin glycoprotein ligand (PSGL)-1, which extrudes membrane tethers from the surfaces of both leukocytes and endothelial cells. Membrane tether extrusion has been suggested to regulate leukocyte rolling. Here we examine several factors that may affect forces required to initiate membrane tethers, or initial tether force. It was found that initial tether forces were similar regardless of the presence or absence of the cytoplasmic tail of P-selectin and regardless of whether the tethers were extruded via binding to PSGL-1 or Fcγ receptors. Initial tether forces were found to depend on the cell types tested and were greatly reduced by treatment of latrunculin A, which inhibits actin polymerization. These data provide additional insights to the control of membrane tether extrusion, which should be taken into account when cellular functions such as rolling where tether extrusion plays a regulatory role are compared using different cell types expressing the same molecule.     

Preparation of enantiomerically pure (S)-flurbiprofen by an esterase from Pseudomonas sp. KCTC 10122BP

Esterase PF1-K from Pseudomonas sp. KTCC 10122BP was overproduced by the fed-batch culture of Escherichia coli. The soluble expression of esterase PF1-K was achieved by shifting the culture temperature from 37 to 25 °C at the time of IPTG induction. The enzyme was partially purified to about 75% purity by a single-step hydrophobic interaction column chromatography. The purified enzyme exhibited a fairly high enantioselectivity towards the hydrolysis of rac-flurbiprofen ethyl ester. The enzymatic chiral resolution was further improved by optimizing the reaction conditions in terms of reaction rate and enantioselectivity. The optimal reaction conditions were found to be 40 °C, pH 10.5 and 600 mM of initial rac-flurbiprofen ethyl ester. After 90 min of batch reaction under the optimal conditions, 50% of the initial rac-flurbiprofen was hydrolyzed with an enantiomeric excess of 99%.     

Astrocytic contributions to blood-brain barrier (BBB) formation by endothelial cells: A possible use of aortic endothelial cell for In vitro BBB model

Astrocytic contribution of endothelial cell monolayer permeability was examined in two blood-brain barrier (BBB) models, using the coculture in a double chamber system: rat astrocytes and bovine aortic endothelial cells (BAECs) or bovine brain endothelial cells (BBECs). In system 1, where astrocytes were separated from endothelial cells, a 40% reduction in -glucose permeability of the BBEC monolayer, but not the BAEC monolayer, was observed by cocultivation with astrocytes. Although several passages of BBEC in culture elicited morphological transformation from spindle-shapes to cobblestone-like features, the passaged BBECs remained responsive to astrocytes in coculture in system 1 (37% reduction of the -glucose permeability). By contrast, in system 2, where respective endothelial cells and astrocytes layered on the upper and lower surfaces of a membrane, the permeability of both BAEC and BBEC monolayers was reduced by cocultivation with astrocytes (75% reduction for BAEC and 40% reduction for BBEC). BAECs in this contiguous coculture (system 2) with astrocytes showed numerous tight junction-like structures characteristic of the BBB in vivo. These results suggest that primary cultured BBECs, which had been primed by astrocytes in vivo, retain a higher sensitivity to astrocytes possibly through an astrocytic soluble factor (s) to exhibit BBB-specific phenotypes, and that even BAEC from extra-neural tissues, when cultured with astrocytes in close proximity in vitro, may acquire the similar phenotypes and serve for an extensive use of BBB model in vitro.     

Staining Mitochondria With Hematoxylin After Formalin-Sublimate Fixation; A Rapid Method

Mitochondria were stained in liver, kidney, pancreas, adrenal and intestinal mucosa of rat and mouse. Tissues 1 mm thick, were fixed in a mixture of saturated aqueous HgCl₂, 90 ml; formalin (37-38% HCHO), 10 ml, at room temperature (25°C) for 1 hr. Deparaffinized sections 3-4μ thick were treated with Lugol's iodine (U.S.P.) followed by Na₂S₂O₃ (5%), rinsed in water and the ribonucleic acid removed by any of the following procedures: 0.2 M McIlavaine's buffer, pH 7.0, 2 hr, or 0.2 M phosphate buffer, pH 7.0, 2 hr at 37°C; 0.1% aqueous ribonuclease, 2 hr at 37°C; 5% aqueous trichloracetic acid overnight at 37°C; or 1% KOH at room temperature for 1 hr. After washing in water, sections were treated with a saturated solution of ferric ammonium alum at 37°C for 8-12 hr and colored by Regaud's ripened hematoxylin for 18 hr. They were then differentiated in 1% ferric ammonium alum solution while under microscopic observation.     

快速减压对豚鼠外周微循环和大脑血流量的影响

目的：探讨动物处于减压病（ＤＣＳ）临界发病状态时微及其血流动力作用的改变。方法：采用小型化激光微综在数测量仪及ＬＤＦ－３微区血流量仪,以检测动物高压暴露前及快速减压后微循环和血流动力作用的改变。结果：快速减压后动物微血管明显收缩;毛细血管开放数量减少;微循环中可见气泡并有血栓形成;白细胞、血小板与血管内皮粘附;血流中有料多白色微小血栓;细动脉血流速度平均比正常状态减慢０．９ｍｍ／ｓ,细静脉流速减慢     

Tannic acid, Iron hematoxylin, Alcian blue, and Basic fuchsin for staining islets and reticular fibers of the pancreas

In this technique alpha cells are stained by basic fuchsin, beta cells by iron-hematoxylin, reticular fibers by ferric tannate, and much by alcian blue. Among 6 commonly used fixatives tested, Bouin's fluid fixation (8-12 hr) gave the best staining results. Procedure: paraffin sections to water; 0.5% Li₂CO₃ to remove picric acid; 20% tannic acid, 15 min; wash well; 2-4 sec in 0.5% basic fuchsin containing 10% alcohol; rinse, then differentiate in 1% aniline in 90% alcohol until alpha cells are red and beta cells pink; 1% phosphomolybdic acid, 1 min; 5% hematoxylin in 2% iron alum, 0.5 min; wash well; 1% filtered alcian blue SGX, 15 sec; rinse, dehydrate, clear, and mount in synthtic resin. Results: reticular fibers, black; acinar cells, orange to gray; alpha cells, red; collagenous fibers, red; beta cells, gray granules; ducts, bluish-green. The method was tested on rat, rabbit, dog, hamster, cow and man.     

Quantitative analysis of the process and propagation of cortical granule breakdown in sea urchin eggs

Cortical granule breakdown in sea urchin eggs has been investigated with a video microscope system using Nomarski differential interference contrast optics, when induced by fertilization, microinjecting inositol 1,4,5-trisphosphate (IP3) or Ca-EGTA buffer solution into the egg, or perfusing a medium containing 1 mM Ca2+ to isolated cortices. The cortical granule increased up to 1.2 times in diameter and broke down within 40 msec. These values were almost constant among the three methods used to induce cortical granule breakdown. Upon fertilization, the cortical granule breakdown propagated over the egg surface at a speed of 3.3 microns/sec in Clypeaster japonicus eggs, which indicates that cortical granule breakdown propagated through the 3.3-microns-wide egg surface within 1 sec. In such a small area of the egg surface, however, it took much more than 1 sec for all cortical granules to break down because the maximal rate of breakdown was 7.6%/sec; that is, it took 9 sec and 18 sec for 50% and 90% respectively, of cortical granules to break down. Moreover, the rate did not simply decrease with time, and a shoulder was found during the reducing phase, which suggests that cortical granules are divided into fast and slow breakdown groups according to the responsiveness to the breakdown stimulus. The cortical granule breakdown induced by microinjecting the Ca-EGTA buffer and IP3 solutions propagated at 68 microns/sec and 35 microns/sec, respectively. The stimulus for cortical granule breakdown is discussed concerning the transient intracellular Ca2+ increase.     

A novel beta 1 integrin-dependent mechanism of leukocyte adherence to apoptotic cells

Adherence of leukocytes to cells undergoing apoptosis has been reported to be dependent on a variety of recognition pathways. These include alpha V beta 3 (CD51/CD61, vitronectin receptor), CD36 (thrombospondin receptor), macrophage class A scavenger receptor, phosphatidylserine translocated to the outer leaflet of apoptotic cell membranes, and CD14 (LPS-binding protein). We investigated the mechanism by which leukocytes adhere to apoptotic endothelial cells (EC). Peripheral blood mononuclear leukocytes and U937 monocytic cells adhered to human or bovine aortic EC induced to undergo apoptosis by withdrawal of growth factors, treatment with the promiscuous protein kinase inhibitor staurosporine, with the protein synthesis inhibitor and protein kinase activator anisomycin, or with the combination of cycloheximide and TNF-alpha. Expression of endothelial adherence molecules such as CD62E (E-selectin), CD54 (ICAM-1), and CD106 (VCAM-1) was not induced or increased by these treatments. A mAb to alpha V beta 3, exogenous thrombospondin, or blockade of phosphatidylserine by annexin V did not inhibit leukocyte adherence. Further, leukocyte binding to apoptotic EC was completely blocked by treatment of leukocytes but not EC with mAb to beta 1 integrin. These results define a novel pathway for the recognition of apoptotic cells.     

The fluid shear stress distribution on the membrane of leukocytes in the microcirculation

Recent in-vivo and in-vitro evidence indicates that fluid shear stress on the membrane of leukocytes has a powerful control over several aspects of their cell function. This evidence raises a question about the magnitude of the fluid shear stress on leukocytes in the circulation. The flow of plasma on the surface of a leukocyte at a very low Reynolds number is governed by the Stokes equation for the motion of a Newtonian fluid. We numerically estimated the distribution of fluid shear stress on a leukocyte membrane in a microvessel for the cases when the leukocyte is freely suspended, as well as rolling along or attached to a microvessel wall. The results indicate that the fluid shear stress distribution on the leukocyte membrane is nonuniform with a sharp increase when the leukocyte makes membrane attachment to the microvessel wall. In a microvessel (10 microns diameter), the fluid shear stress on the membrane of a freely suspended leukocyte (8 microns diameter) is estimated to be several times larger than the wall shear stress exerted by the undisturbed Poiseuille flow, and increases on an adherent leukocyte up to ten times. High temporal stress gradients are present in freely suspended leukocytes in shear flow due to cell rotation, which are proportional to the local shear rate. In comparison, the temporal stress gradients are reduced on the membrane of leukocytes that are rolling or firmly adhered to the endothelium. High temporal gradients of shear stress are also present on the endothelial wall. At a plasma viscosity of 1 cPoise, the peak shear stresses for suspended and adherent leukocytes are of the order of 10 dyn/cm2 and 100 dyn/cm2, respectively.     

The murine CD99-related molecule CD99-like 2 (CD99L2) is an adhesion molecule involved in the inflammatory response

CD99, a glycoprotein found on the surfaces of leukocytes and concentrated at the borders of endothelial cells, plays a major role in the migration of leukocytes across endothelial cells into sites of inflammation, and has other roles in thymocyte development. The human and mouse genomes encode only two proteins related to CD99. One of these, XGA, is a red blood cell surface antigen. The function of the other, CD99-like 2 (CD99L2), is not known. We cloned mouse CD99L2 and used CD99L2 isolated from transfected cells to raise specific antibodies. Similar to human CD99, CD99L2 was expressed at the borders between transfected cells as well as on mouse leukocytes and vascular endothelial cells in situ. Transfection of L cell fibroblasts with CD99L2 imparted to them the ability to adhere to each other in a divalent cation-dependent, homophilic manner. Anti-CD99L2 antibody blocked influx of neutrophils and monocytes into a site of inflammation in vivo.