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 pararosaniline 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 by bovine polymorphs of glass-adherent Escherichia coli in the presence and absence of serum

The influence of serum on the phagocytosis and killing of Escherichia coli adherent to glass (‘surface phagocytosis’) was investigated by using 2 strains known to require opsonisation when phagocytosis takes place in suspension. Although phagocytosis of the unencapsulated strain occurred in the absence of serum, serum was necessary for the uptake of the encapsulated strain, and improved the uptake of both strains. Killing of ingested bacteria appeared to be independent of serum. Antibodies were not an absolute requirement for the promoting effect of serum, and the necessity for complement (Cp) appeared to depend on the presence of antibodies and on the strain tested. The results suggest that serum components other than opsonins (antibodies and Cp) contributed to the serum-enhanced uptake of bacteria.     

Pharmacokinetics,Tissue Distribution and Metabolism of Intravenously Administered Digalactosyldiacylglycerol and Monogalactosyldiacylglycerol in the Rat

Abstract

A bilayer forming galactolipid, digalactosyldiacylglycerol (DGalDG) has been identified as a tool with suitable physicochemichal properties for pharmaceutical formulation work. One possible application is as a carrier for liposome entrapped drugs for intravenous administration. The fate of intravenously administered galactolipids is not known. In this study liposomal dispersions of galactolipids, containing [³H]fatty acid labelled DGalDG or monogalactosyldiacylglycerol (MGalDG) were injected intravenously in the rat and the disappearance from blood and uptake by tissues were examined. The T1/2 of [³H]DGalDG in plasma was 3 to 5 minutes. Of the tissues examined (liver, spleen, kidneys, lung, heart, stomach, upper and lower small intestine and colon), the liver contained the highest radioactivity per g tissue after both 15 min. and 4 h. Autoradiographic examinations after 15 min, 1 h and 4 h showed that the uptake of radiolabeled DGalDG and MGalDG occurred mainly to the hepatocytes. Less than 6 % of the injected [³H]DGalDG remained in liver and plasma as [³H]DGalDG after 4 h. [³H]MGalDG exhibited a similar pattern of metabolism although the initial disappearance rate was faster than for [³H]DGalDG. The study thus shows that the hepatocytes take up and hydrolyse galactolipids after intravenous administration.     

Involvement of locomotion of granulocytes in the phagocytosis of glass-adherent group B streptococci in the absence of opsonins

Abstract Phagocytosis of glass-adherent group B streptococci (GBS) by bovine polymorphonuclear leukocytes (PMN) was investigated by a fluorochrome microassay. Three out of the four tested strains were taken up in the absence of serum, but phagocytosis was increased when immobilized bacteria were pre-treated by either normal bovine serum (NBS, containing antibodies to GBS), precolostral calf serum (PCS, virtually devoid of antibodies), heat-inactivated PCS (H-PCS), or to a lesser extent by human serumalbumin (HSA) or gelatin. The fourth strain required opsonization by NBS to be ingested. The under-agarose PMN migration assay showed that HSA, PCS, H-PCS, and NBS had locomotion-promoting effects that ranked as for enhancement of phagocytosis. In addition, fixed antigen-antibody complexes inhibited both migration under agarose and surface phagocytosis. These findings suggested that when bacteria are sensitive to surface phagocytosis in the absence of opsonins, the enhancing effect of serum is essentially mediated through promotion of PMN locomotion and not through opsonin-enhanced ingestion.     

Phagocytosis is coupled to the formation of phagosome-associated podosomes and a transient disruption of podosomes in human macrophages

Phagocytosis consists in ingestion and digestion of large particles, a process strictly dependent on actin re-organization. Using synchronized phagocytosis of IgG-coated latex beads (IgG-LB), zymosan or serum opsonized-zymosan, we report the formation of actin structures on both phagocytic cups and closed phagosomes in human macrophages. Their lifespan, size, protein composition and organization are similar to podosomes. Thus, we called these actin structures phagosome-associated podosomes (PAPs). Concomitantly to the formation of PAPs, a transient disruption of podosomes occurred at the ventral face of macrophages. Similarly to podosomes, which are targeted by vesicles containing proteases, the presence of PAPs correlated with the maturation of phagosomes into phagolysosomes. The ingestion of LB without IgG did not trigger PAPs formation, did not lead to podosome disruption and maturation to phagolysosomes, suggesting that these events are linked together. Although similar to podosomes, we found that PAPs differed by being resistant to the Arp2/3 inhibitor CK666. Thus, we describe a podosome subtype which forms on phagosomes where it probably serves several tasks of this multifunctional structure.     

Phagocytosis of spermatozoa by the ovum of the domestic fowl,Gallus gallus at the time of fertilization

Summary Spermatozoa with intact acrosomes, as well as those coming into contact with the ovum at a smaller angle, and morphologically abnormal spermatozoa reach the plasma membrane of the ovum via an extensively dissolved zone of the inner layer of the vitelline membrane. This zone is assumed to be formed by overlapping of two or more tunnels formed by spermatozoa that had previously come into contact with the ovum.When a spermatozoon comes into contact with the plasma membrane of the ovum, many cytoplasmic processes extend outwards and cover it. Thereafter, the plasma membranes of the processes fuse, thereby phagocytizing the spermatozoon. It is assumed that the phagocytized spermatozoa cannot undergo transformation into male pronuclei and that they degenerate soon after phagocytosis.The authors are greatly indebted to Assoc. Prof. Osamu Koga for his valuable advice. The authors also wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advice. This investigation was supported by a grant from the Ministry of Education of Japan (156185)Previous name: Fukashi Okamura     

Phagocytosis and elimination of amelocyte debris by stratum intermedium cells in the transitional zone of the enamel organ of the rat incisor

Summary In the transitional zone of the enamel organ (rat) some of the amelocytes perish. Their debris is phagocytized and digested by stratum intermedium cells and macrophages. These two cell types also seem to remove cytosegresomes expelled from those amelocytes which survive and redifferentiate into transporting amelocytes. Digestion of the amelocyte debris in the stratum intermedium cells is effected rapidly and completely. Degeneration of stratum intermedium cells was not observed in the transitional zone.This work was supported by grants from The Danish Medical Research Council (512-149/69 and 512-1008/71) and The Danish Science Research Council (512-1009/71).     

七氟醚通过影响外周血miR-340水平逆转脓毒症大鼠巨噬细胞吞噬功能抑制状态的机制

摘要 目的：探究七氟醚通过影响外周血miR-340水平逆转脓毒症大鼠巨噬细胞吞噬功能抑制状态的机制。方法：60只雄性Sprague-Dawley大鼠根据研究目的将大鼠分为对照组、脓毒症组和七氟醚组。通过RT-PCR分析各组大鼠外周血miR-340以及促炎因子IL-1β、IL-6和TNF-α的mRNA表达水平。统计各实验组大鼠的存活率。通过血琼脂平板对各组大鼠腹腔液和血液中的细菌进行计数。通过使用荧光显微镜测量吞噬率和吞噬指数。通过蛋白印迹分析p65和NF-κB的蛋白表达。结果：脓毒症组miR-340水平较对照组升高（P<0.05）,七氟醚组miR-340水平较脓毒症组降低（P<0.05）。三组不同时间点存活率相比差异无统计学意义（P＞0.05）;第4 d、8 d脓毒症组大鼠存活率较对照组大鼠降低（P<0.05）,七氟醚组大鼠存活率较脓毒症组升高（P<0.05）。脓毒症组IL-1β、IL-6和TNF-α的mRNA表达较对照组升高（P<0.05）,七氟醚组IL-1β、IL-6和TNF-α的mRNA表达较脓毒症组降低（P<0.05）。脓毒症组腹腔液和血液中的细菌数量较对照组升高（P<0.05）,七氟醚组腹腔液和血液中的细菌数量较脓毒症组减少（P<0.05）。血琼脂平板对各组大鼠腹腔液和血液中的细菌进行计数,脓毒症组腹腔液和血液中的细菌数量较对照组升高（P<0.05）,七氟醚组腹腔液和血液中的细菌数量较脓毒症组减少（P<0.05）。脓毒症组p65和NF-κB的蛋白表达较对照组升高（P<0.05）,七氟醚组p65和NF-κB的蛋白表达较脓毒症组降低（P<0.05）。结论：miR-340参与了脓毒症大鼠巨噬细胞吞噬功能调节,七氟醚通过降低外周血miR-340水平减少内毒素诱导的大鼠促炎细胞因子的释放,并恢复巨噬细胞吞噬功能。     

Cleavage of Mer Tyrosine Kinase (MerTK) from the Cell Surface Contributes to the Regulation of Retinal Phagocytosis

Phagocytosis of apoptotic cells by macrophages and spent photoreceptor outer segments (POS) by retinal pigment epithelial (RPE) cells requires several proteins, including MerTK receptors and associated Gas6 and protein S ligands. In the retina, POS phagocytosis is rhythmic, and MerTK is activated promptly after light onset via the αvβ5 integrin receptor and its ligand MFG-E8, thus generating a phagocytic peak. The phagocytic burst is limited in time, suggesting a down-regulation mechanism that limits its duration. Our previous data showed that MerTK helps control POS binding of integrin receptors at the RPE cell surface as a negative feedback loop. Our present results show that a soluble form of MerTK (sMerTK) is released in the conditioned media of RPE-J cells during phagocytosis and in the interphotoreceptor matrix of the mouse retina during the morning phagocytic peak. In contrast to macrophages, the two cognate MerTK ligands have an opposite effect on phagocytosis and sMerTK release, whereas the integrin ligand MFG-E8 markedly increases both phagocytosis and sMerTK levels. sMerTK acts as a decoy receptor blocking the effect of both MerTK ligands. Interestingly, stimulation of sMerTK release decreases POS binding. Conversely, blocking MerTK cleavage increased mostly POS binding by RPE cells. Therefore, our data suggest that MerTK cleavage contributes to the acute regulation of RPE phagocytosis by limiting POS binding to the cell surface.     

Regulation of leukotriene synthesis by arachidonic acid in human polymorphonuclear leukocyte adhesive interactions is dependent on the presence of albumin

We have previously demonstrated that the pretreatment of polymorphonuclear leukocytes (PMNs) with the chemotherapeutic drug, Suramin, increases both cell attachment and inhibits calcium ionophore A23187-stimulated leukotriene (LT) synthesis. Here, we examined the effects of extracellular arachidonic acid (AA) and albumin on attachment and LT synthesis in the interaction of PMNs with both collagen-coated surfaces and human umbilical vein endothelial cell (HUVEC) monolayers. Suramin decreased the release of radiolabelled AA and 5-lipoxygenase metabolites by [(14)C-AA]-prelabelled PMNs stimulated with A23187, with and without human serum albumin (HSA) in the culture medium. Addition of 1 microM AA together with calcium ionophore stimulated the release of endogenous AA to the same level as control and Suramin-pretreated cells, but attachment was unaffected and LT synthesis was still inhibited with Suramin treatment. Using 24 microM AA, regulation of LT synthesis was dependent on the presence of HSA in the medium. Without HSA, 24 microM AA induced detachment of PMNs and increased LT synthesis in Suramin-treated cells above the control level. In the presence of HSA, 24 microM AA did not influence PMN attachment or abolish Suramin-induced inhibition of LT synthesis. These results suggest that tight attachment of PMNs to a solid surface leads to decreased LT synthesis during subsequent stimulation of the cells by A23187 in the presence or absence of exogenous substrate.     

Phagocytosis of the protozoan parasite, Marteilia sydneyi, by Sydney rock oyster (Saccostrea glomerata) hemocytes

QX disease is a fatal disease in Sydney rock oysters caused by the protozoan parasite Marteilia sydneyi. The current study investigates the phagocytosis of M. sydneyi by Sydney rock oyster hemocytes. It also compares the in vitro phagocytic activities of hemocytes from oysters bred for QX disease resistance (QXR) with those of wild-type oysters. After ingestion of M. sydneyi, hemocyte granules fused with phagosome membranes and the pH of phagosomes decreased. Significantly (p = <0.05) more phagosomes in QXR hemocytes showed obvious changes in pH within 40 min of phagocytosis, when compared with wild-type hemocytes. Phenoloxidase deposition was also evident in phagosomes after in vitro phagocytosis. Most importantly, ingested and melanised M. sydneyi were detected in vivo among hemocytes from infected oysters. Overall, the data suggest that Sydney rock oyster hemocytes can recognise and phagocytose M. sydneyi, and that resistance against QX disease may be associated with enhanced phagolysosomal activity in QXR oysters.     

Inhibition of Inducible Nitric Oxide Synthase Expression by Endothelin in Rat Glial Cells Prepared from the Neonatal Rat Brain

Abstract: In primary cultured rat glial cells, a combination of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) stimulates production of nitrite via expression of the inducible form of nitric oxide synthase (iNOS). In these cells, simultaneous addition of endothelin (ET) decreased iNOS expression and nitrite accumulation induced by TNF-α/IL-1β. The inhibitory effect of ET on TNF-α/IL-1β-stimulated iNOS expression appears to be mediated by ET_B receptors, because (1) both ET-1 and ET-3 inhibited the effects of TNF-α/IL-1β on iNOS expression and nitrite accumulation, (2) a selective ET_B receptor agonist, Suc-[Glu⁹,Ala^11,15]-ET-1 (8–21) (IRL1620), decreased the effects of TNF-α/IL-1β, and (3) a selective ET_B receptor antagonist, N-cis -2,6-dimethylpiperidinocarbonyl- l -γ-methylleucyl- d -1-methoxycarbonyltryptophanyl- d -norleucine, abolished the inhibitory effects of ETs and IRL1620. Incubation of glial cells with lipopolysaccharide (LPS) caused an increase in iNOS expression. Simultaneous addition of ET-3 decreased the effects of LPS (10 and 100 ng/ml) on iNOS expression. Furthermore, cyclic AMP-elevating agents (dibutyryl cyclic AMP and forskolin) inhibited TNF-α/IL-1β-induced and LPS-induced iNOS expression and nitrite accumulation. These findings suggest that ETs can decrease TNF-α/IL-1β-induced and LPS-induced iNOS expression via ET_B receptors and that cyclic AMP may be involved in this process.     

Effects of Cytochalasins B and D on Staphylococcus aureus Adherence to and Ingestion by Mouse Renal Cells from Primary Culture

Cytochalasin B (CB) and cytochalasin D (CD), inhibitors of microfilament function of host cell, were examined for their effects on Staphylococcus aureus Cowan I adherence to and ingestion by several types of the hyperosmolarity-tolerant (HOT) cells obtained from primary culture of mouse kidney. Staphylococcal adherence to the HOT cells with epithelial appearance was extraordinarily enhanced by the treatment of those cells with both 5 μg/ml of CB and CD. In particular, staphylococci adhered to the periphery rather than the center of each cytochalasintreated cell. Staphylococcal ingestion by all types of the HOT cells was markedly inhibited by CD in spite of the enhanced adherence. Contrary to our expectation, inhibition by CB was incomplete, and the enhanced adherence of staphylococci to CB-treated cells resulted in the enhanced ingestion.     

Activation of the Voltage-Sensitive Sodium Channel by a β-Scorpion Toxin in Rat Brain Nerve-Ending Particles

Neurotoxins purified from scorpion venoms previously had been divided into two classes according to their binding properties in rat brain synaptosomes. However, the pharmacological action of beta-scorpion toxin (beta-ScTx) on this preparation has not yet been described. In this report we show that a beta-ScTx induced an increase in 22Na+ uptake through synaptosomal voltage-sensitive sodium channels since this stimulation was abolished by tetrodotoxin (TTX). The increase was smaller than with veratridine and no synergy was observed between beta-ScTx and veratridine, as is the case for alpha-scorpion toxin (alpha-ScTx) and veratridine. The effects of alpha- and beta-ScTx were additive and the concentration-effect curves for each type of toxin were not modified by the other, suggesting that these two types of toxins act through distinct and noninteracting receptor sites. This was confirmed by the absence of mutual modification of the equilibrium and kinetic binding properties. beta-ScTx was shown to inhibit the uptake and to stimulate the release of [3H]gamma-aminobutyric acid. These effects were blocked by TTX, and no synergy was observed with veratridine. It was concluded that all these effects are mediated by the activation of voltage-sensitive sodium channels induced by the binding of beta-ScTx to a receptor site (site 4) distinct from those for other neurotoxins acting on sodium channels.     

Differential cellular response of resistant and susceptible rodents to the early stages of infection of Taenia taeniaeformis

Taenia taeniaeformis infection course in C3H/He and BALB/c mice (susceptible and resistant respectively to T. taeniaeformis) was compared for 10 days post-infection (p.i.). Light microscopic observation of early organisms in BALB/c revealed host cell infiltration as early as 2 days p.i. By day 4 p.i. the larvae presented an inconspicuous acellular halo and were surrounded by a dense mass of leukocytes, mainly eosinophils. The amorphous substance reacted positive for sulphated acid mucopolysaccharides. By day 6 p.i. the parasite-host interface was filled by leukocytes and fibroblasts, staining with alcian blue was limited to small areas and the parasites appeared disrupted and lightly stained by haematoxylin-eosin. At day 10 p.i. the parasite had been destroyed by the host. In rats and C3H/He mice, the parasites at day 2 p.i. were 10 μm in diameter, bounded by a larger amorphous acellular halo that reacted positive to alcian blue stain, an indication of the presence of acidic muco-polysaccharides. By day 4 p.i. the clear halo became larger and reacted strongly positive for acid mucopolysaccharides particularly close to the organism. By day 6 p.i. alcian blue positive material appeared more diffuse, covering a broad area well inside the host tissue. By this time a layer of inflammatory cells were often present at some distance from the parasite tegument. By day 10 p.i. the larvae continued growing and increasing the number of tegumental cells. The presence of sulfated acid mucopoly-saccharides around the larvae probably serves as a protective barrier for the parasite by triggering the alternative pathway of complement, consuming C3 and C5, thus preventing cell attachment and humoral antibody attack. The early destruction of the parasite by cell defense mechanism implies a major role of cellular immunity in T. taeniaeformis elimination.     

Association between the frequency of class II HLA antigens and the susceptibility to intrauterine infection of hepatitis B virus

Multiple factors determine the susceptibility to intrauterine hepatitis B virus (HBV) infection. These factors include the HBV structure, HBV mutation, HBV DNA level, placental barrier, the immune status of the mother, and the genetic make-ups of the newborn infants. Since HLA system is an integral component of the immune response, we hypothesized that the highly polymorphic HLA genes are the key determinants of intrauterine HBV infection. In this study, we selected newborn infants of HBsAg-positive mothers, and divided the infants into 2 groups: intrauterine infection group and non-intrauterine infection group according to the status whether or not they were infected at birth. Each infected infant was compared with 2 controls from the same birth cohort. HLA-DR allele typing was performed using a PCR-sequence specific primer (PCR-SSP) for 24 subjects with intrauterine infection and 48 controls without infection. We found that, among the fifteen (15) HLA-DR alleles assessed, HLA-DRB1*07 was the one, and the only one, significantly in excess (OR = 6.66, P = 0.004) in the intrauterine infection group compared to the non-intrauterine infection group. Our findings thus suggest that high frequency of HLA class II molecules, e.g. HLA-DRB1*07, is associated with the susceptibility of the infants to intrauterine HBV infection.     

Regulation of the adhesion ofPseudomonas fluorescens cells to glass by extracellular volatile compounds

The effect of the gaseous metabolites of onePseudomonas fluorescens culture on the attachment of cells of anotherP. fluorescens culture to glass was studied. Gaseous metabolites increased the number of unattached cells by 10–30% and the mean residence time of cells attached to glass by 100%. These effects were presumably due to the yet unidentified compound, which we called volatile antiadhesin. This compound could be adsorbed by activated charcoal and HAYESEP-Q adsorbent.     

Adhesion of human ovarian adenocarcinoma IGROV1 cells to endothelial cells is partly mediated by the alphav integrins-vitronectin adhesive system and induces an alteration of endothelial integrity

Several lines of evidence suggest that vascularization plays an important role in the growth, local expansion and dissemination of ovarian epithelial tumours. However, the interaction of ovarian carcinoma cells with the endothelium remains poorly understood. To investigate adhesive events underlying this process, we used an in vitro model of cocultures between the IGROV1 human ovarian adenocarcinoma cell line and human umbilical vein endothelial cells (HUVECs). IGROV1 cells were shown to adhere rapidly on the HUVECs monolayer. Adhesion was inhibited by anti-alphav integrin and anti-Vn blocking antibodies, but not by anti-beta1 integrin antibodies. Anchorage of carcinoma cells led to the rupture of endothelial integrity, as revealed by the formation of holes in the monolayer and by the disappearance of the interendothelial VE-Cadherin network. Considering the ability of ovarian carcinoma to disseminate by a haematogenous way, these in vitro events could mimic a preliminary step for carcinoma cells crossing the endothelial barrier to extravasate.     

Adhesion to extracellular materials by neural crest cells at the stage of initial migration

Summary Trunk-level neural anlagen bearing neural crest cells at the stage of initiation of migration were isolated from chick embryos and explanted in serum-free medium onto glass substrates which had previously been treated with extracellular materials. After 0.5–2 h incubation, the expiants were dislodged with a stream of culture medium and the substrate examined for adherent crest cells. Crest cells adhered to collagen gels, and adhered to and spread on adsorbed fibronectin; antiserum to fibronectin prevented adhesion to fibronectin but not to collagen gels. Air-dried collagen gels and collagen solutions were less adhesive, the adhesivity declining with longer drying time and lower collagen concentration. Crest cells adhered poorly to dried gelatin and not at all to adsorbed collagen. Fibronectin increased the adhesion to dried collagen and gelatin. Pretreatment of collagen gels with hyaluronate retarded adhesion. Hyaluronate pretreatment also retarded adhesion to adsorbed fibronectin but only when adsorbed collagen was also present. Pretreatment of collagen gels with the proteoglycan monomer from bovine nasal cartilage had no effect of the adhesion of crest cells, but the proteoglycan almost completely inhibited adhesion to adsorbed fibronectin, but only when absorbed collagen was also present. The results are discussed in terms of the control of migration of neural crest cells by extracellular materials.     

The effect of cytochalasin B,colchicine and vinblastine on the adhesion of Trichomonas vaginalis to glass surfaces

The attachment of Trichomonas vaginalis to glass surfaces was studied in the presence of cytochalasin B, colchicine and vinblastine. Marked inhibition of adhesion was noted with high concentrations of cytochalasin B. Colchicine and vinblastine were without effect. These findings suggest that Trichomonas vaginalis adhesion is at least partially mediated by the extension of cellular probes, due to the action of cytochalasin-sensitive microfilaments.