思连康对小鼠腹腔巨噬细胞吞噬率和吞噬指数的影响

目的 研究双歧杆菌四联活菌片（商品名：思连康）对小鼠腹腔巨噬细胞吞噬鸡红细胞吞噬率及吞噬指数的影响。方法 将SPF小鼠30只随机分成三组,每组10只,Ⅰ组灌胃生理盐水,Ⅱ组灌胃婴儿双歧杆菌菌悬液,Ⅲ组灌胃双歧杆菌四联活菌片菌悬液,每天给药0.5 mL,菌液浓度为1.0×108 CFU/mL,连续给药10 d后小鼠腹腔注入2%鸡红细胞悬液1 mL（红细胞数量为2×108个/mL）,30 min后处死,取小鼠腹腔洗液,观察并记录吞噬鸡红细胞的巨噬细胞数及被吞噬的鸡红细胞数,计算吞噬率及吞噬指数。结果 与Ⅰ组相比,Ⅱ组和Ⅲ组小鼠腹腔巨噬细胞吞噬鸡红细胞的吞噬率和吞噬指数均显著升高（Ps＜0.05）,其中Ⅲ组高于Ⅱ组（P＜0.05）。结论 双歧杆菌四联活菌片及其婴儿双歧杆菌通过提高小鼠腹腔巨噬细胞的吞噬率和吞噬指数提高机体的免疫力。     

Flow Cytometric Measurement of Bovine Milk Neutrophil Phagocytosis

A flow cytometric method for the evaluation of the phagocytic capacity of bovine milk polymorphonuclear neutrophils (PMN) is described. Milk PMN were isolated from stripping milk collected from udder quarters fitted with abraded intramammary devices (AIMD). A significant increase in the milk somatic cell count was observed in the stripping milk after the insertion of AIMD (308×103 and 1447×l03 cells/ml milk before respectively after the insertion of the AIMD, p < 0.001). PMN were also isolated from blood by a discontinous gradient of Percoli. Blood and milk PMN were incubated for 15 min with FITC-labeled bacteria in a ratio of 1 PMN:20 bacteria and a final serum concentration of 10 %. The number of extracellular bacteria and the percentage of phagocytic cells were measured by a flow cytometer. Percentage of phagocytized bacteria by milk PMN was significantly lower than that by blood PMN (p < 0.05). A smaller number of active phagocytes was present among cells isolated from milk than among cells isolated from blood. The phagocytic capacity of milk PMN reflects that of blood PMN in the same animal. A large variation in the phagocytic capacity of blood and milk PMN among animals was observed.     

Phagocytosis by catfish neutrophils

Channel catfish peripheral blood leucocytes were separated on a Percoll gradient to establish the phagocytic function of the neutrophils. Four fractions of leucocytes were formed on the Percoll gradient, including a fraction that contained 50–80% neutrophils at a density of 1.08–1.09 g ml⁻¹ and a fraction that contained 10% monocytes at a density of 1.071–1.074 g ml⁻¹. Phagocytic assays, using ³H-uridine, showed that the two fractions had similar phagocytic indices, although neutrophils were less phagocytic than monocytes. Neutrophils were confirmed to be phagocytic when examined with transmission electron microscopy. Staining with 3,3-diaminobenzidine-tetrahydrochloride demonstrated peroxidase-positive granules in the cytoplasm of actively phagocytic cells as well as peroxidase reaction products in a number of phagosomes containing bacteria. Phagocytosis of bacteria by channel catfish neutrophils was further confirmed by differential staining of external bacteria and cell surfaces with ruthenium red during the fixation process.     

Influence of HIV-infection on the phagocytic activity of monocytes/macrophages and granulocytes

Because of the great importance of phagocytosis as a key process in host defence, the influence of HIV-infection on the phagocytic activity of monocytes/macrophages (M0/MAC) and granulocytes was investigated. Therefore, blood samples from the peripheral blood of 70 HIV-infected individuals were incubated with fluorescein isothiocyanate (FITC) labeled Escherichia coli. The uptake of the bacteria was monitored by flow cytometer analysis. A strong and significant increase in the relative number of phagocytic granulocytes was observed ranging from 12.8% in an uninfected control collective to over 30% in AIDS patients. This effect was obtained for all patients and independent of the stage of disease. For monocytes, only marginal changes were found in their phagocytic function. These data suggest that the high susceptibility of HIV patients for secondary infections is not linked to a loss of phagocytic ability of monocytes/macrophages and/or granulocytes.     

Flow Cytometric Characterization of Bovine Blood Neutrophil Phagocytosis of Fluorescent Bacteria and Zymosan Particles

A Flow Cytometric method for the evaluation of the phagocytic capacity of bovine blood neutrophils is described. The neutrophils were isolated from bovine blood by a one step discontinuous gradient of Percoll. By this technique of isolation, 90 ± 2.8 % (mean ± s) of the granulocytes in the whole blood were recovered. Isolated neutrophils were incubated with FITC labeled S. aureus or zymosan particles in a ratio of 1:20 and 1:10, respectively, and a final serum concentration of 10 %. Phagocytosis was terminated after 15 min and the number of extracellular bacteria or zymosan particles and the percentage of phagocytic granulocytes were registered by Flow Cytometry (FCM). FCM and microscopic studies revealed that eosinophils play a minor role in the phagocytosis of bacteria. The neutrophils were the main population of the granulocytes which were actively phagocytic. Variation among cows in the ability of their blood neutrophils to phagocytize bacteria was evident.     

Quantitative assessment of phagocytic activity of hemocytes in the prawn, Penaeus merguiensis, by flow cytometric analysis

BACKGROUND: The blood cells of crustaceans are involved in phagocytosis of invading microorganisms, contributing to their defense mechanisms. In this study, phagocytic activity of hemocytes of the prawn, Penaeus merguiensis, was quantitated by means of flow cytometric analysis. METHOD: This study was done in vitro. Hemolymph, which was extracted from prawns, was mixed with an equal volume of anticoagulant. Heat-killed Escherichia coli prestained with propidium iodide (PI) was then added. Hemocytes were fixed at various time intervals for flow cytometric analysis. This study was supplemented with electron micrographs using transmission electron microscopy (TEM), which showed three populations of hemocytes. RESULTS: It was observed that those hemocytes that were more active engulfed and digested bacteria readily, thus having higher red fluorescence intensity. The phagocytic activity was expressed as fluorescence unit or engulfed E. coli number per hemocyte. CONCLUSIONS: With this approach, the phagocytic and cellular activity of individual hemocyte populations could be studied quantitatively.     

System for testing the phagocytic capacity of human blood platelets

We have elaborated a system for testing the phagocytic activity of human blood platelets. Blood was sampled to heparin and ACD and centrifuged. This procedure yields platelet rich plasma (PRP). As a substrate for phagocytosis we used Staphylococcus aureus 209P. The ratio of platelets to bacteria is 1:1. Incubation of bacteria and platelets continued up to 10 minutes. Lysostaphine was introduced to the phagocytic system to destroy all bacteria outside the blood platelets. The maximal values for the percentage of phagocytizing blood platelets were 0.7% and the phagocytosis index of 1.0 was reached at 6 minutes with incubation of bacteria and platelets. The authors pay attention to the role of blood platelets in antibacterial mechanisms.     

Changes in the leukocyte phagocytic activity of donor blood after its UV irradiation. I. Its relation to the irradiation dose and initial level of phagocytic activity

Phagocytic activity of human mono- and granulocytes increased markedly after UV blood irradiation in the apparatus "Izolda" used in hospitals of the USSR for medical treatment. With the rise of irradiation dose the ratio of cells ingesting latex particles increased, although the average number of particles ingested per cell decreased. The integrative phagocytic index poorly depended on the irradiation dose. In patients with a low initial level of phagocytic index, after UV blood irradiation it became more pronounced than in those with the initial elevated level. The enhancement of phagocytic activity is the result of a direct UV-stimulation of cells. This stimulation not mediated by irradiated blood plasma is known to inhibit the phagocytic activity of leucocytes. A possible mechanism of phagocytic activity stimulation is discussed.     

Role of several physico-chemical surface properties of bacteria in the machanism of the initial phases of phagocytosis]

An attempt was made on E. coli pattern to reveal the role played by the surface charge and the extent of the surface hydration in the mechanism of the initial phagocytosis phases (attraction and submersion). Phagocytosis experiments with washed bacteria and washed rabbit leukocytes demonstrated a marked direct dependence of the electrophoretic velocities of escherichia and the intensity of their phagocytic ingestion, and a strong reverse relationship between the extent of hydration and the phagocytic activity. Treatment of bacteria with rabbit plasma with subsequent washing led to a significant reduction of hydration and to an insignificant reduction of the electrophoretic velocities with parallel increase of the phagocyte activity. Correlation between the phagocytosis intensity and the acquired physico-chemical properties of bacteria became weak.     

The unsuitability of the uridine incorporation assay for the measurement of phagocytosis of Escherichia coli

The uridine incorporation technique for assaying phagocytosis is based on the fact that polymorphonuclear leucocytes are impermeable to labelled uridine, and therefore ingested bacteria inside phagocytic vacuoles will be unable to take it up. Extracellular bacteria, including those adherent to the phagocytic cell surface, can do so however. Differences in uptake between bacteria alone and in the presence of phagocytic cells can be used to measure ingestion. The present paper describes the application of this technique to Escherichia coli O-86 as the test organism. It appears that with this test species, the method is unsuccessful, because exposure of the non-ingested bacteria to some soluble product of the triggered polymorphonuclear leucocytes causes a large increase in their uridine uptake rates, over that of the control bacteria. The nature of the product responsible is unknown. It is unconnected with change in the pH of the medium, is heat stable, and is only produced by polymorphonuclear leucocytes which are actively phagocytosing. It may be that a release of phagolysosome contents is responsible.     

Physicochemical consequences of opsonization: perturbation of liposomal membranes by Salmonella typhimurium 395 MS opsonized with IgG antibodies.

When phagocytosis-resistant Salmonella typhimurium 395 MS bacteria sensitized with anti-MS IgG antibodies were incubated with liposomes composed of phosphatidylcholine, cholesterol, and dicetylphosphate, a previously sequestered liposomal marker (4-methylumbelliferylphosphate) was released. Unsensitized or F(ab')2-sensitized bacteria had no such effect. This perturbation was neither dependent upon negatively charged dicetylphosphate nor upon cholesterol. It was further evident that palmitoyl-poly(ethyleneglycol) caused release of the trapped marker in a similar way as sensitized bacteria. These findings demonstrate a similarity between sensitized bacteria and a hydrophobic probe and lend support to the hypothesis that the perturbation was brought about by hydrophobic interaction. The observations indicate that liposomes, like phagocytes, possess "receptor sites" for the activated part of IgG and raise the possibility that phagocytic effectors can operate in a relatively nonspecific manner.     

The specificity of immune priming in silkworm,Bombyx mori,is mediated by the phagocytic ability of granular cells

In the past decade, the phenomenon of immune priming was documented in many invertebrates in a large number of studies; however, in most of these studies, behavioral evidence was used to identify the immune priming. The underlying mechanism and the degree of specificity of the priming response remain unclear. We studied the mechanism of immune priming in the larvae of the silkworm, Bombyx mori, and analyzed the specificity of the priming response using two closely related Gram-negative pathogenic bacteria (Photorhabdus luminescens TT01 and P. luminescens H06) and one Gram-positive pathogenic bacterium (Bacillus thuringiensis HD-1). Primed with heat-killed bacteria, the B. mori larvae were more likely to survive subsequent homologous exposure (the identical bacteria used in the priming and in the subsequent challenge) than heterologous (different bacteria used in the priming and subsequent exposure) exposure to live bacteria. This result indicated that the B. mori larvae possessed a strong immune priming response and revealed a degree of specificity to TT01, H06 and HD-1 bacteria. The degree of enhanced immune protection was positively correlated with the level of phagocytic ability of the granular cells and the antibacterial activity of the cell-free hemolymph. Moreover, the granular cells of the immune-primed larvae increased the phagocytosis of a previously encountered bacterial strain compared with other bacteria. Thus, the enhanced immune protection of the B. mori larvae after priming was mediated by the phagocytic ability of the granular cells and the antibacterial activity of the hemolymph; the specificity of the priming response was primarily attributed to the phagocytosis of bacteria by the granular cells.     

The professional phagocytes of sea bass (Dicentrarchus labrax L.): cytochemical characterisation of neutrophils and macrophages in the normal and inflamed peritoneal cavity

In order to identify the phagocytic cells of sea bass, the peritoneal leucocyte population of fish injected intraperitoneally with Photobacterium damselae subspecies piscicida was studied by light microscopy using cytocentrifuge preparations stained by the Antonow technique for peroxidase detection. Among the leucocytes present in the peritoneal exudate of the infected fish (macrophages, neutrophils, eosinophilic granular cells, lymphocytes and thrombocytes), macrophages and neutrophils were the only phagocytic cells. Neutrophils were easily distinguished from macrophages in Antonow stained preparations by the pattern of peroxidase positivity. Using ultrastructural cytochemistry, neutrophils were found to have abundant cytoplasmic granules positive for peroxidase and arylsulphatase and were negative for alpha-naphthyl butyrate (ANB) esterase. In contrast, ANB esterase activity was detected in macrophages. These leucocytes were typically negative for peroxidase, but ocasionally, some macrophages with peroxidase or arylsulphatase-positive vacuoles were observed. Both phagocytes had cytoplasmic granules positive for acid phosphatase. Glycogen particles were found in the cytoplasm of the two phagocytic cells, but they were much more abundant in neutrophils. Macrophages were much more abundant than neutrophils in the peritoneal cavity of non-injected sea bass but early after the intraperitoneal injection of bacteria, the number of neutrophils increased quickly and extensively. Higher numbers of intraperitoneally injected bacteria were found inside macrophages as compared to neutrophils because macrophages strongly predominated in the peritoneal population at the time of injection. However, when the bacteria were injected into peritoneal cavities with high numbers of neutrophils (attracted by a previous injection of 12% casein), the percentage of neutrophils with phagocytosed bacteria increased, approaching that of infected macrophages. Taken together, these results show that in sea bass, as in many other organisms, in addition to macrophages, neutrophils are important phagocytic cells, the relative participation of each of the two phagocytes in defense mechanisms against infection depending on the opportunity to encounter the invading infectious agents.     

Sex differences in the impact of ozone on survival and alveolar macrophage function of mice after Klebsiella pneumoniae infection

Background

Sex differences have been described in a number of pulmonary diseases. However, the impact of ozone exposure followed by pneumonia infection on sex-related survival and macrophage function have not been reported. The purpose of this study was to determine whether ozone exposure differentially affects: 1) survival of male and female mice infected with Klebsiella pneumoniae, and 2) the phagocytic ability of macrophages from these mice.

Methods

Male and female C57BL/6 mice were exposed to O₃ or to filtered air (FA) (control) and then infected intratracheally with K. pneumoniae bacteria. Survival was monitored over a 14-day period, and the ability of alveolar macrophages to phagocytize the pathogen in vivo was investigated after 1 h.

Results

1) Both male and female mice exposed to O₃ are significantly more susceptible to K. pneumoniae infection than mice treated with FA; 2) although females appeared to be more resistant to K. pneumoniae than males, O₃ exposure significantly increased the susceptibility of females to K. pneumoniae infection to a greater degree than males; 3) alveolar macrophages from O₃-exposed male and female mice have impaired phagocytic ability compared to macrophages from FA-exposed mice; and 4) the O₃-dependent reduction in phagocytic ability is greater in female mice.

Conclusion

O₃ exposure reduces the ability of mice to survive K. pneumoniae infection and the reduced phagocytic ability of alveolar macrophages may be one of the contributing factors. Both events are significantly more pronounced in female mice following exposure to the environmental pollutant, ozone.     

Differentiation of immune cells challenged by bacteria in the common European starfish,Asterias rubens (Echinodermata)

Amoebocytes are the main effector cells of the echinoderm immune system. In starfishes, a taxon in which bacterial diseases have been rarely reported, amoebocytes are considered to be the only circulating and immune cell type. The present paper addresses the question of amoebocyte differentiation in the starfish Asterias rubens when challenged by bacteria. Starfishes were injected with FITC-coupled bacteria (Micrococcus luteus). Amoebocytes were collected at regular time intervals for 24 h. The cytometric characteristics and the phagocytic activity were studied by flow cytometry. Three amoebocyte groups of different size were identified. The cell concentrations of the two largest and more numerous of these groups (G2 and G3) were modulated by immune stimulation while the group of smallest, less numerous, cells (G1) was unaffected. All of these cell groups were phagocytic but their kinetics of cell activation and bacteria ingestion differed. G1 cells showed the lowest phagocytic activity while G3 cells had the highest and fastest phagocytic activity. Starfish amoebocytes appear to be segregated in three groups, two of them (G2 and G3) being immunomodulated and one of them presenting a very fast reaction to bacteria. It is suggested that the high efficiency of the immune system in starfishes is related to this fast reaction.     

Methodological aspects of assessing phagocytosis of Vibrio anguillarum by leucocytes of gilthead seabream (Sparus aurata L.) by flow cytometry and electron microscopy

In this paper we optimize a flow cytometric method for evaluating the phagocytic activity of leucocytes in gilthead seabream (Sparus aurata L.) and characterize the phagocytic cells observed. Optimal conditions were established for the fluorescein-labelling and analysis of the bacterium Vibrio anguillarum by flow cytometry. Head-kidney leucocytes were incubated with the heat-killed fluorescein isothiocyanate (FITC)-labelled bacteria for different periods, during which the kinetics of phagocytosis was studied. Attached and interiorized bacteria were distinguished. Although phagocytic ability reached a maximum after 60 min, phagocytic capacity reached its maximum at 20 min. The amount of ingested bacteria per phagocyte was estimated from the mean fluorescence of the leucocytes. Cytochalasin B or colchicine was used to inhibit phagocytosis. Monocyte-macrophages and acidophilic granulocytes showed phagocytic activity as demonstrated by transmission electron microscopy. In conclusion, the technique presented allows the screening of thousands of cells, and individual cell evaluation, by quantifying interiorized particles in fish phagocytes. Our ultrastructural results demonstrate that V. anguillarum is actively phagocytized by seabream macrophages and acidophilic granulocytes.     

Recognition of pathogenic microbes by the Drosophila phagocytic pattern recognition receptor Eater

Non-opsonic phagocytosis is a primordial form of pathogen recognition that is mediated by the direct interaction of phagocytic receptors with microbial surfaces. In the fruit fly Drosophila melanogaster, the EGF-like repeat containing scavenger receptor Eater is expressed by phagocytes and is required to survive infections with gram-positive and gram-negative bacteria. However, the mechanisms by which this receptor recognizes different types of bacteria are poorly understood. To address this problem, we generated a soluble, Fc-tagged receptor variant of Eater comprising the N-terminal 199 amino acids including four EGF-like repeats. We first established that Eater-Fc displayed specific binding to broad yet distinct classes of heat- or ethanol-inactivated microbes and behaved similarly to the membrane-bound, full-length Eater receptor. We then used Eater-Fc as a tool to probe Eater binding to the surface of live bacteria. Eater-Fc bound equally well to naive or inactivated Staphylococcus aureus or Enterococcus faecalis, suggesting that in vivo, Eater directly targets live gram-positive bacteria, enabling their phagocytic clearance and destruction. By contrast, Eater-Fc was unable to interact with live, naive gram-negative bacteria (Escherichia coli, Serratia marcescens, and Pseudomonas aeruginosa). For these bacteria, Eater-Fc binding required membrane-disrupting treatments. Furthermore, we found that cecropin A, a cationic, membrane-disrupting antimicrobial peptide, could promote Eater-Fc binding to live E. coli, even at sublethal concentrations. These results suggest a previously unrecognized mechanism by which antimicrobial peptides cooperate with phagocytic receptors to extend the range of microbes that can be targeted by a single, germline-encoded receptor.     

Thein vitro phagocytic activity of guinea-pig macrophages toSalmonella typhi andSalmonella enteritidis

The engulfing, bactericidal and degrading activities toSalmonella typhi, strain ty2-4446 and 0-901 and toSalmonella enteritidis of guinea pig macrophages obtained from peritoneal exudate, spleen and bone marrow that were cultivated for 2–7 days, were studied. The phagocytic activity was expressed as a total number of phagocytosed microbes and the number of viable bacteria, released from mechanically disrupted macrophages. The ratio of phagocytosed bacteria to the original number of bacteria that were introduced to macrophage cultures, were evaluated in per cents. No significant difference in phagocytic activity was found between macrophages submitted to thein vitro cultivation and macrophages freshly isolated from the organism. Profound variations in phagocytic activity of cells were found which were partially dependent on the dose of microbes employed for the infection of cultures. Furthermore, both the engulfing and bactericidal activity of peritoneal macrophages toSalmonella typhi were found to be higher than in bone morrow macrophages.Salmonella typhi 0-901 microbes were phagocytosed by macrophages from bone marrow and peritoneal exudate much better thanSalmonella typhi ty2. In addition, a significant delay in bactericidal activity toSalmonella typhi ty2 of bone marrow macrophages in comparison to peritoneal macrophages was observed. The spleen macrophages possessed better phagocytic and killing activity toSalmonella enteritidis than bone marrow macrophages. A striking difference was found as regards the intracellular growth ofSalmonella typhi andSalmonella gertneri: no multiplication ofSalmonella typhi within the peritoneal and bone marrow macrophages was observed during the 3–5 h cultivation, whereas on the other hand,Salmonella gertneri started to grow intracellularly within the 5 h cultivation in the bone marrow macrophages.     

The dps gene of symbiotic "Candidatus Legionella jeonii" in Amoeba proteus responds to hydrogen peroxide and phagocytosis

To survive in host cells, intracellular pathogens or symbiotic bacteria require protective mechanisms to overcome the oxidative stress generated by phagocytic activities of the host. By genomic library tagging, we cloned a dps (stands for DNA-binding protein from starved cells) gene of the symbiotic "Candidatus Legionella jeonii" organism (called the X bacterium) (dps(X)) that grows in Amoeba proteus. The gene encodes a 17-kDa protein (pI 5.19) with 91% homology to Dps and DNA-binding ferritin-like proteins of other organisms. The cloned gene complemented the dps mutant of Escherichia coli and conferred resistance to hydrogen peroxide. Dps(X) proteins purified from E. coli transformed with the dps(X) gene were in oligomeric form, formed a complex with pBlueskript SKII DNA, and protected the DNA from DNase I digestion and H(2)O(2)-mediated damage. The expression of the dps(X) gene in "Candidatus Legionella jeonii" was enhanced when the host amoeba was treated with 2 mM H(2)O(2) and by phagocytic activities of the host cell. These results suggested that the Dps protein has a function protective of the bacterial DNA and that its gene expression responds to oxidative stress generated by phagocytic activities of the host cell. With regard to the fact that invasion of Legionella sp. into respiratory phagocytic cells causes pneumonia in mammals, further characterization of dps(X) expression in the Legionella sp. that multiplies in a protozoan host in the natural environment may provide valuable information toward understanding the protective mechanisms of intracellular pathogens.     

Step-wise loss of bacterial flagellar torsion confers progressive phagocytic evasion

Phagocytosis of bacteria by innate immune cells is a primary method of bacterial clearance during infection. However, the mechanisms by which the host cell recognizes bacteria and consequentially initiates phagocytosis are largely unclear. Previous studies of the bacterium Pseudomonas aeruginosa have indicated that bacterial flagella and flagellar motility play an important role in colonization of the host and, importantly, that loss of flagellar motility enables phagocytic evasion. Here we use molecular, cellular, and genetic methods to provide the first formal evidence that phagocytic cells recognize bacterial motility rather than flagella and initiate phagocytosis in response to this motility. We demonstrate that deletion of genes coding for the flagellar stator complex, which results in non-swimming bacteria that retain an initial flagellar structure, confers resistance to phagocytic binding and ingestion in several species of the gamma proteobacterial group of Gram-negative bacteria, indicative of a shared strategy for phagocytic evasion. Furthermore, we show for the first time that susceptibility to phagocytosis in swimming bacteria is proportional to mot gene function and, consequently, flagellar rotation since complementary genetically- and biochemically-modulated incremental decreases in flagellar motility result in corresponding and proportional phagocytic evasion. These findings identify that phagocytic cells respond to flagellar movement, which represents a novel mechanism for non-opsonized phagocytic recognition of pathogenic bacteria.