两种不同培养基对巨噬细胞培养的影响

以无血清RPMI-1640培养液和DMEM培养液分别灌洗小鼠腹腔,10min后分别吸出灌洗液于100mL／L胎牛血清和DMEM培养液中培养．巨噬细胞吞噬试验检测其活性、台盼蓝测定体外培养细胞的存活率和成层率。结果表明．DMEM培养基体外培养的巨噬细胞存活率和成层率高、吞噬能力强,与RP—MI-1640相比,DMEM可作为一种简单而实用的体外分离培养巨噬细胞的培养基。     

Phagocytosis and destruction of Staphylococcus aureus

Summary

A review is presented of the phagocytosis and intracellular killing of Staphylococcus aureus by polymorphonuclear and mononuclear leukocytes. Recruitment of adequate numbers of leukocytes to the site of infection occurs through the process of chemotaxis. Recognition of invading staphylococci by the phagocytic cells is mediated through bacterial opsonization. Both processess depend upon the activation of the heat‐labile complement system which generates the majority of chemotactic (C5a) and opsonic (C3b) molecules for S. aureus phagocytosis. The key role of peptidoglycan in the cell wall of staphylococci in these events is stressed. Attachment and ingestion of opsonized staphylococci occurs via poorly‐defined receptors for opsonins in the membrane of the leukocyte. The greater phagocytic capacity of neutrophils as compared to monocytes is not reflected in differences in their membrane receptors for staphylococcal opsonins. Once ingested, staphylococci are rapidly destroyed by oxygen‐dependent and oxygen‐independent bactericidal mechanisms of the phagocytes. Small numbers of S. aureus may survive within the leukocyte. Special attention is focused on the numerous ways S. aureus is able to hinder, evade, and directly damage the phagocytic defense mechanisms of the host.     

Fragment Bb of bovine complement factor B: stimulatory effect on the microbicidal activity of bovine monocytes.

We have previously shown that the Bb fragment of bovine complement factor B activates bovine monocytes and neutrophils. The activation was demonstrated by the enhanced uptake of 3H-deoxyglucose. To investigate the potential effect of fragment Bb on the microbicidal activity of bovine monocytes, a direct method was used. This method involves an initial ingestion period at 37 degrees C followed by repeated washing. The decrease in the total number of viable intracellular Staphylococcus aureus during the reincubation of the bacteria with bovine monocytes determines the intracellular killing. Maximal intracellular killing was seen when the monocytes containing the ingested S. aureus was incubated with fresh bovine serum (mean +/- SEM = 73.4 +/- 1.4%). On incubation of the monocytes, containing the ingested bacteria with heat-inactivated bovine serum, 32.5 +/- 0.7% of the intracellular bacteria were killed. When affinity-purified bovine factor Bb was added to the heat-inactivated serum, the intracellular killing capacity was almost restored (65.8 +/- 1.5%). When monocytes were incubated with medium alone, they killed 22.4% of the intracellular microorganisms. When fragment Bb (25 micrograms/mL) was added to the medium, the intracellular killing of S. aureus doubled (46 +/- 1.29%). We conclude that the Bb fragment of bovine complement factor B stimulates bovine monocytes in their microbicidal activity.     

A simple method for the in vitro determination of phagocytic activity of bovine polymorphonuclear leukocytes

A simple method for the in vitro determination of the phagocytic activity of bovine polymorphonuclear leukocytes (PMNL) is described. An enriched PMNL population was obtained from peripheral blood of healthy cattle and mixed with opsonized Candida utilis or with opsonized bovine red blood cells on BSA treated coverglass slides. The phagocytosis and killing of C. utilis was determined simultaneously on the coverglass slides stained with acridine orange. Phagocytosis of bovine erythrocytes was determined by microscopic examination of the Wright-Giemsa stained slides. This method is appropriate under limited laboratory conditions because it does not require highly sophisticated equipment and materials, it is easy and rapid to perform, reproducible and inexpensive. Therefore, it could be used as a first evaluation of the phagocytic activity of bovine PMNL.     

Long-term storage of blood prior to whole-blood lymphocyte culturing

Studies were conducted to investigate the effect on blastogenesis of long-term storage of blood prior to whole-blood lymphocyte culturing. Peripheral blood from normal cattle was utilized. Blood from each animal was divided into 2 parts, A and B, following collection. Part A was left intact while RPMI-1640 culture medium was added to part B immediately following collection. Both parts were then kept at room temperature for a total of 7 days and a portion of each blood was tested every day. The cultures were incubated and assayed for [³H] thymidine incorporation into their DNA. It was observed that intact blood (A) was good for 4 days but deteriorated thereafter. Addition of RPMI-1640 to B prolonged the keeping quality of blood for 7 days prior to culturing. The possible application of these findings are discussed.     

Decreased neutrophil bactericidal activity during phagocytosis of a slime-producing Staphylococcus aureus strain

Phagocytosis and intracellular killing by bovine polymorphonuclear leukocytes (PMN) are important host defence mechanisms against mastitis caused by Staphlylococcus aureus. We compared the phagocytosis and overall killing of a non slime-producing (NSP) S. aureus and its slime-producing (SP) variant by blood PMN, using an in vitro bacteriological assay. Seven clinically healthy Holstein-Friesian dairy cows in mid-lactation stage were used for this purpose. The percentages of overall killing for the NSP and SP variant were 34+/-3% and 21+/-4% (P < 0.05) and the corresponding percentages of phagocytosis were 40+/-4% and 31+/-4%, respectively. A significant positive correlation (r = 0.79; P < 0.001) was found between phagocytosis and overall killing. These results suggest that the presence of slime was responsible for a decreased phagocytic ingestion and overall killing.     

多房棘球绦虫-泡球蚴的体外培养研究

按照多房棘球绦虫幼虫-泡球蚴培养的培养基（RPMI-1640、M199和MEM）分为3组：Ⅰ组为含10％胎牛血清的RPMI-1640;Ⅱ组为含10％胎牛血清的MEM;HI组为含10％胎牛血清的M199。将泡球蚴在3种细胞培养液中进行培养,观察其存活、生长以及发育情况。结果显示,培养9d的泡球蚴的成活率分别为：Ⅰ组90．10％、Ⅱ组50．25％、Ⅲ组22．03％;成囊率分别为：Ⅰ组57．12％、Ⅱ组63．15％、Ⅲ组48．17％;头节外翻率分别为：Ⅰ组98．28％、Ⅱ组88．65％、Ⅲ组75．50％。可见,大多数虫体在早期向囊发育,一部分虫体头节外翻,并伴有规律的伸缩运动,但随时间的延长虫体运动减缓,又向囊蚴发育。通过对多房棘球绦虫泡球蚴的体外培养,初步表明合有10％小牛血清的细胞培养基RPMI-1640较适合泡球蚴的生长发育,为研究寄生虫发育提供了最基本的数据资料。     

Polymorphonuclear neutrophil leukocyte function in clinical bovine patients and in cows with or withoutStaphylococcus aureus mastitis

A fluorochrome microassay was used to investigate peripheral blood polymorphonuclear leukocyte (PMNL) function in cattle. Glass-adherent PMNL were reacted withStaphylococcus aureus princubated in 20% bovine serum for 30, 60 and 90 min. Coverslips were stained with acridine organge (AO) followed by crystal violet to quench extracellular bacterial fluorescence. PMNL function was evaluated by counting the number of dead (stained red with AO) and live (stained green with AO)S. aureus contained within 100 PMNL. A phagocytic index was calculated as the average number of bacteria contained within PMNL. The percentage killing ofS. aureus was calculated from the average proportion ofS. aureus within PMNL that were dead.Six clinically normal Holstein calves, 3–4 months of age, were sampled on 6 consecutive days. PMNL phagocytosis and killing did not vary significantly (p>0.05) among repeated samplings per calf. PMNL function increased with increasing time of incubation of PMNL withS. aureus. Means (± SD) for percentage killing were 46.7±13.1, 57.4±11.6, and 62.1±9.8% for 30, 60 and 90 min of reaction, respectively. Means (± SD) for the phagocytic index were 2.9±0.8, 3.6±1.0, and 4.2±1.1 bacteria/PMNL for 30, 60 and 90 min of reaction, respectively. PMNL function was determined in 30 normal cattle of various breeds, age and sex, and these values were pooled to provide normal values for PMNL function.When values for bovine clinical patients (n=25) with various diagnoses were compared with normal values (defined by the mean ± 2SD for the 30 normal cattle) for PMNL function, only one patient was observed to exhibit PMNL hypofunction. A cow with disseminated intravascular coagulation in association with peracute coliform mastitis exhibited decreased PMNL killing capacity. Abnormal PMNL function was uncommon in the hospital population studied.Peripheral blood PMNL function was evaluated in lactating Holstein cows with (n=15) or without (n=15) chronic subclinicalS. aureus mastitis. There was no significant (p>0.05) difference in PMNL function among these cows.     

Effect of antibiotics and vehicles on bovine mammary polymorphonuclear leukocyte morphologic features, viability, and phagocytic activity in vitro

Effects of antibiotics and antibiotic vehicles on polymorphonuclear leukocytes (PMNL) isolated from bovine mammary glands were studied in vitro. Amikacin, dicloxacillin, erythromycin, gentamicin, lincomycin, nitrofurantoin, novobiocin-penicillin, polymyxin B, rifampin, tetracycline, or tiamulin was added to culture medium at 1 mg/ml and chloramphenicol was added at 4 mg/ml. Drug concentrations were equivalent to those detected in milk immediately after injection into the mammary gland. Vehicles included mineral oil and peanut oil, each at a dilution of 1:100 in culture medium. The PMNL morphologic features, viability, and phagocytic activity were evaluated. In comparison with the phosphate-buffered saline solution (PBSS) control, significant (P less than 0.05) alterations in normal cell morphologic features were observed in PMNL cultured with tetracycline, nitrofurantoin, erythromycin, polymyxin B, rifampin, novobiocin-penicillin, chloramphenicol, tiamulin, or peanut oil. Viabilities of PMNL cultured with chloramphenicol, novobiocin-penicillin, or tiamulin were significantly (P less than 0.05) reduced when compared with those of PBSS controls. Addition of Staphylococcus aureus to culture medium enhanced morphologic alterations and reduced viabilities of PMNL. Phagocytosis of S aureus by PMNL was significantly (P less than 0.05) depressed in medium containing novobiocin-penicillin, amikacin, rifampin, chloramphenicol, tiamulin, or peanut oil in comparison with that of PMNL incubated in PBSS.     

Development of a migration inhibitory factor assay under agarose of bovine mononuclear leukocytes, using an antigen of Brucella abortus

A study was conducted to develop a migration inhibitory factor assay under agarose of bovine mononuclear leukocytes, with an antigen of Brucella abortus. Different concentrations of mononuclear leukocytes were prepared by the Ficoll-Hypaque technique from the blood of nonvaccinated calves and from calves previously vaccinated with strain 19. Concentrations of 0.5, 1, 2, 3, 4, and 5 x 10(6) leukocytes were suspended in RPMI-1640 medium and various dilutions (20, 10, 1, and 0.1 microgram) of B abortus-soluble antigen, dispensed in triplicate wells cut in 1% agarose containing minimal essential medium and 10% bovine fetal serum. These agarose plates were incubated for 4-, 8-, 12-, 16-, 20-, and 24-hour periods and then were fixed; leukocytes were stained with Wright's stain. Migration distances were measured, and statistical analyses of the data revealed a concentration of 2 x 10(6) cells/well and an antigen concentration of 10 microgram/well. An incubation period of 20 hours was optimal for the assay.     

In vitro effects of beta-hydroxy-beta-methylbutyrate (HMB) on cell-mediated immunity in fish

beta-Hydroxy-beta-methyl butyrate(HMB) has been shown to counteract many of the negative effects of intensive animal production methods and results in increased growth and protection against diseases. In the present study, the effect of HMB on the immunocompetence cell activity in rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio) was examined. Pronephric phagocytes and lymphocytes were isolated from the fish and grown in culture medium (RPMI-1640) containing either 0, 0.1, 1, 5, 10, 25, 50 or 100 microg HMB/ml of medium. The effects of HMB on the respiratory burst activity (RBA) stimulated by phorbol myristate acetate (PMA), the potential killing activity (PKA) and lymphocyte proliferation stimulated by either concanavalin A (Con-A) or lipopolysaccharide (LPS) were examined. The addition of HMB to the culture medium increased the RBA by up to 84% (p<0.01) over that of cells grown without HMB. Similarly, the PKA of the phagocytes was also increased with HMB addition to the medium by up to 140% (p<0.01) over that of cells grown without HMB. Lymphocyte proliferation stimulated by both ConA and LPS was also increased approximately two-fold (p<0.01) when HMB was added to the culture medium at concentrations between 10 and 100 microg HMB/ml in both rainbow trout and carp. The greatest effects of HMB on RBA and PKA activities were observed at a concentration >50 microg HMB/ml while lymphocyte proliferation was maximally stimulated at 25 microg HMB/ml. In conclusion, the current study shows that HMB could potentially improve immunocompetence cell activity in fish through increased cell proliferation and functionality.     

Phagocytic and bactericidal activity of blood and milk-resident neutrophils against Staphylococcus aureus in primiparous and multiparous cows during early lactation

To examine the effect of parity on polymorphonuclear neutrophils (PMN) function, phagocytic and bactericidal activity of the PMN isolated from blood and milk against Staphylococcus aureus was compared between groups of 6 primiparous and 6 multiparous healthy dairy cows during early lactation using bacteriological and PMN-pathogen interaction assays. Latex-stimulated luminol-amplified chemiluminescence (CL) and viability of these PMN were also investigated. The phagocytosis and killing of S. aureus by blood were remarkably higher than those of milk PMN. Similarly, the CL and viability in blood PMN were markedly higher than in milk PMN. Both in blood and in milk the phagocytosis of S. aureus by PMN in primiparous cows was substantially higher than in multiparous cows. The killing activity of blood PMN against S. aureus was 42.3+/-3.4% and 23.2+/-1.7% in primiparous and multiparous, respectively. Milk PMN killed only 20.7+/-2% S. aureus in primiparous and 10.2+/-1.3% in multiparous cows. Blood and milk PMN CL and milk PMN viability were significantly higher in primiparous cows. The pronounced reduction in phagocytic and bactericidal activity in blood and milk-resident PMN from multiparous cows, in part, resulted from the pronounced decrease of PMN viability and free radicals production capacity; this suggests that heifers' udders could be more protected against S. aureus, which remains to be tested in the field.     

Continuous in vitro culture of erythrocytic stages of Babesia gibsoni and virulence of the cultivated parasite

Babesia gibsoni infected erythrocytes were collected from the blood of an experimentally infected dog. The parasite isolated could be continuously cultivated in vitro, with an average parasitemia of 18.2 +/- 2.4% on day 3 of culture, in RPMI-1640 medium supplemented with 7.5% normal dog serum in a humidified atmosphere containing 5% CO(2) at 37 degrees C. The parasites in the original culture were morphologically similar to those found in the peripheral blood of dogs, however, on the 4th generation of subculture, the large oval parasites, erythrocytes including many parasites and extracellular parasites were frequently observed. The B. gibsoni isolate was injected to the dog to test its infectivity after maintained in vitro for 738 days at the 214th subculture. The cultivated parasite did not cause a severe clinical sign in the dog.     

硫酸化20（S）-人参皂苷Rh2体外对鸡免疫活性细胞功能的影响

为了评价硫酸化人参皂苷Rh2化学修饰前后免疫活性变化，采用MTT法及流式细胞术研究体外对ConA促鸡外周血淋巴细胞增殖的影响，采用乳酸脱氢酶释放法研究体外对鸡外周血NK细胞杀伤活性的影响。结果表明：Rh2、Rh2溶剂及硫酸化Rh2低浓度时都能促进淋巴细胞增殖，高浓度时能抑制淋巴细胞增殖，Rh2溶剂及硫酸化Rh2都显著（P〈0．05）或极显著（P〈0．01）高于同浓度的Rh2。Rh2及Rh2溶剂都能显著促进鸡外周血NK细胞杀伤活性，但Rh2极显著（P〈0．01）低于同浓度Rh2溶剂，而硫酸化Rh2表现一定促进作用。结果提示：Rb2体外能抑制免疫活性，化学修饰后的硫酸化Rh，体外能促进免疫活性。     

Response of heifer mammary gland macrophages and neutrophils to interferon-gamma stimulation in vitro.

The phagocytic and killing abilities of heifer mammary gland macrophages (M phi) and neutrophils were evaluated after exposure to recombinant bovine interferon-gamma (rBoIFN-gamma) stimulation in vitro. Macrophages or neutrophils were cultured for 2 h with 0, 10(2), 10(4) and 10(5) units rBoIFN-gamma/mL. Phagocytosis assays were performed by incubation with Staphylococcus aureus at a leukocyte:bacteria ratio of 1:10. After 45 min, cells were stained with acridine-orange and phagocytic and killing abilities were determined. Although rBoIFN-gamma had no effect on M phi phagocytic activity, neutrophil phagocytic activity after incubation in 10(4) units rBoIFN-gamma (41.62%) was significantly higher than 0 (25.24%) or 10(2) units rBoIFN-gamma (24.73%). Neutrophil and M Phi killing abilities were not affected by any dose of rBoIFN-gamma. Results suggested that rBoIFN-gamma promoted neutrophil phagocytic activity, but did not affect neutrophil killing or overall M phi function in vitro.     

In vitro phagocytosis and killing of Corynebacterium equi by alveolar macrophages of foals

Bronchoalveolar lavage was performed 5 times, sequentially, on 3 healthy foals while each foal was 6 to 63 days of age. Phagocytosis and bactericidal assays were performed on recovered alveolar macrophages. Corynebacterium equi and alveolar macrophages at a ratio of 10:1 were incubated for 1 hour in medium containing 1% heat-inactivated rabbit anti-C equi serum. After incubation, greater than 90% of the alveolar macrophages contained at least 1 ingested bacterium and each alveolar macrophage contained 9.4 +/- 1.0 bacteria (mean +/- SE). After alveolar macrophages and C equi were incubated for 1 hour in medium containing heat-inactivated pooled normal horse serum, approximately 24% of the alveolar macrophages contained at least 1 bacterium and each alveolar macrophage contained 0.8 +/- 0.7 bacteria. From 6 to 61 days of age, each foal had significantly (P less than 0.05) decreased phagocytic activity by alveolar macrophages, but a significant change in killing of C equi by alveolar macrophages was not found in the foals from 21 to 61 days of age. After incubating alveolar macrophages and C equi for 4 hours in vitro, approximately 75% of ingested C equi remained viable.     

Phagocytic and bactericidal properties of bovine macrophages from non-lactating mammary glands

Macrophages were isolated from the mammary glands of non-lactating (dry) cows and their ability to phagocytose and kill staphylococci in vitro assessed. Normal bovine serum enhanced the uptake of staphylococci and was required for optimal killing in the bactericidal test. Dry gland secretion interfered with uptake. Secretions taken progressively into the dry period became more inhibitory. The phagocytic ability of macrophages was significantly less than that of neutrophils present in the same gland preparation when tested in the presence of dry gland secretion. A marked variation in the antibacterial activity of macrophages from different cows was noted.     

Phagocytosis and intracellular killing of Pasteurella multocida by porcine alveolar macrophages after infection with pseudorabies virus

The purpose of this study was to determine if pseudorabies virus (PrV) interfered with normal alveolar macrophage phagocytic functions. Porcine alveolar macrophages (PAM) obtained by pulmonary lavage were exposed to PrV. At 1 hour postinfection, cells were challenged with Pasteurella multocida labeled with 3[H] thymidine. The phagocytosis assay was performed by measuring total radioactivity 1 hour after Pm challenge in a soft-beta spectrophotometer. Intracellular killing was measured by counting viable bacteria 3 hours after P. multocida challenge. Phagocytic values of PrV-infected and control PAM ranged from 11% to 20%, a non-significant difference. Values for intracellular killing for PrV-infected PAM ranged from 7.1 X 10(5) to 1 X 10(6) in contrast to 5.1 X 10(1) to 1.8 X 10(2) for the control PAM. This difference in killing function was significantly lower in PrV-infected PAM than in control cells (P less than 0.01). This alteration of macrophage function may be a factor in the pathogenesis of PrV-Pm mediated pneumonia in pigs.     

The interaction of Rhodococcus equi and foal neutrophils in vitro

Polymorphonuclear neutrophil leukocytes (PMNL) from 8 healthy foals (2-14 weeks of age) and 2 foals with bacterial pneumonia were separated from whole blood using a 2 step Percoll gradient. Purified PMNL were tested for bactericidal function against Rhodococcus equi and Staphylococcus aureus in the presence of normal horse serum. The percentage uptake after a 15-min pre-incubation of PMNL and bacteria was also calculated. Ultrastructural examination of the interaction of R. equi and normal foal PMNL was performed after 15 min incubation. Results indicated that foal PMNL effectively phagocytose and destroy R. equi and S. aureus in the presence of normal horse serum. The mean percent uptake for R. equi was 99.3 +/- 0.4% and for S. aureus 99.9 +/- 0.1%. Further, 97.8 +/- 0.1% ingested R. equi and 98.4 +/- 0.1% ingested S. aureus were destroyed in the 15-min incubation period. Over the 3-h incubation, 91.9% of remaining R. equi were killed, but only 49.2 +/- 31.9% of S. aureus (P less than 0.01). Total bactericidal effect of foal PMNL, however, was 99.3 +/- 0.4% against R. equi and 99.9 +/- 0.1% against S. aureus. The percentage uptake and total bactericidal efficacy of neutrophils from sick foals was greater than 95%. Ultrastructural examination of the PMNL-R. equi interaction after 15 min incubation revealed phagocytosis of the bacteria and morphologic changes consistent with neutrophil degranulation. This study suggests that a defect in PMNL bactericidal capability is not likely to be a contributing factor in the pathogenesis of R. equi pneumonia in foals.     

The chemotactic properties of porcine seminal components toward neutrophils in vitro

Our objectives were to investigate the mechanisms of postbreeding inflammation in swine by examining the chemotactic properties of polymorphonuclear neutrophilic granulocytes (PMN) and of various populations of spermatozoa and seminal plasma. Epididymal spermatozoa from two boars obtained under sterile conditions, washed ejaculated spermatozoa from two boars, and pooled seminal plasma from eight boars of known fertility were examined for chemotaxis to PMN. The chemotaxis of blood-derived PMN in response to sperm and seminal plasma was evaluated and expressed as a percentage of a positive control (lipopolysaccharide-activated blood plasma). The mean chemotactic effect of washed sperm alone (4.4+/-0.04) and of epididymal sperm alone (3.4+/-0.06) was not different from that of the negative controls (3.1+/-0.05) of McCoy's medium with 10% heat-inactivated fetal calf serum. A marked chemotactic effect was detected when washed ejaculated and epididymal sperm were incubated with blood plasma, compared with blood plasma without spermatozoa (P < 0.001). Washed sperm in blood plasma (86.2+/-5.6) and epididymal sperm in blood plasma (83.9+/-7.7) were different from blood plasma alone (11.2+/-1.5), but no differences were detected between the two populations of sperm. This effect, however, was not completely inhibited by heat inactivation of the blood plasma. The chemotactic response of washed ejaculated and epididymal spermatozoa incubated in lipopolysaccharide-treated, heat-inactivated blood plasma were greater than that of the negative control (P < 0.05). Polymorphonuclear neutrophilic granulocyte migration toward seminal plasma was similar to the negative control (4.0+/-0.04 vs 3.1+/-0.05). It seems that porcine epididymal sperm and ejaculated sperm activate chemotactic components in porcine blood plasma and heat-inactivated blood plasma, suggesting that, at least partially, a heat-stable (noncomplement) blood plasma component may be involved in sperm-induced PMN chemotaxis. In contrast, porcine seminal plasma was not chemotactic to PMN. These results support the hypothesis that spermatozoa play an active role in initiating postbreeding endometritis.