Cell death and CD14 expression in resident and inflammatory polymorphonuclear leukocytes from virgin bovine mammary gland

This paper investigates the association between expression of CD14 and occurrence of apoptosis in blood, resident (_RESPMN) and inflammatory (_INFPMN) polymorphonuclear leukocytes (PMN) from heifer mammary glands. The fresh population of _RESPMN contained a statistically significant higher proportion of CD14+, apoptotic and necrotic cells than did populations of _INFPMN and blood PMN. In vitro cultivation of _RESPMN, _INFPMN and blood PMN led to concurrent increase of apoptotic, necrotic and CD14+ cells. A positive correlation was found between the proportions of both apoptotic and necrotic PMN and CD14+ PMN as determined by three-color flow cytometry analysis. Our study confirmed that expression of CD14 in blood PMN, _RESPMN and _INFPMN from heifer mammary glands was accompanied by apoptosis and necrosis.     

Effect of apoptosis on phagocytosis, respiratory burst and CD18 adhesion receptor expression of bovine neutrophils

Polymorphonuclear neutrophil leukocytes (PMN) play an important role in intramammary defense against infections by Escherichia coli. During mastitis, PMN are confronted with various inflammatory mediators that can modulate their function. In severely diseased cows, increased concentrations of lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha (TNF-alpha) are detected in plasma. Binding of LPS to membrane bound CD14 molecules on monocytes cause release of inflammatory mediators such as TNF-alpha. Because apoptosis of PMN promotes resolution of inflammation and because the LPS and TNF-alpha response in milk and blood is related to the severity of E. coli mastitis, the effect on apoptosis of bovine PMN of increased concentrations LPS and TNF-alpha was studied together with the functionality of apoptotic PMN.Bovine PMN apoptosis, as determined with annexin-V, was induced with high concentrations of either LPS (1000 and 10,000ng/mL) or TNF-alpha (10,000ng/mL) in whole blood following a 6h incubation at 37 degrees C. The apoptosis inducing effect of LPS on PMN was not inhibited following coculture with either anti-bovine TNF-alpha or anti-ovine CD14 monoclonal antibodies. When compared to controls, apoptotic PMN had a similar level of CD18 expression but lacked phagocytic and respiratory burst activity. This is the first study reporting the effects of apoptosis on bovine PMN function. These functional impairments in apoptotic PMN could be important in contributing to the establishment of intramammary infection. Well functioning PMN could finally determine the severity of mastitis following an invasion of bacteria in the mammary gland.     

Apoptosis and necrosis of blood and milk polymorphonuclear leukocytes in early and midlactating healthy cows.

Increased milk somatic cell counts (SCC) are used as an indicator for bovine mastitis. During mastitis, polymorphonuclear leukocytes (PMN) become the predominant cell type. Shortly after parturition, the severity of mastitis is increased and several PMN functions are downregulated. Apoptotic and necrotic processes of PMN could influence SCC and PMN functions. In this study, the percentages of apoptotic and necrotic PMN in blood and milk from early and midlactating healthy cows were compared. Apoptosis and necrosis of PMN were quantified using a dual-color flow cytometric procedure with fluorescein labeled annexin-V (green) and propidium iodide (red). Using this technique three different subpopulations of bovine PMN could be detected: apoptotic cells (high intensive green fluorescence), necrotic cells (high intensive green and high intensive red fluorescence) and viable cells (low intensive green and low intensive red fluorescence). Following a 4 h incubation of blood from both groups of cows at 37 degrees C to induce apoptosis, the mean percentage of apoptotic blood PMN was significantly higher (P < 0.01) in early lactating cows (15.1%, n = 9) compared with midlactating cows (5.3%, n = 10). The mean percentage of necrotic PMN remained lower than 5% in all cows. In contrast to blood, no significant difference was found between the percentage of apoptotic PMN in milk from early (41.2%, n = 7) and midlactating cows (34.0%, n = 8). The percentage of necrotic PMN in milk from early lactating cows (25.9%, n = 7) was significantly higher than that in midlactating cows (14.2%, n = 8) (P < 0.05). Higher percentages of apoptotic as well as necrotic PMN were consistently found in milk compared to blood in all cows. From these results, it can be concluded that spontaneously induced apoptosis was higher in blood PMN from early lactating cows than in blood PMN from midlactating cows. The higher percentage of necrotic milk PMN in early lactating cows than in midlactating cows could be explained by the induction of secondary necrosis.     

Apoptosis of polymorphonuclear leukocytes of the juvenile bovine mammary gland during induced influx

The dynamics of apoptosis of polymorphonuclear leukocytes (PMN) during induced influx of PMN into the cavity system of the juvenile bovine mammary gland in order to investigate the role of apoptosis of PMN in the resolution of mastitis was studied. The instillation of a synthetic analogue of muramyl dipeptide into teat sinus of the sixteen mammary glands was followed by a massive influx of PMN culminating after 24 h and resolving after 96 h. Every 24 h following the influx, apoptotic PMN were microscopically detected, based on morphological characteristics. Twenty four hours after the stimulation, apoptotic PMN were already observed, and peak counts of apoptotic PMN were reached 48 h after the stimulation. The lowest differential count of apoptotic PMN, corresponding to the pre-stimulation value, was found 96 h after the stimulation. The presence of macrophages (MAC) containing phagocytized apoptotic PMN was observed by histochemical staining for myeloperoxidase (MPO) and electron microscopy. The percentage of MPO-positive macrophages increased during the resolution phase to reach peak values 48 h after the stimulation. Apoptosis of PMN and phagocytosis by macrophages may represent a removal mechanism that is important in the resolution of the induced influx of PMN in the cavity system of juvenile bovine mammary gland.     

The bovine neutrophil: Structure and function in blood and milk

Migration of polymorphonuclear neutrophil leukocytes (PMN) into the mammary gland provide the first line of defense against invading mastitis pathogens. Bacteria release potent toxins that activate white blood cells and epithelial cells in the mammary gland to secrete cytokines that recruit PMN that function as phagocytes at the site of infection. While freshly migrated PMN are active phagocytes, continued exposure of PMN to inhibitory factors in milk such as fat globules and casein, leads to altered PMN morphology and reduced phagocytosis. In the course of phagocytosing and destroying invading pathogens, PMN release chemicals that not only kill the pathogens but that also cause injury to the delicate lining of the mammary gland. This will result in permanent scarring and reduced numbers of milk secretory cells. The life span of PMN is limited by the onset of apoptosis. To minimize damage to mammary tissue, PMN undergo a specialized process of programmed cell death known as apoptosis. Macrophages quickly engulf and phagocytose apoptotic PMN, thereby minimizing the release of PMN granular contents that are damaging to tissue. The PMN possess an array of cell surface receptors that allow them to adhere and migrate through endothelium and to recognize and phagocytose bacteria. One receptor found on phagocytes that is receiving considerable attention in the control of infections by Gram-negative bacteria is CD14. Binding of lipopolysaccharide (LPS) to membrane bound CD14 causes release of tumor necrosis factor-alpha and sepsis. Binding of LPS to soluble CD14 shed from CD14-bearing cells results in neutralization of LPS and rapid recruitment of PMN to the site of infection. Recent advances in the fields of genomics and proteomics should greatly enhance our understanding of the PMN role in controlling intramammary infections in ruminants. Further, manipulation of PMN, through either recombinant proteins such as soluble CD14 that enhance PMN response or agents that mediate PMN apoptosis, may serve as novel therapeutics for the treatment of mastitis.     

In vitro effect of recombinant human tumor necrosis factor-alpha on canine neutrophil apoptosis

Neutrophils (polymorphonuclear leukocytes: PMNs) are essential for the host defense against various infections and are often injurious to the host, causing inflammatory diseases where tumor necrosis factor-alpha (TNF-alpha) is suggested to play an important role. Since an effect of TNF-alpha on canine PMN apoptosis has not been studied, canine PMNs were stimulated with recombinant human (rh)TNF-alpha in the present study to investigate the effect of TNF-alpha on canine PMN apoptosis. PMN apoptosis and function to produce ROS were assessed by flow cytometry. Delayed apoptosis was observed in the PMNs treated with rhTNF-alpha at 100 ng/ml, accompanied by retention of capability to produce ROS. However, PMN apoptosis was accelerated by rhTNF-alpha combined with cycloheximide. Therefore, it is indicated that TNF-alpha is able to activate anti- and pro-apoptotic pathways in PMNs and that the inhibition of PMN apoptosis by TNF-alpha requires protein synthesis in the PMNs.     

Increase in apoptotic polymorphonuclear neutrophils in peripheral blood after intramammary infusion of Escherichia coli lipopolysaccharide

A transient increase in apoptotic polymorphonuclear neutrophils (PMNs) as revealed by the terminal deoxynucleotidyl, transferase-mediated dUTP nick end labeling (TUNEL) technique in bovine jugular and milk vein blood was observed 4 h after intramammary infusion of Escherichia coli lipopolysaccharide (LPS) (jugular vein; before infusion 10.1%, 4h 58.3%: milk vein; before infusion 13.2%, 4 h 76.6%) decrease in PMA-induced oxidative bursts of PMNs was also observed during the same period and continued until 8 h after the infusion. TUNEL-positive cells showed an intention of a Comet tail as detected by a single-cell gel electrophoresis assay (Comet assay) and the morphological apoptotic future, though DNA fragmentation was not clearly detected. A definite decrease in peripheral PMNs and a marked increase in PMNs in the LPS-infused teat cistern were observed during the same period. The migration of milk vein blood-derived PMN and the expression of adhesion receptors (L-selectin and CD18) on PMN were suppressed, accompanied by an increase in apoptotic cells. TUNEL-positive PMN observed in normal animals showed a reduced migration capacity. The increase in apoptotic PMNs observed in the LPS-infused cattle was thought to be due to the remaining intravenous spontaneous apoptotic cells existing under the normal condition (the aging cell), and this increase appeared to lower the expression of adhesion receptors and the migration capacity. Decreased PMA-induced oxidative burst activity in PMN was thought to be derived from these aging cells and immature band cells appearing in the circulation as a subsequent event of leukopenia and/or severe stress associated with mastitis. The results from the present study indicate the possibility that the function of PMN in the circulation at early stages of bovine mastitis is regulated by the kinetics of PMN aging.     

Decreased apoptotic polymorphonuclear leukocyte rate in dogs with pyometra

Polymorphonuclear neutrophil (PMN) apoptosis was examined in three dogs with pyometra by TUNEL assay in a 24-hr incubation period and compared with that in healthy control dogs (n=5). The incidence of apoptotic PMNs in dogs with pyometra was 26.4 +/- 5% and that in healthy dogs was 54.3 +/- 7%. The results indicated that apoptotic PMN rates in dogs with pyometra were significantly lower than those in control dogs (p<0.05), suggesting the prolongation of PMN survival.     

In vitro effects of very low levels of aflatoxin B₁ on free radicals production and bactericidal activity of bovine blood neutrophils

As one of the most potent and hazardous feed/food-originated mycotoxins, aflatoxin (AF) B? is regarded as a potent immunosuppressor in dairy cows. Neutrophils (PMN), as key effector cells against pathogens, have a high potential to kill engulfed microbes. To investigate the in vitro effects of very low doses of AFB? on blood PMN functions, we examined the effects of biologically relevant concentrations of AFB? on the phagocytosis and non-phagocytosis dependent luminol, representative of mainly intracellular free radicals, and isoluminol, representative of mainly extracellular free radicals, chemiluminescence (CL), necrosis and apoptosis of PMN. Isolated blood PMN from healthy dairy cows (n=12) were exposed to 0, 0.01, 0.05 and 0.5 ng/ml of AFB? for 0.5 and 18 h depending on the assay. Further, blood PMN of healthy dairy cows (n=8) were exposed to 0.5 ng/ml of AFB? for 3h and myeloperoxidase (MPO) activity, superoxide anion (O??) production, phagocytosis and killing activities against Staphylococcus (S.) aureus and Escherichia (E.) coli, were examined. Though the effect of extremely low doses of AFB? were less pronounced, at 0.5 ng/ml the production of free radicals was greatly enhanced, especially extracellularly. In contrast to isoluminol CL, the AFB?-treated PMN showed a remarkably impaired phagocytosis-depended luminol CL. PMN necrosis and apoptosis were not affected by AFB?. MPO activity, O?? production, phagocytosis rates and killing of E. coli and S. aureus by AFB?-treated PMN were significantly lower than those of non-treated ones. Our results show the extracellularly pro-oxidant and antiphagocytic properties of very low doses of AFB? for bovine PMN. The scope of the suppressive effects of the in vitro AFB? levels on cellular innate immune functions should be considered for high yielding dairy cows.     

Caprine herpesvirus-1 (CapHV-1) induces apoptosis in goat peripheral blood mononuclear cells

Programmed cell death (PCD), or apoptosis, is initiated in response to various stimuli, including virus infection. A number of studies have shown that deregulation of apoptosis is an important feature of virus-induced immunosuppression for various viral diseases. In the present study, CapHV-1 was found to cause apoptosis in mitogen-stimulated as well as nonstimulated caprine peripheral blood mononuclear cells (PBMC). Apoptotic index, as quantified by fluorescent dyes, revealed a significant increase in the percentage of apoptotic cells at 24 and 48 h postinfection as compared to their respective noninfected controls. Apoptosis specific internucleosomal laddering in DNA from CapHV-1 infected PBMC was seen in agarose gel electrophoresis. No DNA fragmentation was observed in control noninfected PBMC. Virus-induced apoptosis was reduced by Z-VAD-FMK, an aspecific caspase inhibitor, by AC-DEVD-CHO (caspase-3-specific) and AC-VEID-CHO (caspase-6-specific) treatment. PCD in CapHV-1 infected peripheral blood mononuclear cells occurs at the G0/G1 phase of the cell cycle. However, penetration of virus particles and infection was not required for PCD, as UV-inactivated CapHV-1 induced apoptosis of mitogen-stimulated bovine peripheral blood mononuclear cells in vitro.     

High sensitivity of thymocytes of LEC strain rats to induction of apoptosis by X-irradiation

It is known that physical disruption of cell contacts induces apoptosis of thymocytes. When thymocytes from LEC and WKAH rats were incubated in vitro at 37 degrees C for 0-6 hr and then the proportion of apoptotic cells was determined using a flow cytometer, it was found that the percentages of apoptotic thymocytes from both LEC and WKAH rats increased with incubation time and that the proportion of apoptotic cells from LEC rats was significantly higher than that from WKAH rats at each incubation time. The fact that cycloheximide, an inhibitor of protein synthesis, did not show significant inhibitory effects on induction of apoptosis of thymocytes indicates that induction of apoptosis during in vitro cultivation did not require de novo protein synthesis. When thymocytes from LEC and WKAH rats were X-irradiated in vitro at 4 and 8 Gy, the percentages of radiation-induced apoptotic cells increased with post-incubation time after X-irradiation in both LEC and WKAH rat thymocytes and the proportions of apoptotic cells from LEC rats were significantly higher than those from WKAH rat cells at 2 and 4 hr post-incubation after X-irradiation. When thymocytes from LEC and WKAH rats were X-irradiated in the presence of cycloheximide, the induction of apoptosis was substantially inhibited, indicating that radiation-induced apoptosis of thymocytes from LEC and WKAH rats required de novo protein synthesis. The present results showed high sensitivities of thymocytes of LEC rats to induction of apoptosis during in vitro cultivation and by X-irradiation.     

Testicular dynamics in Syrian hamsters exposed to both short photoperiod and low ambient temperature

The object of this study was to determine the details of morphological dynamics of spermatogenesis in Syrian hamsters exposed to both short photoperiod and low ambient temperature. Eight-week-old male hamsters, kept in a long photoperiod (14 h L, 10 h D), were transferred to a short photoperiod (6 h L, 18 h D) and kept there for 13 weeks to induce testicular regression. Some hamsters were then transferred from the room at 23 degrees C to that at 5 degrees C (5 degrees C group). Remaining hamsters were continuously kept at 23 degrees C (23 degrees C group). Thereafter, the morphology was examined. As a result, it took only 8 weeks until spermatogenesis recovered in the 23 degrees C group. However, it was not until 20 weeks that spermatogenesis was recognized in the 5 degrees C group. As the regulation of seasonal testicular activity is characterized by coordinated shifts in the relationships among mitosis, meiosis, and apoptosis, the changes in the proliferative and apoptotic activities were examined. Although no significant difference in proliferative activity of spermatogonia between the 5 degrees C and the 23 degrees C groups was confirmed, a notable increase in the rate of apoptosis was observed in the 5 degrees C group. Furthermore, this increase was more salient during the hibernation period. These findings suggest that both cold ambient temperature and hibernation caused the delay of testicular recrudescence and this delay arose from the increase of apoptotic activity but not the change in proliferative activity in spermatogonia in the 5 degrees C group.     

Effect of follicle-stimulating hormone on ZPC protein secretion by quail granulosa layer

In avian species, ZPC, a glycoprotein of the inner layer of the vitelline membrane, is synthesized in the granulosa cells of developing follicles. We have previously reported that follicle‐stimulating hormone (FSH) is one of the factors that stimulate ZPC production in quails. In the present study, we examined the effect of FSH on ZPC secretion, and the mode of ZPC secretion from pieces of granulosa layer. Western blot analysis demonstrated that, in the absence of FSH, the intensity of ZPC bands in the medium faded after 6 h of incubation. The addition of FSH caused an increase in intensity of ZPC bands when incubated for more than 6 h. The ZPC secretion during the first 6 h of incubation was depressed by the addition of either cycloheximide or actinomycin D. These results show that the ZPC found in the medium during short‐term incubation is newly synthesized protein and is secreted from the granulosa cells by the constitutive secretory pathway.     

In vitro study of neutrophil apoptosis in dogs

In the present study, to investigate the apoptosis of the polymorphonuclear neutrophil (PMN) from healthy dogs, we carried out TUNEL assay and DNA analysis by electrophoresis on dog PMNs. The TUNEL assay indicated that apoptotic PMNs in dogs were 0.15+/-5% before incubation, 0.3+/-5% at 4h incubation, 1+/-6% at 8h, 9+/-4% at 12h and 28+/-5% at 24h, respectively. The ladder formation was much more clearly observed in DNA from PMNs after 24h incubation at 37 degrees C than that before incubation. The results in this study indicated that healthy dog PMNs undergo apoptosis spontaneously within hours to days, and that the apoptosis of PMNs might be related to the high turnover of these circulating cells in dogs.     

Local and systemic effects of endotoxin mastitis on the chemiluminescence of milk and blood neutrophils in dairy cows

The local and systemic effects of intramammary lipopolysaccharide (LPS) injection on the chemiluminescence (CL) of milk and blood polymorphonuclear leukocytes (PMN) were investigated in six healthy early lactation cows. Clinical signs of acute mastitis such as fever, increased heart rate and a decreased milk production were observed in all cows. Before LPS challenge, the CL activity of milk PMN was significantly lower than that of blood PMN (P < 0.01). A significant negative correlation was found between pre-challenge milk and blood PMN CL and, the decreased milk production in unchallenged quarters. The CL activity of milk PMN from LPS-injected quarters increased following LPS challenge, whereas it remained unchanged in control quarters. The CL activity of blood PMN showed a biphasic increase, with two peaks and a valley below pre-challenge CL activity (P < 0.01). At post-challenge hours (PCH) 6 and 12, the CL activity of milk PMN from LPS-injected quarters exceeded that of blood PMN (P < 0.05 and P < 0.001, respectively). The decreased CL activity of blood PMN and the enhanced CL activity of milk PMN during endotoxin-induced mastitis was reflected by changes in the shape of the CL curve. In blood PMN, a decrease of the second peak of the CL curve suggests that the myeloperoxidase (MPO)-H2O2 system is impaired during endotoxin-induced mastitis. In contrast, the MPO-H2O2 system was enhanced in milk PMN from challenged quarters. The highest duration and intensity of reactive oxygen intermediate (ROI) production was observed in milk PMN from LPS-injected quarters at PCH 12. The increased viability of PMN in LPS-injected quarters and to a lesser extent in control quarters suggests possible effects of both facilitated diapedesis and inflammatory mediators on milk PMN survival. In conclusion, our results suggest that a combination of local and systemic action of E. coli endotoxin is involved in the priming of milk PMN during mastitis.     

Blood and Intrauterine Leukocyte Profile and Function in Dairy Cows that Spontaneously Recovered from Postpartum Endometritis

The profile and function of blood and uterine leukocytes were evaluated in 14 dairy cows that spontaneously recovered from postpartum endometritis (mild, n=6 and heavy, n=8; general health not affected). From a minimum of 2 weeks before parturition until 6 weeks postpartum, blood samples were obtained twice weekly for leukocyte counts and leukogram determination and once weekly for flow cytometry assessment of polimorphonuclear neutrophils (PMN) phagocytic capacity and oxidative burst activity. Uterine fluid‐stained smears, obtained twice weekly from parturition until fluid was present in the uterus, were used for determination of the percentage of PMN, of phagocytizing PMN (phago‐PMN) and of the mean number of phagocyted bacteria per phagocytizing PMN (phagocytic index; PI). Uterine swabs were obtained twice weekly from parturition until 35 days postpartum for bacteriological examination. The time of endometritis diagnosis was similar in cows with mild or heavy endometritis but the latter cows had a significantly longer persistence of the infection and of the isolation of Gram‐negative anaerobes from the uterus. However, the effect of group (mild versus heavy) was not significant for all the blood and uterine parameters analysed. The effect of sampling day (within group effect) was significant (p<0.01 to p<0.00001) for all parameters, except for the blood monocyte count and the blood PMN phagocytic capacity, in which only a tendency for significance was observed (p<0.1). The effect of the interaction group × sampling day was significant only for the blood monocyte count. The phago‐PMN and the PI were significantly correlated (r=0.70, p<0.001). A significant correlation was also observed between the uterine fluid phago‐PMN and the blood PMN oxidative burst activity (r=?0.41, p<0.05). At the spontaneous recovery, the blood PMN oxidative burst activity was significantly higher (p<0.05) and the percentage of intrauterine phago‐PMN and the PI were significantly lower (p<0.001 and p<0.01, respectively) than at diagnosis of endometritis. These results suggest that a decrease in blood PMN oxidative burst activity until the first week postpartum could be associated with an increased susceptibility to early postpartum endometritis. The later increase in this parameter as well as the increase in the intrauterine fluid phago‐PMN and PI, might favour the spontaneous resolution of endometritis.     

Evaluation of the influence of meloxicam and flunixin meglumine on the apoptosis of peripheral blood CD4+ and CD8+ T cells in calves

The aim of the study was to determine whether treatment with recommended doses of meloxicam or flunixin had an effect on the apoptosis of peripheral blood T lymphocytes in calves. The study was carried out on 4-5 months old calves (n = 24, 8 per group). Experimental animals were injected subcutaneously with a single dose of 0.5 mg x kg(-1) of meloxicam or intravenously with 3 doses of 2.2 mg x kg(-1) day(-1) of flunixin. The non-treatment animals served as control. Blood samples were taken at day 0 and at days 1, 2, 3, 5, 7 and 14 after the first NSAIDs injection. Apoptosis was determined by flow cytometry using Annexin V-PE/7-AAD staining. The kinetic analysis of apoptosis in the total lymphocyte population, as well as in the CD4+ and CD8+ subsets did not reveal significant differences in the frequency of early apoptotic cells between control and experimental groups throughout the period studied. Although, 24 h after administration of the first dose of NSAIDs, late-stage apoptosis/necrosis was significantly increased in the total lymphocyte population (the meloxicam group), as well as in the CD4+ (the meloxicam group and the flunixin group) and CD8+ (the flunixin group) subsets of T cells. However, this disturbance was transient, relatively poorly expressed and, thus, unlikely to be of clinical significance. Our results indicate that the use of meloxicam or flunixin in accordance with the recommended dosage regimen in cattle do not have a clinically significant influence on apoptosis of peripheral blood T cells.     

Simultaneous measurement of phagocytosis and respiratory burst of leukocytes in whole blood from bottlenose dolphins (Tursiops truncatus) utilizing flow cytometry

Phagocytic and respiratory burst activity was simultaneously measured by flow cytometry in polymorphonuclear leukocytes (PMN) and monocytes in whole blood from bottlenose dolphins (Tursiops truncatus). Blood was collected from 16 adult dolphins, 12 males (6-34 years of age) and 4 females (11-30 years) and subsequently incubated with a bacteria-to-leukocyte ratio of 25:1 and 10 μl of 500 μM 2',7'-dichlorofluorescein diacetate for 70 min at 37°C. PMN (44.5 ± 3.2%) and monocytes (33.5 ± 3.0%) were positive for propidium iodide-labeled Staphylococcus aureus, indicating phagocytosis. Respiratory burst activity after 70 min as measured by the mean fluorescence intensity (MFI) was 68.0 ± 14.4 in PMN and 47.0 ± 10.3 in monocytes. There were no significant differences in MFI or percentage of phagocytizing PMN (p > 0.094) or monocytes (p > 0.275) after storage at 4°C for 24h when compared to activity measured in fresh blood. Nor was there an effect of storage on respiratory burst activity (MFI or percentage) in PMN (p > 0.420) or monocytes (p > 0.301). This assay may be particularly useful to assess the ability of dolphins to effectively combat bacterial pathogen challenges with minimal amounts of blood.     

人工感染鸭病毒性肠炎急性病例细胞凋亡的电镜研究

应用透射电镜和超薄切片技术观察鸭病毒性肠炎病毒（Duck Enteritis Virus，DEV）CH强毒株人工感染成年鸭各组织器官的形态学特征。结果表明，脾脏、胸腺、法氏囊、十二指肠固有层中淋巴细胞除了坏死变化外还有很明显的凋亡变化，两者往往同时存在。疾病过程中，淋巴细胞凋亡数量明显增多。凋亡细胞的形态学改变是细胞体积缩小，细胞器结构正常，染色质初期浓集成团，聚集于核膜的周边，随后出现染色质凝聚，核碎裂以及凋亡小体形成等现象。淋巴细胞的坏死和凋亡共同造成了淋巴细胞的大量损耗，淋巴细胞的这种变化可能在鸭病毒性肠炎的发病机制中起着重要的作用。研究结果表明DEV急性感染可诱导成年鸭体内淋巴细胞的凋亡。     

Relationship of Cell Proliferating Marker Expressions with PGE(2) Receptors in Regenerating Rat Renal Tubules after Cisplatin Injection

Cisplatin, an anticancer drug, is well known to have nephrotoxicity as an adverse effect. We investigated the expressions of cell cycle markers and prostaglandin E(2) (PGE(2)) receptors (EP) in the affected renal tubules in rats injected with a single dose (6 mg/kg body weight) of cisplatin. On days 1-3 after dosing, the affected renal epithelial cells were almost desquamated, showing necrosis. On day 5 onwards, the renal tubules were rimmed by flattened or cuboidal epithelial cells with basophilic cytoplasm; BrdU-immunopositive cells began to significantly increase, indicating regeneration. Simultaneously, TUNEL-positive apoptotic cells were also seen. On days 1-5, cyclin D1-immunopositive cells were decreased with an increased expression in p21 mRNA, indicating G(1) arrest in the cell cycle. The affected renal epithelial cells began to react to EP4 receptor, but not to EP2 receptor. Some EP4 receptor-reacting epithelial cells gave a positive reaction to BrdU or cyclin D1. Collectively, the affected renal tubules underwent various alterations such as necrosis, apoptosis, regeneration and G(1) arrest; the aspects might be influenced by endogenous PGE(2) through EP4 receptor.