-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. polymorphonuclear leukocyte / macrophage / apoptosis / juvenile bovine mammary gland Résumé -Apoptose des leucocytes polymorphonucléaires de la glande mammaire juvénile des bovins durant l'influx induit. Afin de déterminer le rôle de l´apoptose dans la résolution des mammites, la dynamique de l'apoptose des leucocytes polymorphonucléaires (PMN) durant l'influx induit dans le système des cavités de la glande mammaire juvénile des bovins a été étudiée. L'application du dérivé synthétique du muramyl dipeptide dans le sinus du trayon de la glande mammaire a été suivi de l'influx massif de PMN avec un pic à 24 h et une résolution 96 heures après la stimulation. Au cours des 24, 48, 72 et 96 h après la stimulation, les suspensions cellulaires ont été examinées Vet. Res. 31 (2000) 553-563 553

Identification of specific cell death is of a great value for many scientists. Predominant types of cell death can be detected by flow-cytometry (FCM). Nevertheless, the absence of cellular morphology analysis leads to the misclassification of cell death type due to underestimated oncosis. However, the definition of the oncosis is important because of its potential reversibility. Therefore, FCM analysis of cell death using annexin V/propidium iodide assay was compared with holographic microscopy coupled with fluorescence detection - “Multimodal holographic microscopy (MHM)”. The aim was to highlight FCM limitations and to point out MHM advantages. It was shown that the annexin V+/PI− phenotype is not specific of early apoptotic cells, as previously believed, and that morphological criteria have to be necessarily combined with annexin V/PI for the cell death type to be ascertained precisely. MHM makes it possible to distinguish oncosis clearly from apoptosis and to stratify the progression of oncosis.

-The objective of this study was to determine whether neutrophil apoptosis and their consequent elimination by macrophages from the mammary gland is modulated by an infection caused by Staphylococcus aureus (S. aureus). The study was performed on twenty mammary glands of 5 virgin heifers. A buffered physiological solution (PBS) was administered as a means of control into the mammary glands of the heifers and after 168 h, the glands were inoculated with S. aureus. The samples of cell populations were obtained by lavages of the mammary glands in 4 intervals (24, 48, 72 and 168 h) after the experimental infection. Flow cytometry was used for determination of Annexin-V positivity and propidium iodide (PI) negativity of neutrophils. Light microscopy was used for determination of neutrophil karyopyknosis. Cytochemistry was used for the detection of myeloperoxidase-positive (MPO+) macrophages. Instillation of S. aureus resulted in an intramammary infection which persisted during the following experimental period. The total number of both Annexin-V-positive and PI negative neutrophils and karyopyknotic neutrophils peaked at 24 h after both of PBS and S. aureus administration. The highest percentages of Annexin-V-positive and PI negative neutrophils and karyopyknotic neutrophils were detected 48 and 168 h after PBS and S. aureus administration, respectively. The total number of MPO+ macrophages was the highest 24 h and 48 h after PBS and S. aureus administration, respectively; the percentage of MPO+ macrophages was the highest at 72 h in both cases. The dynamics of resolution of mastitis caused by S. aureus was very similar to the resolution of inflammatory response of the mammary gland after PBS administration. Mechanisms of cell pathogen elimination as well as inflammation resolution were very intensively involved; nevertheless, the mammary gland infection persisted. An early inclusion of the mechanisms of an acute inflammatory resolution thus paradoxically led to chronic infection.

-The object of the study was the comparative assessment of phagocyte activation during initiation and resolution of mammary gland injury induced by lipopolysaccharide (LPS) or buffered salt solution (PBS) on the basis of the CD14 receptor positivity. The experiments were carried out in 15 clinically normal Holstein × Bohemian Red Pied crossbred heifers, aged 14 to 18 months. Noninflammatory and inflammatory mammary gland injury were induced by intramammary administration of PBS (10 mL) and LPS (10 mL, 1 µg/mL), respectively. Samples of the cell populations were obtained by mammary lavages at 24 h intervals. Flow cytometry was used to determine the CD14+ neutrophils, monocytes, and macrophages. The percentage of CD14+ neutrophils was only 1.2% and 1.3% 24 h after the treatment with PBS and LPS, respectively. The resolution was accompanied by an increase in proportion of CD14+ neutrophils. The proportion of CD14+ neutrophils returned to initial values in the PBS-treated, but not in the LPS-treated mammary glands till 96 h. Percentage of CD14+ monocytes increased after 24 h and the effect was more pronounced in the LPS-treated than in the PBS treated mammary glands (P < 0.05). The percentage of CD14+ macrophages decreased highly significantly at 24 h in the LPS-treated, but not in the PBS-treated mammary glands (P < 0.01). The resolution of mammary gland injury (48 to 96 h) was characterised by an increase in CD14+ macrophages proportion, which was greater in the LPS-treated than PBS-treated mammary glands (P < 0.01). The activation of macrophages during resolution of mammary gland injury can be interpreted as an important mechanism of restitution. Résumé -Activation des phagocytes au cours de l'initiation et de la résolution de lésion de la glande mammaire induite par des lipopolysaccharides chez des génisses. L'objet de la présente étude est l'analyse de l'activation des phagocytes au cours de l'initiation et de la résolution de lésion de la glande mammaire induite par des lipopolysaccharides (LPS), ou par la solution physiologique (PBS), et cela sur la base de la positivité du CD14 récepteur. L'essai fut réalisé sur 15 génisses, cliniquement en bonne santé, issues du croisement des races Holstein × Pie rouge tchèque, âgées de 14 à 18 mois. Des lésions non-inflammatoires et inflammatoires de la glande mammaire ont été induites par l'administration intra-mammaire de PBS (10 mL) et LPS (10 mL, 1 µg/mL), respectivement. Les échantillons de populations cellulaires ont été obtenus par lavage des glandes mammaires, à des intervalles de 24 heures. On a utilisé la cytométrie de flux pour détecter les neutrophiles, les monocytes et les macrophages CD14+. Pendant l'induction de la lésion de glande mammaire (24 h), on a détecté seulement 1,2 % des neutrophiles CD14+ après traitement par le PBS et 1,3 % après traitement par le LPS. La résolution fut caractérisée par une augmentation de la proportion de neutrophiles CD14+. Jusqu'à 96 heures, après injection de PBS, à la différence de celle de LPS, la proportion de ne...

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