Cryopreservation, culture, and transplantation of human fetal mesencephalic tissue into monkeys

Studies in animals suggest that fetal neural grafts might restore lost neurological function in Parkinson's disease. In monkeys, such grafts survive for many months and reverse signs of parkinsonism, without attendant graft rejection. The successful and reliable application of a similar transplantation procedure to human patients, however, will require neural tissue obtained from human fetal cadavers, with demonstrated cellular identity, viability, and biological safety. In this report, human fetal neural tissue was successfully grafted into the brains of monkeys. Neural tissue was collected from human fetal cadavers after 9 to 12 weeks of gestation and cryopreserved in liquid nitrogen. Viability after up to 2 months of storage was demonstrated by cell culture and by transplantation into monkeys. Cryopreservation and storage of human fetal neural tissue would allow formation of a tissue bank. The stored cells could then be specifically tested to assure their cellular identity, viability, and bacteriological and virological safety before clinical use. The capacity to collect and maintain viable human fetal neural tissue would also facilitate research efforts to understand the development and function of the human brain and provide opportunities to study neurological diseases.     

The image of exocytosis during neutrophils and macrophages phagocytic activities in inflammation of mammary gland triggered by experimental Staphylococcus aureus infection

The experiments were carried out in five clinically normal virgin heifers. Before the experimental infection and at 24, 48, 72 and 168 h after the infection, respective mammary glands were rinsed with phosphate buffered saline. Neutrophils as well as macrophages underwent a classic exocytosis accompanied by translocation of lysosomal granules. The granules filled the protuberances of the plasmalemma and after the protuberances separated from the cell, they entered the extracellular space in the shape of round bodies of different sizes. After exocytosis, neutrophils displayed a smaller nucleus/cytoplasm ratio, a greater chromatin density of the nucleus, and an overall smaller size. Macrophages phagocytosed bacteria and/or neutrophils with and without signs of apoptosis (early and late apoptotic respectively) and neutrophils after exocytosis. Macrophages underwent cytolysis that was accompanied by extrusion of granules, phagosomes and phagolysosomes containing phagocytosed bacteria or neutrophils. Confluences were formed in which the process of digestion continued. Apoptosis of neutrophils gradually appeared and intensified in resolution of inflammation. The macrophages contributed to the inactivation of bacterial noxa as well as of histotoxic contents of neutrophils. Nevertheless, macrophages often underwent cytolysis at the site of inflammation.     

Effect of Staphylococcus aureus and Streptococcus uberis on apoptosis of bovine mammary gland lymphocytes

The aim of this study was to determine whether lymphocyte apoptosis is modulated by infections caused by Staphylococcus aureus and Streptococcus uberis. Samples of cell populations were obtained by lavage of the mammary glands at 4 intervals (24, 48, 72 and 168 h) following infection. The percentage of apoptotic lymphocytes peaked at 168 h after challenge with S. aureus or S. uberis. Subsequent experiments focused on in vitro cultivation of mammary gland lymphocytes with S. aureus and S. uberis. These experiments showed a lower percentage of apoptotic lymphocytes following 3 h of cultivating cells with bacteria than after cultivation without bacteria. The results demonstrate that during both experimental infection of bovine mammary glands with S. aureus or S. uberis and during in vitro cultivation of lymphocytes with S. aureus or S. uberis, apoptosis of lymphocytes is delayed.     

Neutrophil apoptosis during experimentally induced Staphylococcus aureus mastitis

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.     

Fetal research

This article reviews some of the significant contributions of fetal research and fetal tissue research over the past 20 years. The benefits of fetal research include the development of vaccines, advances in prenatal diagnosis, detection of malformations, assessment of safe and effective medications, and the development of in utero surgical therapies. Fetal tissue research benefits vaccine development, assessment of risk factors and toxicity levels in drug production, development of cell lines, and provides a source of fetal cells for ongoing transplantation trials. Together, fetal research and fetal tissue research offer tremendous potential for the treatment of the fetus, neonate, and adult.     

Neural transplantation: a call for patience rather than patients

Research into embryonic cell grafting in human subjects suffering from Parkinson's disease should not repeat the mistakes made by adrenal autograft investigators, who operated on far more humans than on nonhuman primates because of a single unconfirmed report of dramatic improvement in two Mexican patients. Citing extensive data from experimental transplants conducted as early as 1944, the authors argue that, until a sufficient number of animal studies have been performed to answer many crucial questions about human fetal tissue transplants, researchers should refrain from experimenting on human patients.     

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.     

Activation of phagocytes during initiation and resolution of mammary gland injury induced by lipopolysaccharide in heifers

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 x 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 microg/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.     

The role of CD14 during resolution of experimentally induced Staphylococcus aureus and Streptococcus uberis mastitis

This study was undertaken to investigate the time course of surface expression of CD14 on neutrophils and macrophages and to determine their association with resolution of inflammatory responses during Staphylococcus aureus and Streptococcus uberis experimental mastitis. Infections of the mammary gland induce a local immune response characterized by an increase in the total counts of CD14+ neutrophils and CD14+ macrophages particularly. On the other hand, resolution is accompanied by an increase in relative counts of CD14+ neutrophils, CD14+ vacuolized macrophages and apoptotic neutrophils. Following the immune reaction of mammary gland against Gram-negative/positive bacteria is very similar. Between the apoptotic and CD14+ neutrophils a high correlation was measured during the whole experimental period (S. aureus: r=0.64; S. uberis: r=0.61; P<0.05). Using anti-CD14 monoclonal antibodies in vitro suggested the involving of the CD14 surface receptor in recognition of apoptotic neutrophils by macrophages.