Search

Phagocytosis of apoptotic cells is a key step in the completion of programmed cell death that occurs throughout life in multicellular organisms. The molecular events involved in clearance of apoptotic cells are just beginning to be elucidated. Recently, CED-6, an adapter protein involved in engulfment has been cloned in Caenorhabditis elegans and in humans. CED-6 is composed of a phosphotyrosine-binding (PTB) domain and a proline-rich C-terminal domain with no apparent catalytic domain. Since PTB domains, originally identified in Shc, mediate intracellular signaling downstream of cell surface receptors, CED-6 has also been proposed to mediate intracellular signals leading to engulfment. In this report, we demonstrate that CED-6 dimerizes through a leucine zipper domain that is immediately adjacent to the PTB domain. Several lines of evidence based on co-immunoprecipitation studies, yeast two-hybrid assays, and gel filtration studies suggest that CED-6 exists as a dimer in vivo. Through mutational analyses, we show that the leucine zipper is necessary and sufficient for CED-6 dimerization and that this dimerization is conserved among C. elegans, rodent, and human CED-6 proteins. We propose that dimerization may have unique implications for ligand binding via CED-6 and its function during the phagocytosis of apoptotic cells.

epithelium. Neutrophil migration across mammary arterial endothelial cells was almost completely dependent on CD18, the beta-chain of the beta(2) integrins, and to a lesser extent on CD11b, one of the alpha-chains of the beta(2) integrins. Neutrophil migration across collagen was partially blocked by monoclonal antibodies to CD18. No inhibition was observed by monoclonal antibodies to CD11b. Conversely, neutrophil diapedesis across mammary epithelial cells was dependent to a greater extent on CD11b. These results provide evidence for different CD11b/CD18-dependent mechanisms for neutrophil diapedesis across the various cell layers of the blood-milk barrier.

A key feature of the process of programmed cell death (apoptosis) is the efficiency with which the dying cells are recognized and engulfed by phagocytes [1]. Apoptotic cells are rapidly cleared either by neighbouring cells acting as semi-professional phagocytes or by experts of the macrophage line, so that an inflammatory response is avoided [2]. The Caenorhabditis elegans gene ced-6 is required for efficient engulfment of apoptotic cells [3] and is one of a group of genes that define two partially redundant parallel pathways for the engulfment process [4] [5]. These pathways may be conserved across evolution, as two other engulfment genes have human homologues. A CED-5 homologue is part of a human CrkII-DOCK180-Rac signaling pathway proposed to mediate cytoskeletal reorganization [6] [7] [8] and a CED-7 homologue is similar to the ABC transporters [9] [10]. Here, we report the cloning and characterization of human CED-6, a human homologue of C. elegans CED-6. The 34 kDa hCED-6 protein is expressed in most tissues, some human cancer cells, and in primary human macrophages. We developed an assay that quantitates the phagocytic activity of mammalian macrophages: the number of apoptotic cells that have been internalized is measured by the uptake of lacZ-positive apoptotic cells by adherent transgenic macrophages. The results of this assay demonstrate that overexpression of hCED-6 promotes phagocytosis only of apoptotic cells and suggest that hCED-6 is the mammalian orthologue of C. elegans CED-6 and is a part of a highly conserved pathway that specifically mediates the phagocytosis of apoptotic cells.

The effect of diapedesis on the phagocytic and oxidative burst activity of polymorphonuclear neutrophil (PMN) was examined, using an in vitro cell culture model consisting of a monolayer of primary mammary epithelial cells. Isolated blood PMN from 10 cows were added to the basal side of the epithelial cell monolayer. Diapedesis was induced by the addition of complement factor C5a to the apical side of the monolayer. PMN phagocytosis of Staphylococcus aureus and oxidative burst were measured before diapedesis on PMN that were non-activated and activated by incubation with C5a and on PMN after diapedesis, using flow cytometry. The percentages of PMN fluorescing due to phagocytosis of S. aureus and oxidative burst were reduced by 21.2 and 14.4%, respectively, after diapedesis. Pre-incubation in the presence of C5a had no effect on percentage PMN fluorescing due to phagocytosis or oxidative burst. The capacity for individual migrated PMN to phagocytose S. aureus and to produce an oxidative burst, as measured by the intensity of fluorescence, decreased by 34.2 and 30.3%. Activation of PMN with C5a increased intensity due to the oxidative burst, but had no effect on intensity due to phagocytosis. These data show that PMN diapedesis across mammary epithelium results in decreased phagocytosis and oxidative burst of the PMN.

Bacterial clearance, L-selectin adhesion receptor expression and neutrophil recruitment during experimentally induced Escherichia coli mastitis were investigated. Acute mastitis was induced by injection of 10(4) cfu E. coli into both left quarters of 12 clinically healthy lactating cows, 2-6 weeks after parturition. Half the cows were treated intravenously 10 h after infection, and subcutaneously 30 h after infection with enrofloxacin. In both groups, bacterial count, milk serum albumin, milk somatic cell count, circulating leukocyte count and L-selectin expression on neutrophils were determined. Both groups responded to challenge with udder inflammation and fever. Treatment with enrofloxacin affected the elimination rate of bacteria at hours +14, +18, +24, +48, and +72 after challenge, but not the bacteriological cure rate (five out of six for treated and three out of six for nontreated cows). The drop in L-selectin expression found following challenge did not differ between groups throughout the study. No effect of treatment was found on SCC. Based on these findings, it may be concluded that when treatment is administered 10 h after infection, the number of bacteria in milk is not correlated with L-selectin expression on circulating PMN during experimentally induced E. coli mastitis. The initial bacterial load probably dictates the extent of the decrease in L-selectin expression and milk somatic cells.

The expression of adhesion receptors and diapedesis by polymorphonuclear neutrophils (PMN) were studied before and during experimentally induced Streptococcus uberis mastitis. Both quarters of the left half of the udders of five midlactation cows were inoculated with a suspension containing approximately 500 CFU of S. uberis 0140J. Clinical signs of an inflammatory reaction and leukocyte influx were observed 24 h after challenge. The expression of CD11b/CD18 adhesion receptors, determined by flow cytometry, was upregulated 24 h after challenge. A confluent monolayer of bovine secretory mammary epithelial cells on collagen-coated inserts was used to study PMN diapedesis. Bovine C5a was used as the chemoattractant. An 80% decrease in PMN diapedesis was observed 24 h after challenge. The decrease in diapedesis continued for 3 weeks after challenge.

Polymorphonuclear neutrophils (PMN) are important in host defence against bacterial infection in the bovine mammary gland and techniques are needed to evaluate their functional activities. A rapid and sensitive two-color flow cytometric method for simultaneous measurement of phagocytosis rate and oxidative burst activity of bovine PMN in small blood samples is described. The method utilizes the oxidation of intracellular dihydrorhodamine 123 to green fluorescent rhodamine 123 by oxidative burst products that were generated by incubating the PMNs with red fluorescent propidium iodide labeled Staphylococcus aureus. Phagocytosis and oxidative burst both increased with time of incubation and with increasing bacteria concentration. A 20 min phagocytosis time and a ratio of 25 bacteria to one cell were considered optimal for assaying bovine blood PMN activity. To further evaluate the proposed two-color flow cytometric method, blood samples were treated with factors known to interfere with phagocytosis and oxidative burst metabolism of human neutrophils. Incubation of whole bovine blood with cytochalasin B at 10 micrograms ml-1 resulted in an approximate 70% decrease in the phagocytosis rate with a concommitant decrease in oxidative burst activity. Staurosporine (2 micrograms ml-1) inhibited bovine neutrophil oxidative burst formation for 95% while the phagocytosis was unaffected. PMNs in whole blood samples of ten cows differed significantly in their ability to phagocytose Staphylococcus aureus and to produce reactive oxygen products. However, only a weak correlation was detected between the burst activity:phagocytosis ratio of ten individual cows as indicated by the ROD/PI index.

Cross-correlograms between voluntarily active soleus (SOL) and tibialis anterior (TA) motor units were generated from seven control subjects and six subjects with spastic cerebral palsy (CP). Short-duration central peaks were observed in three subjects with spastic diplegia only. All subjects demonstrated reciprocal inhibition in TA following electrical stimulation of group I afferents to SOL, and all subjects with CP demonstrated strong activation of both TA and SOL in response to transcranial magnetic stimulation. Responses in SOL were stronger than those observed from controls. These data support the existence of abnormal corticospinal projections to soleus motor neurons in individuals with spastic CP. In spastic diplegia, short-term discharge synchrony between SOL and TA motor units may reflect abnormal interneuronal modulation at the spinal level. Abnormal corticospinal projections and/or modulation of spinal interneurons may contribute to the disordered movement patterns and co-activation observed in this population.

Neutrophils are the major defense against bacterial infection in the bovine mammary gland. Neutrophils migrate from blood into the lumen of the gland in response to inflammatory stimuli. This study describes the development of a system of cell culture that can be used to study neutrophil diapedesis through secretory and ductal mammary epithelial barriers. The culture system consists of successive layers of collagen, fibroblasts, collagen, and a confluent monolayer of secretory or ductal epithelial cells layered on a porous membrane. Confluence was determined by electrical resistance and trypan blue diffusion. Neutrophil diapedesis occurred from the basal to the apical surface of the monolayers. Purified complement C5a, fetal bovine serum that had been activated by zymosan, and fetal bovine serum that had been activated by Escherichia coli induced neutrophil diapedesis. Neutrophil diapedesis was greater across ductal cell monolayers. Blood neutrophils from five cows differed in their ability to migrate through the multilayered culture system in response to C5a. Monoclonal antibodies to C5a blocked diapedesis induced by purified C5a but had no effect on diapedesis induced by fetal bovine serum that had been activated by zymosan or by fetal bovine serum that had been activated by E. coli endotoxin, indicating that factors other than C5a were chemotactic for neutrophils. Monomeric IgG2, immune complexes, and E. coli endotoxin did not induce neutrophil diapedesis.

Although nomograms to convert readings of resistance (R) and reactance (Xc) into estimates of body composition have begun to emerge for children as well as adults, there has been reluctance to use bioelectrical impedance analysis (BIA) in very young children due to the irritability of children, measurement instability, positioning of electrodes, and, of course, reproducibility of measurements. The precision of completely independent measurements of BIA indices was evaluated by two observers by comparing a series of 40 measurements of R and Xc and 36 measurements for weight in four malnourished children on 10 different days during the course of their nutritional recuperation. For weight, the CV (coefficient of variation) was 3.5% for observer A and 3.8% for observer B. There were no differences in the means across observers with respect to weight, R, and Xc. The results obtained in this study illustrate that properly trained observers following the same standards can produce equivalent data for BIA and weight measurements even in sick children.

Self-directed work teams are innovative tools used in industry to improve productivity and quality. Work teams are complementary to the process of Total Quality Management (TQM) and build on the same foundations of customer satisfaction, goal setting, and staff education and training (Arikian, 1991; Sheehy & Musselwhite, 1990). The Collaborative Healthcare Utilization Model (CHUM) encompasses a self-directed team which enables nurse managers and physicians to lead a busy, tertiary care surgical service. The opportunity to develop CHUM arose when a middle management (director) position was removed from the nursing division. Development of the self-directed team included "selling" the idea and piloting the venture in an environment where work teams are not part of the organizational structure. The authors describe their experience in introducing a self-directed team into a hospital setting.