SUMMARY Neutrophils from newborn calves have been shown to be deficient in ability to generate superoxide anion (O 2 - ) after stimulation of the respiratory burst enzyme with the phorbol ester, phorbol 12-myristate 13-acetate ( pma ). This compound activates the O 2 - -generating enzyme of bovine neutrophils through a pathway involving protein kinase C ( pkc ). To investigate the biochemical basis underlying this functional difference between neutrophils from newborn and adult cattle, we measured and compared the activity of the enzyme pkc in nonstimulated and pma -stimulated bovine neutrophils. Neutrophils from newborn calves (n = 5) and adult cows (n = 5) were stimulated with various concentrations of pma (0, 10, 100, and 500 ng/ml) for 3 minutes, and pkc activity was assayed in the cytosolic and the membrane fractions. In nonstimulated cells, most pkc activity was detected in the cytosolic fraction of neutrophils from newborn and adult cattle. Activity of pkc in the cytosol was dependent on the presence of added calcium and phospholipids, whereas membrane-associated pkc in nonstimulated cells did not have such dependence. Significant differences in pkc activity were not observed between newborn and adult cattle in either the cytosolic or the membrane fractions from nonstimulated cells. Stimulation with pma caused redistribution of pkc activity in the cell (translocation) in newborns and adults, consisting of decrease in cytosolic pkc activity and increase in membrane-associated pkc activity. Similar to that in nonstimulated cells, pkc activity in cytosolic fractions from pma -stimulated neutrophils was dependent on the presence of cofactors (calcium and phospholipids), whereas pkc activity in the membrane did not have such requirement. Translocation of pkc from the cytosol to the membrane was expressed as percentage of decrease of activity in the cytosol and increase of activity in the membrane, compared with that in nonstimulated cells. Overall, significant differences were not evident in the ability of neutrophils from newborn and adult cattle to translocate pkc after stimulation with various concentrations of pma . Results indicate that differences in redistribution of pkc activity are not responsible for the difference observed in O 2 - production between neutrophils from newborn calves and adult cattle after cells are stimulated with pkc agonists.
Skin cancer is the most common tumor type in Caucasians, with an incidence that approaches the lifetime risk for all other cancer subtypes combined. The most common predisposing factor in the development of non-melanoma skin cancer is exposure to ultraviolet (UV) radiation in sun-light. UV radiation activates c-Jun amino-terminal kinases (JNK); this kinase pathway is involved in UV-mediated apoptosis and phosphorylation of c-Jun, all of which are part of the cellular stress response. Transforming growth factor-beta1 (TGF-beta1) is an important negative regulator of keratinocyte proliferation and has other pleiotropic effects in these cells. The purpose of these investigations was to decide whether TGF-beta1 activated c-Jun amino-terminal kinases in a spontaneously immortalized human keratinocyte cell line, HaCaT, and if TGF-beta1 modulated the activation of JNK in keratinocytes exposed to ultraviolet C (UVC) radiation. Results from these investigations showed that TGF-beta1 (10 ng/ml) activated JNK within 5 min. Pretreatment with TGF-beta1 enhanced UV-mediated JNK activation and was time- and UV-dose-dependent. Pretreatment with TGF-beta1 also enhanced activity of the c-Jun promoter-reporter construct, TRE(x5)-CAT. These results suggested that TGF-beta1 modulates the response of keratinocytes to ultraviolet radiation and implicates TGF-beta1 as a potential mediator the cellular of stress response in keratinocytes.
Summary Reasons for performing study: Two studies report variability in proliferation and limited adipocyte differentiation of equine peripheral blood‐derived adult mesenchymal stem cells, thus casting doubt on their adipogenic potential. Peripheral blood can be a valuable source of adult mesenchymal stem cells if cell culture conditions permissive for their adherence, proliferation and differentiation are defined. Hyperbaric oxygen treatment has been reported to mobilise haematopoietic progenitor stem cells into the peripheral blood in humans and mice, but similar experiments have not been done in horses. Objectives: To optimise cell culture conditions for isolation, propagation and differentiation of adult stem cells from peripheral blood and to assess the effect of hyperbaric oxygen treatment on adult stem cell concentrations. Methods: Peripheral blood was collected from the jugular vein of 6 research mares, and mononuclear cells were isolated. They were subjected to cell culture conditions that promote the adherence and proliferation of adult stem cells. The cells were characterised by their adherence, expression of cellular antigen markers, and trans‐differentiation. Each horse was subjected to 3 hyperbaric oxygen treatments, and stem cells were compared before and after treatments. Stem cells derived from adipose tissue were used as controls. Results: One‐third of the horses yielded viable stem cells from peripheral blood, positive for CD51, CD90 and CD105, and demonstrated osteocyte, chondrocyte and adipocyte differentiation. Hyperbaric oxygen treatment resulted in a significant increase in CD90‐positive cells. Horses that did not yield any cells pretreatment did so only after 3 hyperbaric oxygen treatments. Conclusions and potential relevance: Peripheral blood can be a valuable source of adult stem cells, if one can identify reliable equine‐specific markers, provide methods to increase the number of circulating progenitor cells and optimise cell culture conditions for growth and viability. Our findings are important for further studies towards technological advances in basic and clinical equine regenerative medicine.
Bovine leukocyte adhesion deficiency (BLAD) was identified in a two-month-old Holstein heifer calf using DNA-polymerase chain reaction analysis of the affected calf and other clinical parameters. Neutrophil integrin expression (CD18, CD11a, CD11c), aggregation, and transendothelial migration were studied in vitro. Neutrophils were isolated from the affected calf and from normal, healthy, age-matched control Holstein calves. Neutrophils isolated from the affected BLAD calf had decreased expression of leukocyte integrins on their cell surface, decreased ability to aggregate in response to chemotactic stimuli, and decreased ability to migrate across bovine endothelial cell monolayers in vitro. Transendothelial migration of neutrophils from normal calves was reduced to levels comparable to the BLAD neutrophils by treatment with an anti-CD18 monoclonal antibody (MAb 60.3). Peripheral-blood lymphocytes from the BLAD calf also expressed negligible levels of leukocyte integrins, similar to their neutrophil counterparts. Our experimental findings in vitro correlate well with the clinical observations of decreased leukocyte trafficking and diminished host defense in leukocyte adhesion-deficient animals. The syndrome of BLAD may be a suitable model for one of the human leukocyte adhesion deficiency disorders.
SUMMARY Newborn calves have a high susceptibility to bacterial infections, which may be related to the impaired neutrophil defense functions in newborns. The oxygen-dependent production of the free radical superoxide anion (O 2 - ) represents an important part of the leukocyte respiratory burst central to neutrophil-directed defenses against bacterial infection. Because protein kinase C ( pkc ) activation is considered to be an important step in the signal transduction pathway for the O 2 - generating system, we compared O 2 - production by newborn and adult bovine neutrophils stimulated with 3 different pkc agonists. When the phorbol ester phorbol 12-myristate 13-acetate ( pma ) was used, pkc -dependent O 2 - generation from newborn neutrophils was significantly reduced ( P < 0.01) for all concentrations of pma tested (10, 100, and 500 ng/ml). In addition, newborn neutrophils had a significantly ( P < 0.01) reduced lag time for O 2 - generation. Similar significantly ( P < 0.01) reduced O 2 - generation from newborn neutrophils was observed with an additional phorbol ester (phorbol 12, 13-dibutyrate); lag times were not calculated for phorbol 12, 13-dibutyrate. When O 2 - generation was stimulated with a synthetic diacylglycerol analogue (1,2-dioctanoyl-sn-glycerol), less O 2 - was generated from both adult and newborn neutrophils than was obtained with the phorbol esters, and newborn neutrophils produced significantly ( P < 0.01) less O 2 - only at 50 μ M 1,2-dioctanoyl-sn-glycerol. To assess the importance of pkc and cyclic nucleotide-dependent protein kinases in the O 2 - -related signal-transduction pathways of bovine neutrophils, we determined the influence of a pkc inhibitor, H-7, and a cyclic nucleotide-dependent protein kinase inhibitor, HA-1004, on the respiratory burst of adult and newborn bovine neutrophils. Preincubation of neutrophils (15 minutes) with 100 or 500 μ M H-7 inhibited subsequent pma -induced O 2 - generation from newborn and adult neutrophils in a dose-dependent fashion, but preincubation with HA-1004 did not affect subsequent pma -induced O 2 - generation from either newborn or adult neutrophils. The pkc inhibitor, H-7 (100 and 500 μ M), induced a significantly ( P < 0.01) prolonged lag time in adult, but not newborn, neutrophils; HA-1004 had no effect on lag time. These results indicate that pkc , but not cyclic nucleotide-dependent protein kinase, is important in the signal-transduction pathway leading to O 2 - generation in appropriately stimulated adult and newborn bovine neutrophils, and that deficient respiratory burst activity in newborn neutrophils may be explained by altered activation of pkc .
Abstract Objective To investigate receptor-mediated intracellular events in bovine alveolar macrophages (AM) stimulated by bacterial lipopolysaccharide (LPS), using tissue factor (TF) expression as the measurable functional endpoint. Sample Population Pulmonary AM harvested from 1- to 4-month-old male Holstein calves. Procedure Alveolar macrophages, acquired by use of volume-controlled bronchopulmonary lavage, were treated with CD14 monoclonal antibody (20 μg/ml), pertussis toxin (300 ng/ml), or 1 of 3 known protein kinase C (PKC) inhibitors (10 μ M chelerythrin, 100 μ M H-7, or 50 n M staurosporin), then were stimulated with LPS alone (0.01, 0.10, 1.0, 10.0 μg/ml) or LPS (0.25, 0.5, 1.0 ng/ml) in combination with concentrated bovine serum fraction 2 (500 ng/ml). Tissue factor expression was quantified by use of a colorimetric assay. Changes in intracellular Ca 2+ concentration and pH were monitored, using Ca 2+ - and pH-sensitive fluorescent dyes, with changes in fluorescent intensity after incubation with LPS measured by spectrophotometry. Results Treatment of AM with a CD14 monoclonal antibody caused profound inhibition of TF expression ( P < 0.0001) after stimulation by LPS combined with bovine serum fraction 2. Pertussis toxin had a significant ( P < 0.0319) inhibitory effect on TF expression when cells were stimulated by LPS alone. Treatment with all 3 PKC inhibitors caused marked reduction in TF expression of cells stimulated with LPS alone or with phorbol myristate acetate. Stimulation of cells by LPS failed to mobilize intracellular Ca 2+ stores or to alter cytosolic pH. Conclusion LPS combined with serum factors binds to CD14 on the surface of AM, and PKC is an important signaling kinase in the pathway utilized by LPS, resulting in enhanced TF expression; a pertussis toxin-sensitive G protein is involved in the signaling pathway utilized by LPS alone; and mobilization of Ca 2+ does not have a role in the signal transduction pathway utilized by LPS nor does LPS affect cytosolic pH of AM. ( Am J Vet Res 1998;59:445–451)
Peripheral nerve injuries cause poor performance in horses. They are difficult to manage and clinicians mostly rely upon physical therapy and anti‐inflammatories. The long term effects are time and personnel consuming. The aim of this research is to test the ability of equine mesenchymal stromal cells (MSCs) to differentiate into neuronal lineage, and trans‐differentiate into Schwann cells for nerve regeneration. Bone marrow‐derived MSCs were obtained from 3 young and 4 adult horses. Firstly, their stemness was demonstrated. Low passaged cells were neuronally induced in 24 well plates and assessed by microscopy at 3, 6, 12, 24 and 48 h. Results showed that the plate PRIMARIA™ supported proliferation and differentiation. Survival was improved by high number of cells. Morphological changes were observed as early as 3 hours; at 12 h approximately 60% of the cells were neuron‐like. Morphology was similar in all horses, but proliferation was variable. Protein concentration was influenced by cell number and method of protein extraction. Expression of neuronal progenitor proteins, vimentin, β3 tubulin and GFAP, was detected by immunoblot analysis; nestin was not evident. Results demonstrate that equine MSCs can differentiate into neuronal lineage. Future work is aimed to commit these cells into Schwann cells. Expression of additional neural‐specific markers, S100B, Krox20 and CD104 will be tested.
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