Nancy Neilsen

Dietary n-3 Polyunsaturated Fatty Acids Enhance Hormone Ablation Therapy in Androgen-Dependent Prostate Cancer

Hormone ablation therapy typically causes regression of prostate cancer and represents an important means of treating this disease, particularly after metastasis. However, hormone therapy inevitably loses its effectiveness as tumors become androgen-independent, and this conversion often leads to death because of subsequent poor responses to other forms of treatment. Because environmental factors, such as diet, have been strongly linked to prostate cancer, we examined the affects of dietary polyunsaturated fatty acids (PUFAs; at 1.5 wt%) on growth of androgen-dependent (CWR22) and androgen-independent (CWR22R) human prostatic cancer xenografts, the acute response of CWR22 tumors to ablation therapy, and their progression to androgen independence. Significant diet-induced changes in tumor n-3 or n-6 PUFA content had no affect on CWR22 or CWR22R tumors growing with or without androgen support, respectively. However, dietary changes that increased tumor eicosapentaenoic acid and linoleic acid content enhanced responses to ablation therapy, measured by cancer cell apoptosis and mitosis. In addition, relapse to androgen-independent growth (measured by renewed increases in tumor volume and serum prostate-specific antigen after ablation) positively correlated with tumor arachidonic acid content. There was no correlation between expression of 15-lipoxygenase isozymes or their products and tumor growth or responses to ablation. In conclusion, dietary n-3 PUFA may enhance the response of prostate cancer to ablation therapy and retard progression to androgen-independent growth by altering tumor PUFA content.

Graphene supports in vitro proliferation and osteogenic differentiation of goat adult mesenchymal stem cells: potential for bone tissue engineering

Current treatments for bone loss injuries involve autologous and allogenic bone grafts, metal alloys and ceramics. Although these therapies have proved useful, they suffer from inherent challenges, and hence, an adequate bone replacement therapy has not yet been found. We hypothesize that graphene may be a useful nanoscaffold for mesenchymal stem cells and will promote proliferation and differentiation into bone progenitor cells. In this study, we evaluate graphene, a biocompatible inert nanomaterial, for its effect on in vitro growth and differentiation of goat adult mesenchymal stem cells. Cell proliferation and differentiation are compared between polystyrene‐coated tissue culture plates and graphene‐coated plates. Graphitic materials are cytocompatible and support cell adhesion and proliferation. Importantly, cells seeded on to oxidized graphene films undergo osteogenic differentiation in fetal bovine serum‐containing medium without the addition of any glucocorticoid or specific growth factors. These findings support graphenes potential to act as an osteoinducer and a vehicle to deliver mesenchymal stem cells, and suggest that the combination of graphene and goat mesenchymal stem cells provides a promising construct for bone tissue engineering. Copyright

Cell proliferation, viability, and in vitro differentiation of equine mesenchymal stem cells seeded on bacterial cellulose hydrogel scaffolds.

The culture of multipotent mesenchymal stem cells on natural biopolymers holds great promise for treatments of connective tissue disorders such as osteoarthritis. The safety and performance of such therapies relies on the systematic in vitro evaluation of the developed stem cell-biomaterial constructs prior to in vivo implantation. This study evaluates bacterial cellulose (BC), a biocompatible natural polymer, as a scaffold for equine-derived bone marrow mesenchymal stem cells (EqMSCs) for application in bone and cartilage tissue engineering. An equine model was chosen due to similarities in size, load and types of joint injuries suffered by horses and humans. Lyophilized and critical point dried BC hydrogel scaffolds were characterized using scanning electron microscopy (SEM) to confirm nanostructure morphology which demonstrated that critical point drying induces fibre bundling unlike lyophilisation. EqMSCs positively expressed the undifferentiated pluripotent mesenchymal stem cell surface markers CD44 and CD90. The BC scaffolds were shown to be cytocompatible, supporting cellular adhesion and proliferation, and allowed for osteogenic and chondrogenic differentiation of EqMSCs. The cells seeded on the BC hydrogel were shown to be viable and metabolically active. These findings demonstrate that the combination of a BC hydrogel and EqMSCs are promising constructs for musculoskeletal tissue engineering applications.

Equine peripheral blood-derived mesenchymal stem cells: isolation, identification, trilineage differentiation and effect of hyperbaric oxygen treatment

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.

Alveolar macrophage phagocytic kinetics following pulmonary parainfluenza-3 virus infection.

Qualitative and quantitative evaluations of the cellular components of bronchoalveolar washings of calves with experimental parainfluenza‐3 virus pneumonitis and control calves were made. Calves were exposed to 109 TCID50 of PI‐3 by intranasal aerosol exposure and bronchoalveolar cells obtained 7 days after infection by volume‐controlled bronchopulmonary lavage. Transient tachypnea and pyrexia occurred in all infected calves, and virus was recoverable at 7 days from nasal swabs and lung tissue. Pulmonary lesions were typical of viral pneumonitis, characterized by patchy alveolitis and bronchiolitis with accumulations of cells and inflammatory debris. The mean total lavage cell yield was elevated in the virus‐infected calves, and the percentage of neutrophils was elevated (P < 0.05). Increased numbers of pulmonary alveolar macrophages (PAM) were also recovered but the difference was not significant. Linear regression equations showed that a decreased proportion of PAM from virus‐infected animals were phagocytic. The mean initial phagocytic rates of macrophages from calves with viral pneumonitis were significantly decreased (P < 0.05) over controls. This difference was concentration dependent and required a phagocytic stimulus in excess of 12.5 × 106beads/ml. Studies of phagocytic kinetics showed that PAM from calves with viral pneumonitis had a lower Vmax than PAM from control calves, but that Km values were comparable. No differences in PAM β‐glucuronidase and acid phosphatase activity were observed. These results indicate depressed phagocytic function in PI‐3 virus‐inflamed lungs relative to controls. In concert with virus‐induced airway lesions, such in vivo depression of PAM phagocytic functions would be expected to depress pulmonary particulate clearance and lung defense mechanisms.

In vitro analysis of equine, bone marrow-derived mesenchymal stem cells demonstrates differences within age- and gender-matched horses

REASONS FOR PERFORMING THE STUDY Stem cell therapies are used routinely in equine practice. Most published reports characterise stem cells derived from younger horses; however, middle-aged horses are often in athletic performance, and experience degenerative medical conditions. Thus, mesenchymal stem cells (MSCs) from this group should be investigated. OBJECTIVE To describe differences in in vitro adherence, proliferation and potential for differentiation of equine bone marrow-derived MSCs (equine BMMSCs) harvested from middle-aged (10-13 years old) female donors. STUDY DESIGN Descriptive study of stem cell characteristics. METHODS Equine BMMSCs from 6 horses were cultured in vitro and evaluated for viability, proliferation, osteogenesis, chondrogenesis, adipogenesis, cluster-of-differentiation markers and gene expression. RESULTS Equine BMMSCs from all 6 donors demonstrated fibroblastic, cellular morphology, adherence to plastic and expression of cluster-of-differentiation markers. They varied in their rate of proliferation and trilineage differentiation. The equine BMMSCs of one of 6 donors demonstrated a higher rate of proliferation, enhanced ability for cell passaging and a more robust in vitro differentiation. Comparatively, equine BMMSCs from 2 donors demonstrated a lower rate of proliferation and lack of osteogenic and chondrogenic differentiation. CONCLUSION The results of this study confirm that donor-to-donor variation in equine BMMSCs exists and this variation can be documented using in vitro assays. Subjective assessment suggests that the rate of proliferation tends to correlate with differentiation potential.

Secretory activity of equine polymorphonuclear leukocytes: stimulus specificity and priming effects of bacterial lipopolysaccharide

Neutrophil (PMN) contributions to the acute inflammatory process and host defense include generation of bioreactive oxygen metabolites and secretion of granule enzymes. We assessed equine PMN secretion using several PMN stimuli, singly and in combination with bacterial lipopolysaccharide (LPS). LPS avidly associated with equine PMN, as shown by strong PMN labeling with FITC-conjugated LPS. LPS alone (1 or 10 micrograms ml-1) was a weak stimulus for PMN superoxide anion (O2-) generation, but preincubation with LPS followed by phorbol ester (PMA, 10 ng ml-1) significantly augmented (P less than 0.01) secretion of O2- (19.38 nmol O2- per 2 x 10(6) PMN per 5 min) over the amount generated by PMA stimulation alone (13.75 nmol O2-). A qualitatively similar, but smaller O2(-)-generation response occurred when either opsonized zymosan or recombinant human C5a was used as the PMN stimulus. Arachidonic acid (ArA; 50-200 microM) was a potent stimulus, with secreted O2- levels similar to those from PMA-stimulated PMN. Preincubation of PMN with either the formyl peptide, fMLP, or platelet-activating factor before stimulation with ArA did not significantly increase O2- generation over levels obtained using ArA alone. Release of PMN granule enzymes was also quantitated. A small amount of lysozyme secretion resulted when PMN were exposed to LPS alone (8.20% of total cell content), and PMA stimulation caused marked release of PMN lysozyme (44.45%). Non-specific proteolytic activity in PMN supernatants, assessed by cleavage of a collagen-rich substrate, was minimal with LPS as a sole stimulus (5.08%). There was significant proteolytic activity (P less than 0.01) in supernatants from PMA-stimulated PMN (27.21%), and preincubation with LPS followed by PMA stimulation slightly enhanced (P less than 0.05) the release of PMN proteases (34.62%). The activities of beta-glucuronidase, acid phosphatase, and alkaline phosphatase were minimal in PMN supernatants when using LPS and PMA as stimuli. The activity of PMN granule enzymes was found to be sensitive to the presence of normal equine serum, and proteolytic activity was markedly reduced (80.13% reduction) in the presence of 10% pooled serum.

Transendothelial migration of neonatal and adult bovine neutrophils in vitro.

We have compared and quantitated transen‐dothelial migration of neonatal neutrophils (N‐PMNs) and adult bovine peripheral‐blood PMNs (A‐PMNs) in vitro using monolayers of endothelium and a two‐chamber apparatus. Bovine aortic endothelial cells were cultured to confluence on polycarbonate filters perforated with 3.0‐μm‐diameter pores. 51Cr‐labeled PMNs were added to the upper chamber, with or without an anti‐CD18 antibody (monoclonal antibody 60.3). Chemotactic stimuli in the lower chambers included recombinant human interleukin‐8 (rhIL‐8; 75 ng/ml), rhC5a (10‐7 M), and zymosan‐activated bovine serum (ZAS; 10%). At 60 min incubation with rhIL‐8, greater numbers (P < .01) of N‐PMNs (24.70 ± 5.95%) than of A‐PMNs (15.77 ± 3.66%) had migrated across the endothelial barrier, and a similar difference was present at 90 min. Migration rates of N‐PMNs and A‐PMNs were similar (P > .05) at all time points when using rhC5a and ZAS as stimuli. Anti‐CD18 monoclonal antibody significantly decreased migration (P < .01) of both N‐PMNs and A‐PMNs to low levels when IL‐8 and ZAS were used as stimuli. Because leukocyte integrin expression on PMNs affects transendothelial migration, we also compared surface expression of CD18, CD11a, and CD11c on PMNs from the two age groups. We found no significant quantitative differences in integrin expression between PMNs from the two age groups, regardless of whether the PMNs were incubated with buffer alone or with chemotaxins (rhIL‐8, rhC5a, ZAS). J. Leukoc. Biol. 55: 43–49; 1994.

Increased adhesiveness of complement-stimulated neonatal calf neutrophils and its pharmacologic inhibition.

Several in vitro functions of neonatal neutrophils (N‐PMN) have been reported to be deficient and may be functionally related to the increased susceptibility of the newborn to infection. To evaluate an in vitro event corresponding to one of the early steps in the sequence of inflammation, we used zymosan‐activated plasma as a source of activated complement fragments (Cf) and measured adherence of normal and Cf‐stimulated bovine N‐PMN to columns of Sephadex G‐25. Adherence of control N‐PMN and adult PMN (A‐PMN) was comparable. When N‐PMN and A‐PMN were stimulated with a subaggregating dose of Cf, both responded with similar increases in adhesiveness. The stimulatory effect of Cf on N‐PMN adhesiveness could be inhibited by pre‐incubation of the N‐PMN with either steroidal (0.05 mM dexamethasone) or non‐steroidal (32 mM phenylbutazone) anti‐inflammatory drugs. Ultrastructural observations correlated well with the results of the adhesiveness assays, and morphometric evaluation revealed an increase in the sectional circumference of Cf‐stimulated N‐PMN. Control cells were round with few short cytoplasmic projections, whereas Cf‐stimulated cells exhibited marked shape irregularity, polarity, and prominent organelle‐free lamellipodia development. There was a highly significant (P < 0.001) increase in the measured circumference of Cf‐stimulated cells. Thus, N‐PMN were highly responsive to Cf stimulation, developed morphologic and functional changes indistinguishable from Cf‐stimulated A‐PMN, and were sensitive to pharmacologic inhibition.

Stimulus-dependent actin polymerization in bovine neutrophils

Polymorphonuclear leukocytes (PMNs) are responsible for much of the first wave of leukocyte-mediated host defense against microbial pathogens. In order to migrate through the endothelium of vessel walls, undergo chemotaxis, and phagocytize microbes, PMNs must modulate their cytoskeletal elements and undergo change of cellular shape. We have used fluorescence flow cytometric analysis and cellular microscopic observations to demonstrate actin polymerization in bovine PMNs and to examine the kinetics of PMN actin polymerization utilizing different PMN stimuli. In addition, we compared temporal relationships between cellular shape and actin polymerization. Actin polymerization occurred rapidly, and the kinetics of actin polymerization were similar for each of the three PMN agonists used, ZAS (10%), PAF (10−6 M), and rhC5a (10−7 M). Actin polymerization was near-maximal by 10 sec poststimulation (95.4% of maximal F-actin content attained by 10 sec poststimulation with ZAS stimulation), and reached peak values by 30 sec. The maximal increase in F-actin content of agonist-stimulated cells as compared to resting cells was 2.8-fold with ZAS; 2.3-fold with PAF; and 2.3-fold with rhC5a. PMN shape change (pseudopodia, membrane raffles) was not as rapid, with only 22.4% of cells attaining visible membrane deformation by 10 sec and requiring 120 sec to reach peak shape-change values. After attaining peak values, the two events also differed. Whereas the percent of shape-changed PMNs remained plateaued up to 5 min poststimulation, the F-actin content gradually decreased after 30 sec, approaching F-actin values of unstimulated PMNs.