The suitability of biodegradable poly(dl-lactide-co-glycolide)75:25 microspheres for the sustained release of antibiotics

Post-operative infections resulting from total hip arthroplasty are caused by bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa entering the wound perioperatively or by haemetogenous spread from distant loci of infection. They can endanger patient health and require expensive surgical revision procedures. Gentamicin impregnated poly (methyl methacrylate) bone cement is traditionally used for treatment but is often removed due to harbouring bacterial growth, while bacterial resistance to gentamicin is increasing. The aim of this work was to encapsulate the antibiotics vancomycin, ciprofloxacin and rifampicin within sustained release microspheres composed of the biodegradable polymer poly (dl-lactide-co-glycolide) [PLCG] 75:25. Topical administration to the wound in hydroxypropylmethylcellulose gel should achieve high local antibiotic concentrations while the two week in vivo half life of PLCG 75:25 removes the need for expensive surgical retrieval operations. Unloaded and 20% w/w antibiotic loaded PLCG 75:25 microspheres were fabricated using a Water in Oil emulsification with solvent evaporation technique. Microspheres were spherical in shape with a honeycomb-like internal matrix and showed reproducible physical properties. The kinetics of in vitro antibiotic release into newborn calf serum (NCS) and Hank's balanced salt solution (HBSS) at 37°C were measured using a radial diffusion assay. Generally, the day to day concentration of each antibiotic released into NCS over a 30 day period was in excess of that required to kill St. aureus and Ps. auruginosa. Only limited microsphere biodegradation had occurred after 30 days of in vitro incubation in NCS and HBSS at 37°C. The moderate in vitro cytotoxicity of 20% w/w antibiotic loaded microspheres to cultured 3T3-L1 cells was antibiotic induced. In conclusion, generated data indicate the potential for 20% w/w antibiotic loaded microspheres to improve the present treatment regimens for infections occurring after total hip arthroplasty such that future work should focus on gaining industrial collaboration for commercial exploitation.

Zinc-α2-glycoprotein, a lipid mobilizing factor, is expressed in adipocytes and is up-regulated in mice with cancer cachexia

Zinc-α2-glycoprotein (ZAG), a 43-kDa protein, is overexpressed in certain human malignant tumors and acts as a lipid-mobilizing factor to stimulate lipolysis in adipocytes leading to cachexia in mice implanted with ZAG-producing tumors. Because white adipose tissue (WAT) is an endocrine organ secreting a wide range of protein factors, including those involved in lipid metabolism, we have investigated whether ZAG is produced locally by adipocytes. ZAG mRNA was detected by RT-PCR in the mouse WAT depots examined (epididymal, perirenal, s.c., and mammary gland) and in interscapular brown fat. In WAT, ZAG gene expression was evident in mature adipocytes and in stromal-vascular cells. Using a ZAG Ab, ZAG protein was located in WAT by Western blotting and immunohistochemistry. Mice bearing the MAC16-tumor displayed substantial losses of body weight and fat mass, which was accompanied by major increases in ZAG mRNA and protein levels in WAT and brown fat. ZAG mRNA was detected in 3T3-L1 cells, before and after the induction of differentiation, with the level increasing progressively after differentiation with a peak at days 8-10. Both dexamethasone and a β 3 agonist, BRL 37344, increased ZAG mRNA levels in 3T3-L1 adipocytes. ZAG gene expression and protein were also detected in human adipose tissue (visceral and s.c.). It is suggested that ZAG is a new adipose tissue protein factor, which may be involved in the modulation of lipolysis in adipocytes. Overexpression in WAT of tumor-bearing mice suggests a local role for adipocyte-derived ZAG in the substantial reduction of adiposity of cancer cachexia.

The effect of the senescence-associated secretory phenotype (SASP) on glucose homeostasis in metabolic cells

Aim: Dysregulated glucose homeostasis is a hallmark of Type 2diabetes. A distinctive feature of ageing is the accumulation ofsenescent cells, defined as cells that have undergone irreversible lossof proliferative capacity. Characteristic of senescent cells is thesenescence-associated secretory phenotype (SASP) involving theproduction of factors which reinforce senescence arrest in neigh-bouring tissue environments. We hypothesise that SASP inducesmetabolic dysfunction in non-senescent cells, impairing glucosemetabolism and propagating insulin resistance. We sought todetermine the effect of SASP on glucose homeostasis in hepatic,adipose and skeletal muscle cell lines. Methods: Human dermal fibroblasts were subjected to a geno-toxic dose of doxorubicin to induce senescence, confirmed using ab-galactosidase assay. Conditioned media containing SASP werecollected post 24h and 48h of inducing senescence and used at20% and 40% concentrations to treat AML-12 hepatocytes, 3T3-L1 adipocytes and C2C12 myocytes for 24h and 48h. Cells andmedia were collected and glucose and lipid concentrations weremeasured before and after the respective incubation periods. Results: Cell media obtained from C2C12 myocytes exposed to40% SASP for 24h and 48h and AML-12 hepatocytes after 48hexhibited significantly higher concentrations of glucose in com-parison to control media (p < 0.0001, p < 0.05) suggesting areduced glucose uptake. Glucose utilisation remained unchanged in3T3-L1 cells. Conclusion: Our data suggest an important role for SASP inaltering glucose homeostasis and identify SASP as a potentialmediator between ageing and the increase in age-related insulinresistance.

Mechanism of action of a tumour derived lipid mobilising factor

Cancer cachexia comprises unintentional and debilitating weight loss associated with certain tumour types. Fat loss in cachexia is mediated by a 43kDa Lipid Mobilising Factor (LMF) sharing homology with endogenous Zinc-α2-Glycoprotein (ZAG). LMF and ZAG induced significant lipolysis in isolated epidydimal adipose tissue. This is attenuated by co-incubation with 10μM of antagonist SR59230A and partially attenuated by 25μM PD098059 (indicating β3-AR and MAPK involvement respectively). LMF/ZAG induced in vitro lipid depletion in differentiated 3T3-L1 adipocytes that seen to comprise a significant increase in lipolysis (p<0.01), with only a modest decrease in lipid synthesis (p=0.09). ZAG significantly increased in vitro protein synthesis (p<0.01) in C2C12 myotubes (without an effect on protein degradation). This increase was activated at transcription and attenuated by co-incubation with 10μM SR59230A. Proteolytic digestion of ZAG and LMF followed by sephadex G50 chromatography yielded active fragments of 6-15kDa, indication the entire molecule was not required for bioactivity. Cachexigenic MAC16 cells demonstrated significant in vitro ZAG expression over non-cachexigenic MAC13 cells (p<0.001). WAT and BAT excised from MAC16 mice of varying weight loss demonstrated increased ZAG expression compared to controls. Dosing of NMRI mice with s/c ZAG failed to reproduce this up-regulation, thus another cachectic factor is responsible. 0.58nM LMF conferred significant protection against hydrogen peroxide, paraquat and bleomycin-induced oxidative stress in the non-cachexigenic MAC13 cell line. This protection was attenuated by 10μM SR59230A indicating a β3-AR mediated effect. In addition, 0.58nM LMF significantly up regulated UCP2 expression (p<0.001), (a mitochondrial protein implicated in the detoxification of ROS) implying this to be the mechanism by which survival was achieved. In vitro, LMF caused significant up-regulation of UCP1 in BAT and UCP2 and 3 in C2C12 myotubes. This increase in uncoupling protein expression further potentiates the negative energy balance and wasting observed in cachexia.

Effect of eicosapentaenoic acid (EPA) on expression of a lipid mobilizing factor in adipose tissue in cancer cachexia

Adipose tissue of mice bearing a cachexia-inducing murine tumour (MAC16) shows increased expression of zinc-α2-glycoprotein (ZAG), a lipolytic factor thought to be responsible for the increased lipolysis. The anti-cachectic agent eicosapentaenoic acid (EPA) (0.5 g/kg) attenuated the loss of body weight in mice bearing the MAC16 tumour, and this was accompanied by downregulation of ZAG expression in both white and brown adipose tissue, as determined by Western blotting. Glucocorticoids may be responsible for the increased ZAG expression in adipose tissue. Dexamethasone (1.68 μM) stimulated lipolysis in 3T3-L1 adipocytes, and this effect was attenuated by EPA (50 μM). In addition the lipolytic action of dexamethasone was attenuated by anti-ZAG antibody, suggesting that the induction of lipolysis was mediated through an increase in ZAG expression. This was confirmed by Western blotting, which showed that dexamethasone (1.68 μM) induced a two-fold increase in ZAG expression in both cells and media, and that this was attenuated by EPA (50 μM). These results suggest that EPA may preserve adipose tissue in cachectic mice by downregulation of ZAG expression through interference with glucocorticoid signalling. © 2005 Elsevier Ltd. All rights reserved.

The role of glucocorticoids in the induction of zinc-α2-glycoprotein expression in adipose tissue in cancer cachexia

Loss of adipose tissue in cancer cachexia in mice bearing the MAC16 tumour arises from an increased lipid mobilisation through increased expression of zinc-α2-glycoprotein (ZAG) in white (WAT) and brown (BAT) adipose tissue. Glucocorticoids have been suggested to increase ZAG expression, and this study examines their role in cachexia and the mechanisms involved. In mice bearing the MAC16 tumour, serum cortisol concentrations increased in parallel with weight loss, and the glucocorticoid receptor antagonist RU38486 (25 mg kg-1) attenuated both the loss of body weight and ZAG expression in WAT. Dexamethasone (66 μg kg-1) administration to normal mice produced a six-fold increase in ZAG expression in both WAT and BAT, which was also attenuated by RU38486. In vitro studies using 3T3-L1 adipocytes showed dexamethasone (1.68 μM) to stimulate lipolysis and increase ZAG expression, and both were attenuated by RU38486 (10 μM), anti-ZAG antibody (1 μ gml-1), and the β3-adrenoreceptor (β3-AR) antagonist SR59230A (10 μM). Zinc-α2-glycoprotein also increased its own expression and this was attenuated by SR59230A, suggesting that it was mediated through the β3-AR. This suggests that glucocorticoids stimulate lipolysis through an increase in ZAG expression, and that they are responsible for the increase in ZAG expression seen in adipose tissue of cachectic mice. © 2005 Cancer Research UK.

Identification of nesfatin-1 in human and murine adipose tissue:a novel depot-specific adipokine with increased levels in obesity

Nesfatin-1 is a recently identified anorexigenic peptide derived from its precursor protein, nonesterified fatty acid/nucleobindin 2 (NUCB2). Although the hypothalamus is pivotal for the maintenance of energy homeostasis, adipose tissue plays an important role in the integration of metabolic activity and energy balance by communicating with peripheral organs and the brain via adipokines. Currently no data exist on nesfatin-1 expression, regulation, and secretion in adipose tissue. We therefore investigated NUCB2/nesfatin-1 gene and protein expression in human and murine adipose tissue depots. Additionally, the effects of insulin, dexamethasone, and inflammatory cytokines and the impact of food deprivation and obesity on nesfatin-1 expression were studied by quantitative RT-PCR and Western blotting. We present data showing NUCB2 mRNA (P < 0.001), nesfatin-1 intracellular protein (P < 0.001), and secretion (P < 0.01) were significantly higher in sc adipose tissue compared with other depots. Also, nesfatin-1 protein expression was significantly increased in high-fat-fed mice (P < 0.01) and reduced under food deprivation (P < 0.01) compared with controls. Stimulation of sc adipose tissue explants with inflammatory cytokines (TNFa and IL-6), insulin, and dexamethasone resulted in a marked increase in intracellular nesfatin-1 levels. Furthermore, we present evidence that the secretion of nesfatin-1 into the culture media was dramatically increased during the differentiation of 3T3-L1 preadipocytes into adipocytes (P < 0.001) and after treatments with TNF-a, IL-6, insulin, and dexamethasone (P < 0.01). In addition, circulating nesfatin-1 levels were higher in high-fat-fed mice (P < 0.05) and showed positive correlation with body mass index in human. We report that nesfatin-1 is a novel depot specific adipokine preferentially produced by sc tissue, with obesity- and food deprivation-regulated expression.

In vivo and ex vivo regulation of visfatin production by leptin in human and murine adipose tissue: role of mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways

Visfatin is an adipogenic adipokine with increased levels in obesity, properties common to leptin. Thus, leptin may modulate visfatin production in adipose tissue (AT). Therefore, we investigated the effects of leptin on visfatin levels in 3T3-L1 adipocytes and human/murine AT, with or without a leptin antagonist. The potential signaling pathways and mechanisms regulating visfatin production in AT was also studied. Real-time RT-PCR and Western blotting were used to assess the relative mRNA and protein expression of visfatin. ELISA was performed to measure visfatin levels in conditioned media of AT explants, and small interfering RNA technology was used to reduce leptin receptor expression. Leptin significantly (P<0.01) increased visfatin levels in human and murine AT with a maximal response at leptin 10(-9) M, returning to baseline at leptin 10(-7) M. Importantly, ip leptin administration to C57BL/6 ob/ob mice further supported leptin-induced visfatin protein production in omental AT (P<0.05). Additionally, soluble leptin receptor levels rose with concentration dependency to a maximal response at leptin 10(-7) M (P<0.01). The use of a leptin antagonist negated the induction of visfatin and soluble leptin receptor by leptin. Furthermore, leptin-induced visfatin production was significantly decreased in the presence of MAPK and phosphatidylinositol 3-kinase inhibitors. Also, when the leptin eceptor gene was knocked down using small interfering RNA, eptin-induced visfatin expression was significantly decreased. Thus, leptin increases visfatin production in AT in vivo and ex vivo via pathways involving MAPK and phosphatidylinositol 3-kinase signaling. The pleiotropic effects of leptin may be partially mediated by visfatin.

Transglutaminase overexpression sensitizes neuronal cell lines to apoptosis by increasing mitochondrial membrane potential and cellular oxidative stress

'Tissue' transglutaminase (tTG) selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Considering the central role played by mitochondria in apoptosis, we investigated the relationships existing amongst tTG expression, apoptosis and mitochondrial function. To this aim we studied the mechanisms of apoptosis in a neuronal cell line (SK-N-BE (2)) in which the tTG-expression was driven by a constitutive promoter. Furthermore, a tet-off inducible promoter was also used in 3T3 fibroblastic cells used as control. Both cell lines, when expressing tTG, appeared 'sensitized' to apoptosis. Strikingly, we found major differences in the morphological features of mitochondria among cell lines in the absence of apoptotic stimuli. In addition, these ultrastructural characteristics were associated with specific functional features: (i) constitutively hyperpolarized mitochondria and (ii) increased reactive oxygen intermediates production. Importantly, after mitochondrial-mediated apoptosis by staurosporine, a rapid loss of mitochondrial membrane potential was found in tTG cells only. Taken together, these results seem to suggest that, via hyperpolarization, tTG might act as a 'sensitizer' towards apoptotic stimuli specifically targeted to mitochondria. These results could also be of pathogenetic relevance for those diseases that are characterized by increased tTG and apoptotic rate together with impaired mitochondrial function, e.g. in some neurodegenerative disease.

Analysis of tissue transglutaminase function in the migration of Swiss 3T3 fibroblasts:the active-state conformation of the enzyme does not affect cell motility but is important for its secretion

Increasing evidence suggests that tissue transglutaminase (tTGase; type II) is externalized from cells, where it may play a key role in cell attachment and spreading and in the stabilization of the extracellular matrix (ECM) through protein cross-linking. However, the relationship between these different functions and the enzyme's mechanism of secretion is not fully understood. We have investigated the role of tTGase in cell migration using two stably transfected fibroblast cell lines in which expression of tTGase in its active and inactive (C277S mutant) states is inducible through the tetracycline-regulated system. Cells overexpressing both forms of tTGase showed increased cell attachment and decreased cell migration on fibronectin. Both forms of the enzyme could be detected on the cell surface, but only the clone overexpressing catalytically active tTGase deposited the enzyme into the ECM and cell growth medium. Cells overexpressing the inactive form of tTGase did not deposit the enzyme into the ECM or secrete it into the cell culture medium. Similar results were obtained when cells were transfected with tTGase mutated at Tyr(274) (Y274A), the proposed site for the cis,trans peptide bond, suggesting that tTGase activity and/or its tertiary conformation dependent on this bond may be essential for its externalization mechanism. These results indicate that tTGase regulates cell motility as a novel cell-surface adhesion protein rather than as a matrix-cross-linking enzyme. They also provide further important insights into the mechanism of externalization of the enzyme into the extracellular matrix.

Cells, surfaces and adhesion

This thesis concerns cell adhesion to polymer surfaces with an experimental emphasis on hydrogels. The thesis begins with a review of the literature and a synthesis of recent evidence to describe the process of cell adhesion in a given situation. The importance of understanding integrin-adhesion protein interactions and adhesion protein-surface interactions is emphasised. The experimental chapters describe three areas of investigation. Firstly, in vitro cell culture techniques are used to explore a variety of surfaces including polyethylene glycol methacrylate (PEGMA) substituted hydrogels, sequence distribution modified hydrogels and worn contact lenses. Cell adhesion to PEGMA substituted gels is found to decrease with increases in polyethylene oxide chain length and correlations are made between sequence distribution and adhesion. Worn contact lenses are investigated for their cell adhesion properties in the presence of antibodies to specific adhesion proteins, demonstrating the presence of vitronectin and fibronectin on the lenses. The second experimental chapter addresses divalent cation regulation of integrin mediated cell adhesion. Several cell types and various cations are used. Zinc, previously not regarded as an important cation in the process, is found to inhibit 3T3 cell adhesion to vitronectin that is promoted by other divalent cations. The final experimental chapter concerns cell adhesion and growth on macroporous hydrogels. A variety of freeze-thaw formed porous gels are investiated and found generally to promote cell growth rate.Interpenetrating networkbased gels (IPN) are made porous by elution of dextrin particles of varying size and loading density. These materials provide the basis for synthetic cartilage. Cartilage cells (chondrocytes) plated onto the surface of the porous IPN materials maintain a rounded shape and hence phenotypic function when a critical pore size and density is achieved. In this way, a prospective implant, made porous at the perpendicular edges contacting natural cartilage can be both mechanically stabilised and encourage the maintenance of normal matrix production at the tissue interface.