The in vivo expression of actin/salt-resistant hyperactive DNase I inhibits the development of anti-ssDNA and anti-histone autoantibodies in a murine model of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is characterised by the production of autoantibodies against ubiquitous antigens, especially nuclear components. Evidence makes it clear that the development of these autoantibodies is an antigen-driven process and that immune complexes involving DNA-containing antigens play a key role in the disease process. In rodents, DNase I is the major endonuclease present in saliva, urine and plasma, where it catalyses the hydrolysis of DNA, and impaired DNase function has been implicated in the pathogenesis of SLE. In this study we have evaluated the effects of transgenic overexpression of murine DNase I endonucleases in vivo in a mouse model of lupus. We generated transgenic mice having T-cells that express either wild-type DNase I (wt. DNase I) or a mutant DNase I ( ash. DNase I), engineered for three new properties - resistance to inhibition by G-actin, resistance to inhibition by physiological saline and hyperactivity compared to wild type. By crossing these transgenic mice with a murine strain that develops SLE we found that, compared to control nontransgenic littermates or wt. DNase I transgenic mice, the ash. DNase I mutant provided significant protection from the development of anti-single-stranded DNA and anti-histone antibodies, but not of renal disease. In summary, this is the first study in vivo to directly test the effects of long-term increased expression of DNase I on the development of SLE. Our results are in line with previous reports on the possible clinical benefits of recombinant DNase I treatment in SLE, and extend them further to the use of engineered DNase I variants with increased activity and resistance to physiological inhibitors.

Leishmaniasis Mimicking Lymphoma and/or Liver Cirrhosis

A 76-year-old man was admitted to hospital with fever, weight loss, pancytopenia, hepatosplenomegaly and a double monoclonal component IgM-IgG-k, suggesting a diagnosis of myeloma. Bone marrow and liver biopsies disclosed the presence of Donovan bodies, and the titre of anti-Leishmania antibodies was extremely high. After treatment with liposomal amphotericin B, the titre of antibodies fell considerably, while monoclonal components, pancytopenia and clinical symptoms slowly disappeared. Polyclonal γ-globulins are made of innumerable monoclonal components, one of which can appear as a recognizable band and be misdiagnosed as myeloma when representing the high titre of an antibody directed towards a specific antigen.

Detection of African Swine Fever Virus Antibodies in Serum and Oral Fluid Specimens Using a Recombinant Protein 30 (p30) Dual Matrix Indirect ELISA

In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence.

Tratamiento del rechazo mediado por anticuerpos en pacientes trasplantados renales revisión sistemática

Antecedentes: El trasplante renal es la mejor alternativa terapéutica para la enfermedad renal crónica terminal. Los medicamentos inmunosupresores previenen el rechazo. El rechazo mediado por anticuerpos es frecuente y disminuye la función y duración del injerto. Objetivo: Evaluar sistemáticamente la evidencia disponible relacionada con la eficacia y seguridad del tratamiento para el rechazo mediado por anticuerpos en pacientes trasplantados renales. Metodologia: Revisión sistemática en bases de datos MEDLINE, EMBASE, Scopus y Biblioteca virtual de la salud. Literatura gris google scholar, google academico, www.clinicaltrialsregister.eu, and https://clinicaltrials.gov/. Búsqueda manual referencias artículos pre-seleccionados así como de revisiones previamente publicadas. Se siguieron las recomendacioes guia PRISMA para la identificacion de artículos potenciales, tamizaje y selección teniendo en cuenta los criterios de inclusion. Extracción datos de acuerdo a las variables, revisión calidad de los artículos elegidos utilizando evaluación riesgo de segos de Cochrane. Resultados: Se seleccionaron 9 ensayos clínicos publicados entre 1980 y 2016, incluyeron 222 pacientes (113 brazo de intervención y 109 en el control), seguimiento promedio 16 meses. Intervenciones evaluadas plasmaféresis, inmunoadsorción y rituximab. Hubo una amplia heterogeneidad en la definición de criterios de inclusión, criterios diagnósticos de rechazo y medidas de evaluación de eficacia de las intervenciones. Tres estudios encontraron diferencias estadísticamente significativas entre los grupos de tratamiento. Conclusiones: La evidencia sobre la eficacia de los tratamientos del rechazo mediado por anticuerpos en injertos renales es de baja calidad. Son necesarios ensayos clínicos controlados para poder definir el tratamiento óptimo de estos pacientes.

Caracterización y prevalencia del compromiso ocular en pacientes con enfermedad autoinmune y autoinflamatoria en un centro de referencia reumatológica en Colombia entre los años 2000 a 2015

RESUMEN Objetivo: Estimar la prevalencia de las diferentes enfermedades oftalmológicas que aparecen en el contexto de una enfermedad autoinmune (EAI) en pacientes de un centro de referencia reumatológica en Colombia, según características clínicas y sociodemográficas durante un período de 15 años, comprendido entre los años 2000 a 2015. Métodos: Se realizó un estudio descriptivo, observacional de prevalencia. El tipo de muestreo fue aleatorio estratificado con asignación proporcional en el programa Epidat 3.4. Los datos se analizaron en el programa SPSS v22.0 y se realizó análisis univariado de las variables categóricas, para las variables cuantitativas se realizaron medidas de tendencia central. Resultados: De 1640 historias clínicas revisadas, se encontraron 634 pacientes (38,65%) con compromiso ocular. Si excluimos los pacientes con SS, que por definición presentan ojo seco, 222 pacientes (13,53%) presentaron compromiso oftalmológico. Del total de pacientes, el 83,3% fueron mujeres. La AR fue la enfermedad autoinmune con mayor compromiso oftalmológico con 138 pacientes (62,2%), y en último lugar la sarcoidosis con 1 solo paciente afectado. La QCS fue la manifestación más común en todos los grupos diagnósticos de EAI, con 146 pacientes (63,5%). De 414 pacientes con Síndrome de Sjögren (SS) y QCS 8 presentaron compromiso ocular adicional, siendo la uveítis la segunda patología ocular asociada en pacientes con SS y la primera causa en las espondiloartropatias (71,4 %). Los pacientes con catarata (4,1%) presentaron la mayor prevalencia de uso de corticoide (88.8%). De 222 pacientes, 28 (12,6%) presentaron uveítis. Del total de pacientes, 16 (7,2%) presentaron maculopatía por antimalaráricos y 6 (18,75%) de los pacientes con LES. Los ANAS se presentaron en el 100% los pacientes con trastorno vascular de la retina. Los pacientes con epiescleritis presentaron la mayor proporción de positivización de anticuerpos anti-DNA. La EAI que más presentó epiescleritis fue LES con 4 pacientes (12,5%) El 22% de paciente con anticuerpos anti-RNP presentaron escleritis y 32,1% de los pacientes con uveítis presentaron HLA-B27 positivo. Las manifestaciones oftalmológicas precedieron a las sistémicas entre un 11,1% y un 33,3% de los pacientes. Conclusión: Las enfermedades oculares se presentan con frecuencia en los pacientes colombianos con EAI (38.65%), siendo la AR la enfermedad con mayor compromiso ocular (62,2%) y la QCS la enfermedad ocular con mayor prevalencia en todas las EAI (63,5%). La uveítis se presentó en 28 pacientes (12,6%). Las manifestaciones oftalmológicas pueden preceder a las sistémicas. El examen oftalmológico debe ser incluido en los pacientes con EAI, por ser la enfermedad ocular una comorbilidad frecuente. Adicionalmente, los efectos oftalmológicos de las medicaciones sistémicas utilizadas en EAI deben ser estrechamente monitorizados, durante el curso del tratamiento.

In vitro and in vivo examination of the cell surface glycoprotein CDCP1

A number of reports have demonstrated the importance of the CUB domaincontaining protein 1 (CDCP1) in facilitating cancer progression in animal models and the potential of this protein as a prognostic marker in several malignancies. CDCP1 facilitates metastasis formation in animal models by negatively regulating anoikis, a type of apoptosis triggered by the loss of attachment signalling from cell-cell contacts or cell-extra cellular matrix (ECM) contacts. Due to the important role CDCP1 plays in cancer progression in model systems, it is considered a potential drug target to prevent the metastatic spread of cancers. CDCP1 is a highly glycosylated 836 amino acid cell surface protein. It has structural features potentially facilitating protein-protein interactions including 14 N-glycosylation sites, three CUB-like domains, 20 cysteine residues likely to be involved in disulfide bond formation and five intracellular tyrosine residues. CDCP1 interacts with a variety of proteins including Src family kinases (SFKs) and protein kinase C ä (PKCä). Efforts to understand the mechanisms regulating these interactions have largely focussed on three CDCP1 tyrosine residues Y734, Y743 and Y762. CDCP1-Y734 is the site where SFKs phosphorylate and bind to CDCP1 and mediate subsequent phosphorylation of CDCP1-Y743 and -Y762 which leads to binding of PKCä at CDCP1-Y762. The resulting trimeric protein complex of SFK•CDCP1•PKCä has been proposed to mediate an anti-apoptotic cell phenotype in vitro, and to promote metastasis in vivo. The effect of mutation of the three tyrosines on interactions of CDCP1 with SFKs and PKCä and the consequences on cell phenotype in vitro and in vivo have not been examined. CDCP1 has a predicted molecular weight of ~90 kDa but is usually detected as a protein which migrates at ~135 kDa by Western blot analysis due to its high degree of glycosylation. A low molecular weight form of CDCP1 (LMWCDCP1) of ~70 kDa has been found in a variety of cancer cell lines. The mechanisms leading to the generation of LMW-CDCP1 in vivo are not well understood but an involvement of proteases in this process has been proposed. Serine proteases including plasmin and trypsin are able to proteolytically process CDCP1. In addition, the recombinant protease domain of the serine protease matriptase is also able to cleave the recombinant extracellular portion of CDCP1. Whether matriptase is able to proteolytically process CDCP1 on the cell surface has not been examined. Importantly, proteolytic processing of CDCP1 by trypsin leads to phosphorylation of its cell surface-retained portion which suggests that this event leads to initiation of an intracellular signalling cascade. This project aimed to further examine the biology of CDCP1 with a main of focus on exploring the roles played by CDCP1 tyrosine residues. To achieve this HeLa cells stably expressing CDCP1 or the CDCP1 tyrosine mutants Y734F, Y743F and Y762F were generated. These cell lines were used to examine: • The roles of the tyrosine residues Y734, Y743 and Y762 in mediating interactions of CDCP1 with binding proteins and to examine the effect of the stable expression on HeLa cell morphology. • The ability of the serine protease matriptase to proteolytically process cell surface CDCP1 and to examine the consequences of this event on HeLa cell phenotype and cell signalling in vitro. • The importance of these residues in processes associated with cancer progression in vitro including adhesion, proliferation and migration. • The role of these residues on metastatic phenotype in vivo and the ability of a function-blocking anti-CDCP1 antibody to inhibit metastasis in the chicken embryo chorioallantoic membrane (CAM) assay. Interestingly, biochemical experiments carried out in this study revealed that mutation of certain CDCP1 tyrosine residues impacts on interactions of this protein with binding proteins. For example, binding of SFKs as well as PKCä to CDCP1 was markedly decreased in HeLa-CDCP1-Y734F cells, and binding of PKCä was also reduced in HeLa-CDCP1-Y762F cells. In contrast, HeLa-CDCP1-Y743F cells did not display altered interactions with CDCP1 binding proteins. Importantly, observed differences in interactions of CDCP1 with binding partners impacted on basal phosphorylation of CDCP1. It was found that HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F displayed strong basal levels of CDCP1 phosphorylation. In contrast, HeLa-CDCP1-Y734F cells did not display CDCP1 phosphorylation but exhibited constitutive phosphorylation of focal adhesion kinase (FAK) at tyrosine 861. Significantly, subsequent investigations to examine this observation suggested that CDCP1-Y734 and FAK-Y861 are competitive substrates for SFK-mediated phosphorylation. It appeared that SFK-mediated phosphorylation of CDCP1- Y734 and FAK-Y861 is an equilibrium which shifts depending on the level of CDCP1 expression in HeLa cells. This suggests that the level of CDCP1 expression may act as a regulatory mechanism allowing cells to switch from a FAK-Y861 mediated pathway to a CDCP1-Y734 mediated pathway. This is the first time that a link between SFKs, CDCP1 and FAK has been demonstrated. One of the most interesting observations from this work was that CDCP1 altered HeLa cell morphology causing an elongated and fibroblastic-like appearance. Importantly, this morphological change depended on CDCP1- Y734. In addition, it was observed that this change in cell morphology was accompanied by increased phosphorylation of SFK-Y416. This suggests that interactions of SFKs with CDCP1-Y734 increases SFK activity since SFKY416 is critical in regulating kinase activity of these proteins. The essential role of SFKs in mediating CDCP1-induced HeLa cell morphological changes was demonstrated using the SFK-selective inhibitor SU6656. This inhibitor caused reversion of HeLa-CDCP1 cell morphology to an epithelial appearance characteristic of HeLa-vector cells. Significantly, in vitro studies revealed that certain CDCP1-mediated cell phenotypes are mediated by cellular pathways dependent on CDCP1 tyrosine residues whereas others are independent of these sites. For example, CDCP1 expression caused a marked increase in HeLa cell motility that was independent of CDCP1 tyrosine residues. In contrast, CDCP1- induced decrease in HeLa cell proliferation was most prominent in HeLa- CDCP1-Y762F cells, potentially indicating a role for this site in regulating proliferation in HeLa cells. Another cellular event which was identified to require phosphorylation of a particular CDCP1 tyrosine residue is adhesion to fibronectin. It was observed that the CDCP1-mediated strong decrease in adhesion to fibronectin is mostly restored in HeLa-CDCP1-Y743F cells. This suggests a possible role for CDCP1-Y743 in causing a CDCP1-mediated decrease in adhesion. Data from in vivo experiments indicated that HeLa-CDCP1-Y734F cells are more metastic than HeLa-CDCP1 cells in vivo. This indicates that interaction of CDCP1 with SFKs and PKCä may not be required for CDCP1-mediated metastasis formation of HeLa cells in vivo. The metastatic phenotype of these cells may be caused by signalling involving FAK since HeLa-CDCP1- Y734F cells are the only CDCP1 expressing cells displaying constitutive phosphorylation of FAK-Y861. HeLa-CDCP1-Y762F cells displayed a very low metastatic ability which suggests that this CDCP1 tyrosine residue is important in mediating a pro-metastatic phenotype in HeLa cells. More detailed exploration of cellular events occurring downstream of CDCP1-Y734 and -Y762 may provide important insights into the mechanisms altering the metastatic ability of CDCP1 expressing HeLa cells. Complementing the in vivo studies, anti-CDCP1 antibodies were employed to assess whether these antibodies are able to inhibit metastasis of CDCP1 and CDCP1 tyrosine mutants expressing HeLa cells. It was found that HeLa- CDCP1-Y734F cells were the only cell line which was markedly reduced in the ability to metastasise. In contrast, the ability of HeLa-CDCP1, HeLa- CDCP1-Y743F and -Y762F cells to metastasise in vivo was not inhibited. These data suggest a possible role of interactions of CDCP1 with SFKs, occurring at CDCP1-Y734, in preventing an anti-metastatic effect of anti- CDCP1 antibodies in vivo. The proposal that SFKs may play a role in regulating anti-metastatic effects of anti-CDCP1 antibodies was supported by another experiment where differences between HeLa-CDCP1 cells and CDCP1 expressing HeLa cells (HeLa-CDCP1-S) from collaborators at the Scripps Research Institute were examined. It was found that HeLa-CDCP1-S cells express different SFKs than CDCP1 expressing HeLa cells generated for this study. This is important since HeLa-CDCP1-S cells can be inhibited in their metastatic ability using anti-CDCP1 antibodies in vivo. Importantly, these data suggest that further examinations of the roles of SFKs in facilitating anti-metastatic effects of anti-CDCP1 antibodies may give insights into how CDCP1 can be blocked to prevent metastasis in vivo. This project also explored the ability of the serine protease matriptase to proteolytically process cell surface localised CDCP1 because it is unknown whether matriptase can cleave cell surface CDCP1 as it has been reported for other proteases such as trypsin and plasmin. Furthermore, the consequences of matriptase-mediated proteolysis on cell phenotype in vitro and cell signalling were examined since recent reports suggested that proteolysis of CDCP1 leads to its phosphorylation and may initiate cell signalling and consequently alter cell phenotype. It was found that matriptase is able to proteolytically process cell surface CDCP1 at low nanomolar concentrations which suggests that cleavage of CDCP1 by matriptase may facilitate the generation of LWM-CDCP1 in vivo. To examine whether matriptase-mediated proteolysis induced cell signalling anti-phospho Erk 1/2 Western blot analysis was performed as this pathway has previously been examined to study signalling in response to proteolytic processing of cell surface proteins. It was found that matriptase-mediated proteolysis in CDCP1 expressing HeLa cells initiated intracellular signalling via Erk 1/2. Interestingly, this increase in phosphorylation of Erk 1/2 was also observed in HeLa-vector cells. This suggested that initiation of cell signalling via Erk 1/2 phosphorylation as a result of matriptase-mediated proteolysis occurs by pathways independent of CDCP1. Subsequent investigations measuring the flux of free calcium ions and by using a protease-activated receptor 2 (PAR2) agonist peptide confirmed this hypothesis. These data suggested that matriptase-mediated proteolysis results in cell signalling via a pathway induced by the activation of PAR2 rather than by CDCP1. This indicates that induction of cell signalling in HeLa cells as a consequence of matriptase-mediated proteolysis occurs via signalling pathways which do not involve phosphorylation of Erk 1/2. Consequently, it appears that future attempts should focus on the examination of cellular pathways other than Erk 1/2 to elucidate cell signalling initiated by matriptase-mediated proteolytic processing of CDCP1. The data presented in this thesis has explored in vitro and in vivo aspects of the biology of CDCP1. The observations summarised above will permit the design of future studies to more precisely determine the role of CDCP1 and its binding partners in processes relevant to cancer progression. This may contribute to further defining CDCP1 as a target for cancer treatment.

Subcellular localization of proteasomes and their regulatory complexes in mammalian cells

Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable c-interferoninducible catalytic b-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit speci®c antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core a-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some signi®cant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immuno¯uorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following cinterferon treatment of cultured cells but c-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes.

The effects of increased endurance training load on biomarkers of heat intolerance during intense exercise in the heat

The effects of increased training (IT) load on plasma concentrations of lipopolysaccharides (LPS), proinflammatory cytokines, and anti-LPS antibodies during exercise in the heat were investigated in 18 male runners, who performed 14 days of normal training (NT) or 14 days of 20% IT load in 2 equal groups. Before (trial 1) and after (trial 2) the training intervention, all subjects ran at 70% maximum oxygen uptake on a treadmill under hot (35 degrees C) and humid (similar to 40%) conditions, until core temperature reached 39.5 degrees C or volitional exhaustion. Venous blood samples were drawn before, after, and 1.5 h after exercise. Plasma LPS concentration after exercise increased by 71% (trial 1, p < 0.05) and 21% (trial 2) in the NT group and by 92% (trial 1, p < 0.01) and 199% (trial 2, p < 0.01) in the IT group. Postintervention plasma LPS concentration was 35% lower before exercise (p < 0.05) and 47% lower during recovery (p < 0.01) in the IT than in the NT group. Anti-LPS IgM concentration during recovery was 35% lower in the IT than in the NT group (p < 0.05). Plasma interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha concentrations after exercise (IL-6, 3-7 times, p < 0.01, and TNF-alpha, 33%, p < 0.01) and during recovery (IL-6, 2-4 times, p < 0.05, and TNF-alpha, 30%, p < 0.01) were higher than at rest within each group. These data suggest that a short-term tolerable increase in training load may protect against developing endotoxemia during exercise in the heat.

In vitro and in vivo studies on protease-activated receptor-2

Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) that is activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. Cleavage of this receptor exposes a neoepitope, termed the tethered ligand (TL), which binds intramolecularly within the receptor to stimulate signal transduction via coupled G proteins. PAR2-mediated signal transduction is also experimentally stimulated by hexapeptides (agonist peptides; APs) that are homologous to the TL sequence. Due to the irreversible nature of PAR2 proteolysis, downstream signal transduction is tightly regulated. Following activation, PAR2 is rapidly uncoupled from downstream signalling by the post-translational modifications phosphorylation and ubiquination which facilitate interactions with â- arrestin. This scaffolding protein couples PAR2 to the internalisation machinery initiating its desensitisation and trafficking through the early and late endosomes followed by receptor degradation. PAR2 is widely expressed in mammalian tissues with key roles for this receptor in cardiovascular, respiratory, nervous and musculoskeletal systems. This receptor has also been linked to pathological states with aberrant expression and signalling noted in several cancers. In prostate cancer, PAR2 signalling induces migration and proliferation of tumour derived cell lines, while elevated receptor expression has been noted in malignant tissues. Importantly, a role for this receptor has also been suggested in prostate cancer bone metastasis as coexpression of PAR2 and a proteolytic activator has been demonstrated by immunohistochemical analysis. Based on these data, the primary focus of this project has been on two aspects of PAR2 biology. The first is characterisation of cellular mechanisms that regulate PAR2 signalling and trafficking. The second aspect is the role of this receptor in prostate cancer bone metastasis. In addition, to permit these studies, it was first necessary to evaluate the specificity of the commercially available anti-PAR2 antibodies SAM11, C17, N19 and H99. The evaluation of the four commercially available antibodies was assessed using four techniques: immunoprecipitation; Western blot analysis; immunofluorescence; and flow cytometry. These approaches demonstrated that three of the antibodies efficiently detect ectopically expressed PAR2 by each of these techniques. A significant finding from this study was that N19 was the only antibody able to specifically detect N-glycosylated endogenous PAR2 by Western blot analysis. This analysis was performed on lysates from prostate cancer derived cell lines and tissue derived from wildtype and PAR2 knockout mice. Importantly, further evaluation demonstrated that this antibody also efficiently detects endogenous PAR2 at the cell surface by flow cytometry. The anti-PAR2 antibody N19 was used to explore the in vitro role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Significantly, use of the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling experiments using two approaches which showed that PAR2 stably expressed by CHO cells is palmitoylated and that palmitoylation occurs on cysteine 361. Another key finding from this study is that palmitoylation is required for optimal PAR2 signalling as Ca2+ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ~9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. Importantly, this study also identified that palmitoylation of this receptor within the Golgi apparatus is required for efficient agonist-induced rab11amediated trafficking of PAR2 to the cell surface. Interestingly, palmitoylation is also required for receptor desensitization, as agonist-induced â-arrestin recruitment and receptor degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. Collectively, these data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor. This project also evaluated PAR2 residues involved in ligand docking. Although the extracellular loop (ECL)2 of PAR2 is known to be required for agonist-induced signal transduction, the binding pocket for receptor agonists remains to be determined. In silico homology modelling, based on a crystal structure for the prototypical GPCR rhodopsin, and ligand docking were performed to identify PAR2 transmembrane (TM) amino acids potentially involved in agonist binding. These methods identified 12 candidate residues that were mutated to examine the binding site of the PAR2 TL, revealed by trypsin cleavage, as well as of the soluble ligands 2f-LIGRLO-NH2 and GB110, which are both structurally based on the AP SLIGRLNH2. Ligand binding was evaluated from the impact of the mutated residues on PAR2-mediated calcium mobilisation. An important finding from these experiments was that mutation of residues Y156 and Y326 significantly reduced 2f-LIGRLO-NH2 and GB110 agonist activity. L307 was also important for GB110 activity. Intriguingly, mutation of PAR2 residues did not alter trypsin-induced signalling to the same extent as for the soluble agonists. The reason for this difference remains to be further examined by in silico and in vitro experimentation and, potentially, crystal structure studies. However, these findings identified the importance of TM domains in PAR2 ligand docking and will enhance the design of both PAR2 agonists and potentially agents to inhibit signalling (antagonists). The potential importance of PAR2 in prostate cancer bone metastasis was examined using a mouse model. In patients, prostate cancer bone metastases cause bone growth by disrupting bone homeostasis. In an attempt to mimic prostate cancer growth in bone, PAR2 responsive 22Rv1 prostate cancer cells, which form mixed osteoblastic and osteolytic lesions, were injected into the proximal aspect of mouse tibiae. A role for PAR2 was assessed by treating these mice with the recently developed PAR2 antagonist GB88. As controls, animals bearing intra-tibial tumours were also treated with vehicle (olive oil) or the prostate cancer chemotherapeutic docetaxel. The effect of these treatments on bone was examined radiographically and by micro-CT. Consistent with previous studies, 22Rv1 tumours caused osteoblastic periosteal spicule formation and concurrent osteolytic bone loss. Significantly, blockade of PAR2 signalling reduced the osteoblastic and osteolytic phenotype of 22Rv1 tumours in bone. No bone defects were detected in mice treated with docetaxel. These qualitative data will be followed in the future by quantitative micro-CT analysis as well as histology and histomorphometry analysis of already collected tissues. Nonetheless, these preliminary experiments highlight a potential role for PAR2 in prostate cancer growth in bone. In summary, in vitro studies have defined mechanisms regulating PAR2 activation, downstream signalling and trafficking and in vivo studies point to a potential role for this receptor in prostate cancer bone metastasis. The outcomes of this project are that a greater understanding of the biology of PAR2 may lead to the development of strategies to modulate the function of this receptor in disease.

Ultra-trace detection of diagnostically important biomarkers using functionalised-Surface Enhanced Raman Spectroscopy (SERS)

Here we report an ultrasensitive method for detecting bio-active compounds in biological samples by means of functionalised nanoparticles interrogated by surface enhanced Raman spectroscopy (SERS). This method is applicable to the recovery and detection of many diagnostically important peptidyl analytes such as insulin, human growth hormone, growth factors (IGFs) and erythropoietin (EPO), as well as many small molecule analytes and metabolites. Our method, developed to detect EPO, demonstrates its utility in a complex yet well defined biological system. Recombinant human EPO (rhEPO) and EPO analogues have successfully been used to treat anaemia in end-stage renal failure, chronic disorders and infections, cancer and AIDS. Current methods for EPO testing are lengthy, laborious and relatively insensitive to low concentrations. In our rapid screening methodology, gold nanoparticles were functionalised with anti-EPO antibodies to provide very high selectivity towards the EPO protein in urine. These “smart sensor” nanoparticles interact with and trap EPO. Subsequent SERS screening allows for the detection and quantisation of ultra trace amounts (<<10-15 M) of EPO in urine samples with minimal sample preparation. We present data showing that the SERS spectrum differentiates between human endogenous EPO and rhEPO in unpurified urine, and potentially distinguishes between purified EPO isoforms. The elimination of sample preparation and direct screening in biological fluids significantly reduces the time required by current methods. Antibody recognition against a variety of biological targets and the availability of portable commercial SERS analysers for rapid onsite testing suggest broad diagnostic applicability in a flexible analytical platform.

The function and mechanisms of action of ghrelin and obestatin in ovarian cancer

In this study, we have demonstrated that the preproghrelin derived hormones, ghrelin and obestatin, may play a role in ovarian cancer. Ghrelin and obestatin stimulated an increase in cell migration in ovarian cancer cell lines and may play a role in cancer progression. Ovarian cancer is the leading cause of death among gynaecological cancers and is the sixth most common cause of cancer-related deaths in women in developed countries. As ovarian cancer is difficult to diagnose at a low tumour grade, two thirds of ovarian cancers are not diagnosed until the late stages of cancer development resulting in a poor prognosis for the patient. As a result, current treatment methods are limited and not ideal. There is an urgent need for improved diagnostic markers, as well better therapeutic approaches and adjunctive therapies for this disease. Ghrelin has a number of important physiological effects, including roles in appetite regulation and the stimulation of growth hormone release. It is also involved in regulating the immune, cardiovascular and reproductive systems and regulates sleep, memory and anxiety, and energy metabolism. Over the last decade, the ghrelin axis, (which includes the hormones ghrelin and obestatin and their receptors), has been implicated in the pathogenesis of many human diseases and it may t may also play an important role in the development of cancer. Ghrelin is a 28 amino acid peptide hormone that exists in two forms. Acyl ghrelin (usually referred to as ghrelin), has a unique n-octanoic acid post-translational modification (which is catalysed by ghrelin O-acyltransferase, GOAT), and desacyl ghrelin, which is a non-octanoylated form. Octanoylated ghrelin acts through the growth hormone secretagogue receptor type 1a (GHSR1a). GHSR1b, an alternatively spliced isoform of GHSR, is C-terminally truncated and does not bind ghrelin. Ghrelin has been implicated in the pathophysiology of a number of diseases Obestatin is a 23 amino acid, C-terminally amidated peptide which is derived from preproghrelin. Although GPR39 was originally thought to be the obestatin receptor this has been disproven, and its receptor remains unknown. Obestatin may have as diverse range of roles as ghrelin. Obestatin improves memory, inhibits thirst and anxiety, increases pancreatic juice secretion and has cardioprotective effects. Obestatin also has been shown to regulate cell proliferation, differentiation and apoptosis in some cell types. Prior to this study, little was known regarding the functions and mechanisms of action ghrelin and obestatin in ovarian cancer. In this study it was demonstrated that the full length ghrelin, GHSR1b and GOAT mRNA transcripts were expressed in all of the ovarian-derived cell lines examined (SKOV3, OV-MZ-6 and hOSE 17.1), however, these cell lines did not express GHSR1a. Ovarian cancer tissue of varying stages and normal ovarian tissue expressed the coding region for ghrelin, obestatin, and GOAT, but not GHSR1a, or GHSR1b. No correlations between cancer grade and the level of expression of these transcripts were observed. This study demonstrated for the first time that both ghrelin and obestatin increase cell migration in ovarian cancer cell lines. Treatment with ghrelin (for 72 hours) significantly increased cell migration in the SKOV3 and OV-MZ-6 ovarian cancer cell lines. Ghrelin (100 nM) stimulated cell migration in the SKOV3 (2.64 +/- 1.08 fold, p <0.05) and OV-MZ-6 (1.65 +/- 0.31 fold, p <0.05) ovarian cancer cell lines, but not in the representative normal cell line hOSE 17.1. This increase in migration was not accompanied by an increase in cell invasion through Matrigel. In contrast to other cancer types, ghrelin had no effect on proliferation. Ghrelin treatment (10nM) significantly decreased attachment of the SKOV3 ovarian cancer cell line to collagen IV (24.7 +/- 10.0 %, p <0.05), however, there were no changes in attachment to the other extracellular matrix molecules (ECM) tested (fibronectin, vitronectin and collagen I), and there were no changes in attachment to any of the ECM molecules in the OV-MZ-6 or hOSE 17.1 cell lines. It is, therefore, unclear if ghrelin plays a role in cell attachment in ovarian cancer. As ghrelin has previously been demonstrated to signal through the ERK1/2 pathway in cancer, we investigated ERK1/2 signalling in ovarian cancer cell lines. In the SKOV3 ovarian cancer cell line, a reduction in ERK1/2 phosphorylation (0.58 fold +/- 0.23, p <0.05) in response to 100 nM ghrelin treatment was observed, while no significant change in ERK1/2 signalling was seen in the OV-MZ-6 cell line with treatment. This suggests that this pathway is unlikely to be involved in mediating the increased migration seen in the ovarian cancer cell lines with ghrelin treatment. In this study ovarian cancer tissue of varying stages and normal ovarian tissue expressed the coding region for obestatin, however, no correlation between cancer grade and level of obestatin transcript expression was observed. In the ovarian-derived cell lines studied (SKOV3, OV-MZ-6 and hOSE 17.1) it was demonstrated that the full length preproghrelin mRNA transcripts were expressed in all cell lines, suggesting they have the ability to produce mature obestatin. This is the first study to demonstrate that obestatin stimulates cell migration and cell invasion. Obestatin induced a significant increase in migration in the SKOV3 ovarian cancer cell line with 10 nM (2.80 +/- 0.52 fold, p <0.05) and 100 nM treatments (3.12 +/- 0.68 fold, p <0.05) and in the OV-MZ-6 cancer cell line with 10 nM (2.04 +/- 0.10 fold, p <0.01) and 100 nM treatments (2.00 +/- 0.37 fold, p <0.05). Obestatin treatment did no affect cell migration in the hOSE 17.1normal ovarian epithelial cell line. Obestatin treatment (100 nM) also stimulated a significant increase in cell invasion in the OV-MZ-6 ovarian cancer cell line (1.45 fold +/- 0.13, p <0.05) and in the hOSE17.1 normal ovarian cell line cells (1.40 fold +/- 0.04 and 1.55 fold +/- 0.05 respectively, p <0.01) with 10 nM and 100 nM treatments. Obestatin treatment did not stimulate cell invasion in the SKOV3 ovarian cancer cell line. This lack of obestatin-stimulated invasion in the SKOV3 cell line may be a cell line specific result. In this study, obestatin did not stimulate cell proliferation in the ovarian cell lines and it has previously been shown to have no effect on cell proliferation in the BON-1 pancreatic neuroendocrine and GC rat somatotroph tumour cell lines. In contrast, obestatin has been shown to affect cell proliferation in gastric and thyroid cancer cell lines, and in some normal cell lines. Obestatin also had no effect on attachment of any of the cell lines to any of the ECM components tested (fibronectin, vitronectin, collagen I and collagen IV). The mechanism of action of obestatin was investigated further using a two dimensional-difference in gel electrophoresis (2D-DIGE) proteomic approach. After treatment with obestating (0, 10 and 100 nM), SKOV3 ovarian cancer and hOSE 17.1 normal ovarian cell lines were collected and 2D-DIGE analysis and mass spectrometry were performed to identify proteins that were differentially expressed in response to treatment. Twenty-six differentially expressed proteins were identified and analysed using Ingenuity Pathway Analysis (IPA). This linked 16 of these proteins in a network. The analysis suggested that the ERK1/2 MAPK pathway was a major mediator of obestatin action. ERK1/2 has previously been shown to be associated with obestatin-stimulated cell proliferation and with the anti-apoptotic effects of obestatin. Activation of the ERK1/2 signalling pathway by obestatin was, therefore, investigated in the SKOV3 and OV-MZ-6 ovarian cancer cell lines using anti-active antibodies and Western immunoblots. Obestatin treatment significantly decreased ERK1/2 phosphorylation at higher obestatin concentrations in both the SKOV3 (100 nM and 1000 nM) and OV-MZ-6 (1000 nM) cell lines compared to the untreated controls. Currently, very little is known about obestatin signalling in cancer. This thesis has demonstrated for the first time that the ghrelin axis may play a role in ovarian cancer migration. Ghrelin and obestatin increased cell migration in ovarian cancer cell lines, indicating that they may be a useful target for therapies that reduce ovarian cancer progression. Further studies investigating the role of the ghrelin axis using in vivo ovarian cancer metastasis models are warranted.

Use of genetically modified muscle and fat grafts to repair defects in bone and cartilage

We report a novel technology for the rapid healing of large osseous and chondral defects, based upon the genetic modification of autologous skeletal muscle and fat grafts. These tissues were selected because they not only possess mesenchymal progenitor cells and scaffolding properties, but also can be biopsied, genetically modified and returned to the patient in a single operative session. First generation adenovirus vector carrying cDNA encoding human bone morphogenetic protein-2 (Ad.BMP-2) was used for gene transfer to biopsies of muscle and fat. To assess bone healing, the genetically modified (“gene activated”) tissues were implanted into 5mm-long critical size, mid-diaphyseal, stabilized defects in the femora of Fischer rats. Unlike control defects, those receiving gene-activated muscle underwent rapid healing, with evidence of radiologic bridging as early as 10 days after implantation and restoration of full mechanical strength by 8 weeks. Histologic analysis suggests that the grafts rapidly differentiated into cartilage, followed by efficient endochondral ossification. Fluorescence in situ hybridization detection of Y-chromosomes following the transfer of male donor muscle into female rats demonstrated that at least some of the osteoblasts of the healed bone were derived from donor muscle. Gene activated fat also healed critical sized defects, but less quickly than muscle and with more variability. Anti-adenovirus antibodies were not detected. Pilot studies in a rabbit osteochondral defect model demonstrated the promise of this technology for healing cartilage defects. Further development of these methods should provide ways to heal bone and cartilage more expeditiously, and at lower cost, than is presently possible.

Targeting cancer with a lupus autoantibody

Systemic lupus erythematosus (SLE) is distinct among autoimmune diseases because of its association with circulating autoantibodies reactive against host DNA. The precise role that anti-DNA antibodies play in SLE pathophysiology remains to be elucidated, and potential applications of lupus autoantibodies in cancer therapy have not previously been explored. We report the unexpected finding that a cell-penetrating lupus autoantibody, 3E10, has potential as a targeted therapy for DNA repair–deficient malignancies. We find that 3E10 preferentially binds DNA single-strand tails, inhibits key steps in DNA single-strand and double-strand break repair, and sensitizes cultured tumor cells and human tumor xenografts to DNA-damaging therapy, including doxorubicin and radiation. Moreover, we demonstrate that 3E10 alone is synthetically lethal to BRCA2-deficient human cancer cells and selectively sensitizes such cells to low-dose doxorubicin. Our results establish an approach to cancer therapy that we expect will be particularly applicable to BRCA2-related malignancies such as breast, ovarian, and prostate cancers. In addition, our findings raise the possibility that lupus autoantibodies may be partly responsible for the intrinsic deficiencies in DNA repair and the unexpectedly low rates of breast, ovarian, and prostate cancers observed in SLE patients. In summary, this study provides the basis for the potential use of a lupus anti-DNA antibody in cancer therapy and identifies lupus autoantibodies as a potentially rich source of therapeutic agents.

Modelling the dynamics of Plasmodium falciparum histidine-rich protein 2 in human malaria to better understand malaria rapid diagnostic test performance

BACKGROUND: Effective diagnosis of malaria is a major component of case management. Rapid diagnostic tests (RDTs) based on Plasmodium falciparumhistidine-rich protein 2 (PfHRP2) are popular for diagnosis of this most virulent malaria infection. However, concerns have been raised about the longevity of the PfHRP2 antigenaemia following curative treatment in endemic regions. METHODS: A model of PfHRP2 production and decay was developed to mimic the kinetics of PfHRP2 antigenaemia during infections. Data from two human infection studies was used to fit the model, and to investigate PfHRP2 kinetics. Four malaria RDTs were assessed in the laboratory to determine the minimum detectable concentration of PfHRP2. RESULTS: Fitting of the PfHRP2 dynamics model indicated that in malaria naive hosts, P. falciparum parasites of the 3D7 strain produce 1.4 x 10(-)(1)(3) g of PfHRP2 per parasite per replication cycle. The four RDTs had minimum detection thresholds between 6.9 and 27.8 ng/mL. Combining these detection thresholds with the kinetics of PfHRP2, it is predicted that as few as 8 parasites/muL may be required to maintain a positive RDT in a chronic infection. CONCLUSIONS: The results of the model indicate that good quality PfHRP2-based RDTs should be able to detect parasites on the first day of symptoms, and that the persistence of the antigen will cause the tests to remain positive for at least seven days after treatment. The duration of a positive test result following curative treatment is dependent on the duration and density of parasitaemia prior to treatment and the presence and affinity of anti-PfHRP2 antibodies.

The glycosphingolipid P1 is an ovarian cancer-associated carbohydrate antigen involved in migration

Background The level of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P1 trisaccharide has previously been shown to significantly discriminate between ovarian cancer patients and healthy women. Here we aim to identify the Ig class that causes this discrimination, to identify on cancer cells the corresponding P1 antigen recognised by circulating anti-P1 antibodies and to shed light into the possible function of this glycosphingolipid. Method An independent Australian cohort was assessed for the presence of anti-P1 IgG and IgM class antibodies using suspension array. Monoclonal and human derived anti-glycan antibodies were verified using three independent glycan-based immunoassays and flow cytometry-based inhibition assay. The P1 antigen was detected by LC-MS/MS and flow cytometry. FACS-sorted cell lines were studied on the cellular migration by colorimetric assay and real-time measurement using xCELLigence system. Results Here we show in a second independent cohort (n=155) that the discrimination of cancer patients is mediated by the IgM class of anti-P1 antibodies (P=0.0002). The presence of corresponding antigen P1 and structurally related epitopes in fresh tissue specimens and cultured cancer cells is demonstrated. We further link the antibody and antigen (P1) by showing that human naturally circulating and affinity-purified anti-P1 IgM isolated from patients ascites can bind to naturally expressed P1 on the cell surface of ovarian cancer cells. Cell-sorted IGROV1 was used to obtain two study subpopulations (P1-high, 66.1%; and P1-low, 33.3%) and observed that cells expressing high P1-levels migrate significantly faster than those with low P1-levels. Conclusions This is the first report showing that P1 antigen, known to be expressed on erythrocytes only, is also present on ovarian cancer cells. This suggests that P1 is a novel tumour-associated carbohydrate antigen recognised by the immune system in patients and may have a role in cell migration. The clinical value of our data may be both diagnostic and prognostic; patients with low anti-P1 IgM antibodies present with a more aggressive phenotype and earlier relapse.