Dissertations / Theses on the topic 'Mesothelial'

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2006.
"June 2006."
Includes bibliographical references (p. 63-64).
In the pleural space, sliding between the lung and chest wall induces shear stress that could damage the delicate mesothelial cells covering the tissue surfaces. Normally, the pleural space, which is filled with fluid, is able to sustain continuous shear loading throughout its lifetime. To understand the mechanisms in preventing frictional damage on mesothelial tissue, we conducted experiments using abdominal tissue excised from a rat. We allowed the tissue to slide against a glass surface, and measured the fluid thickness and shear force between them. We also studied independent variables such as location on the tissue, applied normal load, sliding velocity and direction to determine their effects on mesothelial tissue lubrication. Both thickening and thinning of the fluid layer were observed during sliding. The fluid thickness was found to change with sliding velocity and direction, but invariant with location on tissue surface. In tribological experiments, shear force decreased with increasing velocity until it reached a minimum value varying with different tissue samples. Normal load had a strong effect on shear force, but not on friction coefficient.
(cont.) Overall, the friction curves had similar shape as described by the mixed/elasto-hydrodynamic regions of the Stribeck curve. Results were consistent within each tissue sample, but varied among samples. The dependency on velocity and direction suggested elasto-hydrodynamic lubrication. Taken together, we conclude that elasto-hydrodynamic lubrication is likely to be an important lubrication mechanism for mesothelial tissue sliding in the pleural region. Our findings support the existence of a continuous fluid layer separating the pleural surfaces. The fluid pressure gradient generated by surface roughness redistributes fluid from thick to thin regions preventing surface contact.
by Judy Li-Wen Lin.
S.M.

Metastatic dissemination of ovarian tumors involves the invasion of multi-cellular tumor cell clusters into the mesothelial cell lining of organs in the peritoneal cavity. We developed an in vitro assay that models this initial step of ovarian cancer metastasis to investigate the mechanisms of invasion. Pre-clustered ovarian cancer multicellular spheroids are incubated with GFP-expressing mesothelial monolayers and the extent of mesothelial invasion is monitored by time-lapse video microscopy.

Thesis (Ph.D.)--Boston University
Mesothelium-derived progenitors have been demonstrated to contribute to differentiated mesenchymal components of the heart, liver, and gut during organogenesis. The precise contribution of the mesothelium to lung development, however, has not been fully clarified and the key signals regulating mesothelial cell entry have not been identified. To rigorously address this issue, we employed mice with an inducible Cre expressed from the Wilm's tumor-1 (WT1) locus for high fidelity lineage tracing after confirming that Cre-recombinase was mesothelial-specific and faithfully recapitulated endogenous WT1 gene expression. We visualized WT1+ mesothelial cell entry into the fetal lung by live imaging and identified their progenies in subpopulations of bronchial smooth muscle cells, vascular smooth muscle cells, and desmin+ fibroblasts by lineage tagging. In view of the role of Sonic Hedgehog (Hh) signaling in regulating mesenchymal cell differentiation and epithelial-mesenchymal transition, we hypothesized that this pathway regulates events associated with migration of mesothelial cells into the developing lung. To examine for this, we first used two independent reporter mice to show that Hh signaling is active within the lung mesothelium at time points coinciding with the appearance of mesothelium-derived cells in the lung parenchyma. Using loss-of-function assays in organ cultures, and targeted mesothelial-restricted loss-of hedgehog function mice, we demonstrated that mesothelial cell movement into the lung requires the direct action of Hh signaling. In order to examine whether WT1 interacts with Hh pathway, we conducted ChIP assays on fetal lung mesothelial cells, and found that WT1 directly binds and regulates promoter elements of downstream targets of Hh pathway. Consistent with this observation, Hh pathway gene expression was down-regulated in isolated WT1 deficient fetal lung mesothelial cells. Taken together, these findings lend further support to a paradigm in which mesothelial cells are an important source of progenitors for mesenchymal structures. Our findings also reveal a role for Hh pathway in the early events associated with mesothelial cell entry and indicate that WT1 likely acts upstream of Hh signaling.

This thesis consists of three studies on the synthesis of proteoglycans (PGs) and hyaluronan (HA) in relation to peritoneal dialysis. In the first study, newly synthesized proteoglycans from human peritoneal mesothelial cells were labelled in vitro with either ³⁵S-sulphate, ³H-glucosamine or ³⁵S-methionine for 24h, and were characterised using biochemical and immunological techniques. The following were identified: i). a large chondroitin sulphate proteoglycan (CSPG I), Mr > 1000 kDa, protein core > 400 kDA, which was present in both the culture medium (CM) and the cell layer extract (CL). ii). a minor dermatan sulphate proteoglycan (DSPG I), Mr 200 kDa, protein core 45 kDa, also present in the CM and identified as biglycan. iii). a second small dermatan sulphate proteoglycan (DSPG I), Mr 110 kDa, protein core 43 and 47 kDa, and found solely in theCM. This proteoglycan, identified as decorin, was thepredominant species. iv). a large heparan sulphate proteoglycan (HSPG I), Mr 1200 kDa, isolated in both the CM and in the cytoskeletal-matrix which may be related to perlecan. v). a cell surface hybrid proteoglycan (containing both HS and CS chains), Mr 190-210 kDa, and identified as syndecan. vi). a glycosylphosphatidylinositol-anchored heparan sulphate (HSPF II), Mr ≈190 kDa and identified as glypican. In addition to these proteoglycans, a large amount of free CS/HS GAGs were present (70% of the total CL). Pulse chase data revealed that these GAG chains were undergoing degradation. In the second study, proteoglycans were isolated from CAPD fluid and characterised. The results revealed that decorin was the predominant species with minor amounts of biglycan. Finally, the presence of HA in CAPD fluid was monitored. Results showed that the HA levels are elevated during peritonitis, but with successful treatment, the levels returned to normal. In vitro studies suggested that peritoneal mesothelial cells were the likely source of the HA.

Mesothelial cells have been described to possess progenitor characteristics and contribute to regeneration through differentiation. However, it is not clear what the effects of prolonged culture have on the mesothelial cell properties and relative plasticity; as long-term cultures have not yet been established as a result of early senescence. Understanding the effects of time in culture is crucial for the development of novel therapies. In this thesis, we demonstrated that mesothelial cell cultures isolated from murine omentum could be cultured for more than 40 passages and showed relatively stable population doubling times. While initially, the cells did down-regulated the expression of mesothelial markers Wilm’s tumor protein 1 (Wt1) and mesothelin and epithelial genes; their mesenchymal profile was maintained. This along with the increased Snail2 expression suggested the cells were progressing through the mesothelial to mesenchymal (MMT) transdifferentiation programme. TGF-β and more recently EGF are known mediators of MMT. Targeting signalling through these receptors with small molecule inhibitors LY364947 and PD153035, slowed the rate of gap closure, in vitro and increased zonula occludens 1 accumulation at cell-cell contacts. Which seemed to be mediated through the MEK5/ERK5 pathway. Furthermore, siRNA-mediated transient knockdown of Zeb1 and Zeb2 transcription factors also achieved attenuated the rate of migration. Next, we moved onto to accessing the progenitor properties of the mesothelial cells with time in culture. The expression of stem cells markers Bmi1 (Proto-Oncogene, Polycomb Ring Finger), Sox9 ((Sex Determining Region Y)-Box 9) and CD34 were downregulated with repeated passaging. However, the low passage mesothelial cells exhibited clonogenicity and could differentiate into osteoblasts and adipocytes. Finally, in an embryonic kidney rudiment assay, we could show that the mesothelial cells co-existed with the embryonic kidney cells and allowed renal structures to form in their presence. Ultimately, we have demonstrated that the mesothelial cells from the omentum maintain some mesothelial characteristics with prolonged culturing. The cells showed clonogenic and multi-lineage potential and expressed a number of stem cell genes. The MMT programme is complex and could be partly reversed by targeting TGF-βR1 and EGFR tyrosine kinases, which along with transiently silencing the Zeb transcriptional factors seem likely key targets in ameliorating pathological fibrosis.

Malignant mesothelioma (MM) is a major health problem as it is an invariably fatal disease resulting from occupational exposure to asbestos. The long latency period of this disease means that death rates will continue to rise for 10-20 years before improved exposure regulations take effect. The studies described here were designed to explore mechanisms by which asbestos exposure elicits this malignancy. Cell signalling events germane to malignant transformation were investigated in rat (4/4 RM4) and human (MET5A) mesothelial cells in vitro following exposure to asbestos. The activation state of the MAPK family and Akt were probed, because these pathways are pivotal in determining death or survival of the cell. The results suggest that extracellular signal regulated protein kinase (ERK), p38 and Akt are activated by asbestos exposure. For the former two, at least, this activation depended on oxidative stress. A selective inhibitor of EGFR tyrosine kinase, PKI166, inhibited asbestos-mediated Akt and ERK activation. Asbestos-mediated Akt activation was also inhibited by LY294002, an inhibitor of phosphatidylinositol 3-kinase (P13K). The effect of events triggered by asbestos downstream of these kinases, on the transcription factors activator protein (AP) -1 and nuclear factor-kB (NF-kB) were investigated. Pharmacological inhibition of either the ERK pathway by UO126 or the p38 pathway by SB203580 ameliorated crocidolite-induced AP-1 activation. Inhibition of the Akt pathway by LY294002 or PKI166 reduced crocidolite-induced NF-kB translocation. The same panel of inhibitors were used to investigate the role of these pathways in defined endpoints characteristic of asbestos exposure i.e. cell death and survival/proliferation. In both cell lines the p38 pathway was involved in cell death and the Akt pathway in survival. In 4/4 RM4 cells activation of the ERK pathway also induced cell death. In contrast, in MET5A cells ERK appeared to be involved in survival.

Encapsulating peritoneal sclerosis (EPS) is a rare complication of long-term peritoneal dialysis (PD). EPS is associated with extensive thickening and fibrosis of the peritoneum resulting in the formation of a fibrous cocoon encapsulating the bowel leading to intestinal obstruction. The presence of peritoneal thickening, peritoneal calcification and bowel obstruction is considered to be diagnostic of EPS. The current understanding of the pathogenesis of EPS is through the 'two-hit' fibrosis model. This model, however, does not explain the development of peritoneal calcification in patients with EPS. This thesis addresses the hypothesis that altered bone mineral metabolism in ESRF patients together with the mechanical stress of PD influences mesothelial cells to differentiate into osteoblasts promoting calcification in peritoneal tissue. Peritoneal calcification leads to increased tissue stiffness causing progressive fibrosis and the development of EPS. We compared the temporal evolution of the levels of bone mineral markers during PD between patients who developed EPS and control patients on PD. We found that raised serum levels of calcium, phosphate and alkaline phosphatase during PD increased the risk of development of EPS. We compared peritoneum from patients with EPS with that of PD patients without EPS using histological techniques. We found that calcification, organised fibrillary collagen and elastic fibres were significantly more abundant in the EPS peritoneum. Peritoneal calcification was also generalised and distributed not only on the peritoneal surface but also in the sub-mesothelial zone of fibrosis. EPS peritoneum also exhibited osteocalcin, an osteogenic protein, suggesting a cellular mechanism of calcification. Atomic force microscopy of EPS peritoneum showed increased stiffness when compared to control PD peritoneum with the areas of calcification possibly contributing to the increase in tissue stiffness. Human omental cells (HOMCs) were isolated by protease digestion and characterised using a panel of mesothelial markers. HOMCs were cultured in phosphate rich media and phosphate and calcium rich media. HOMCs when cultured with high extracellular levels of calcium showed accelerated mineralisation with upregulation of osteogenic transcription factor runx-2 suggesting osteoblastic transformation. In summary, this thesis indicates that poorly controlled secondary hyperparathyroidism is a risk factor for the development of EPS. On a background of PD related simple sclerosis, uncontrolled secondary hyperparathyroidism can lead to the transformation of mesothelial cells to osteoblasts. This leads to increased matrix deposition and matrix mineralisation causing increased matrix stiffness. Increase in matrix stiffness leads to progressive fibrosis culminating in EPS. Peritoneal calcification can act as the second hit leading to progressive fibrosis and development of EPS.

Worldwide 100,000 people are dependent on peritoneal dialysis (PD) for treatment of end-stage renal failure. Long term technique survival is limited by peritoneal membrane fibrosis and loss of membrane function. The human peritoneal mesothelial cell (HPMC) is the one of the most abundant cells in the peritoneal cavity, and is in direct contact with the peritoneal dialysate. The aim of these experiments was to investigate the role of the HPMC in the regulation of peritoneal fibrosis in the context of peritoneal dialysis. Hyperosmolar glucose lactate-buffered dialysate is the most widely used dialysis solution. A culture system was developed to grow HPMC from uraemic patients undergoing PD catheter insertion. The effect exposure to a 50:50 mixture of dialysate and M199 for 12 hours was examined. Glucose was varied between 5-40mmol/L. Increases in glucose concentration caused a decrease in cell viability, a decrease in proliferation, and increase in fibronectin mRNA and protein amount. The mechanism responsible for glucose induced increase in fibronectin was examined. Increasing glucose caused an increase in HPMC TGF- protein amount. Exogenous TGF- caused a dose dependent increase in HPMC fibronectin production, and increase mRNA for fibronectin and TGF- itself. An anti TGF- Antibody prevented glucose induced HPMC fibronectin production. Two alternative dialysis solutions were investigated; a different osmotic agent (amino acid dialysate), and a different dialysate buffer (bicarbonate). Amino acid dialysate showed less cytotoxicity, but inhibited proliferation, and caused TGF- mediated fibronectin production. Although amino acid dialysate contains 3.3 mmo1/L L-arginine, NO was not shown to mediate this response. Dialysate with bicarbonate:lactate buffer allowed greater HPMC proliferation, and no inhibition of proliferation with hyperosmolar glucose previously seen with lactate buffered dialysate. These studies suggest the HPMC has a role in the production and regulation of ECM, and that TGF- is an important intermediary.

Malignant Mesothelioma (MM) is a fatal disease with a low median survival between 8 to 12 months after diagnosis. MM has a long latency period (10-60 years), is causally related to asbestos exposure, and is refractory to all available modes of therapy. Despite the causal association between asbestos exposure and MM however, the mechanisms by which asbestos induces this deadly disease remain unclear. Chronic inflammation due to the presence of asbestos fibers is believed to play an important role in all aspects of MM pathogenesis, from development to progression and resistance. Chronic inflammation has been shown to promote dysregulated wound repair, fibrosis and epithelial to mesenchymal transition (EMT). One of the inflammatory pathways that asbestos activates is the inflammasome (a multiprotein scaffold that assembles in response to various stimuli to facilitate the activation of caspase-1), which has been implicated in several chronic inflammatory diseases and disorders. The nucleotide binding oligomerization domain (NOD) - like receptor containing a pyrin domain 3 (NLRP3) inflammasome, both as a whole or via its components [NLRP3, apoptosis related speck-like protein containing a CARD (caspase activating and recruitment domain) (ASC) and caspase-1] as well as its products, IL-1β and IL-18, has been implicated in the development of EMT during chronic inflammation. Asbestos fibers, especially the amphiboles, are non-biodegradable and thus persist in tissues of the body for years after exposure. In mesothelial cells, the squamous epithelial-like cells that line the serosal cavities of the body, from which MM originates, asbestos chronically activates the NLRP3 inflammasome. Asbestos also activates the NLRP3 inflammasome in human macrophages that can lead to the establishment of a chronic inflammation environment. We therefore hypothesized that asbestos dependent regulation of the inflammasome played a role in mesothelial to fibroblastic transition to facilitate eventual neoplastic transformation of the mesothelial cells. Using in vitro models, siRNA knockdown approaches as well as in vivo models of asbestos exposure utilizing inflammasome component knockout mice, we demonstrate that asbestos-induced reactive oxygen species generation modulates the redox state of the endogenous antioxidant, thioredoxin, causing its dissociation from thioredoxin interacting protein to promote activation of the inflammasome. We also show that the inflammasome plays a role in asbestos-induced mesothelial to fibroblastic transition (MFT) (a form of EMT occurring in the mesothelial cells) both in vitro and in vivo with a requirement for caspase-1 in vivo to promote thickening of the submesothelium. Through our studies, we have identified tissue factor pathway inhibitor 2 (TFPI2) and fibroblast growth factor 2 (FGF2) as molecules that are upregulated in response to asbestos exposure with potential roles in the progression of asbestos-induced MFT. There is a dearth of diagnostic biomarkers that enable early detection of MM, thus with further studies these two molecules could be explored as biomarkers of asbestos exposure/disease progression. TFPI2 levels were downregulated in response to blockage of IL-1β signaling and thus could be harnessed as a potential marker for therapy efficiency with further studies.

Post-operative adhesion is a common complication after abdominal surgery, with high impact on patient wellbeing and healthcare costs. The repair of peritoneum is a complex process involving orderly phases which share some common features to normal wound healing. These include coagulation, infiltration of inflammatory cells, cell proliferation, extracellular matrix (ECM) deposition and remodelling, often with overlap between phases. The unique feature of peritoneal repair is that both small and large peritoneal wounds heal in a similar time. The peritoneum is a monolayer of elongated, flattened, squamous-like peritoneal mesothelial cells (PMC). Local mesothelial cell proliferation, centripetal cell migration from the wound edge, as well as incorporation of free-floating mesothelial cells may all contribute to repair of injured peritoneum. To date, the only well-characterised pathologic mechanism underlying post-operative adhesion formation at the molecular level is the formation of the fibrin layer and regulation of peritoneal fibrinolytic capacity. However, the contributions of collagen deposition and ECM remodelling to the peritoneal repair mechanism are not well understood. This thesis focuses on the role of PMC in the regulation of ECM deposition and remodelling in response to inflammatory stimuli in both in vivo and in vitro models, aiming to identify other key pro-fibrotic factors involved in the development of post-operative adhesion. We first identified that lysyl oxidase (LOX) played a key role in the progression of peritoneal fibrosis by regulating collagen cross-linking and deposition in vivo. The inhibition of LOX enzyme activity prevented the formation of fibrotic tissue by reducing collagen deposition. Meanwhile, dexamethasone (DEX) treatment also minimized the fibrotic response. Furthermore, in vitro studies showed that the induction of collagen deposition factors in PMC, including LOX and pro-collagen I, required both IL-1 and TGF-β signalling pathways. Thus, the combination of IL-1 + TGF-β was adopted in an in vitro model to mimic the inflammatory environment during peritoneal repair. Treatment of PMC with IL-1+TGF-β caused an epithelial-to-mesenchymal transition (EMT). These transformed PMC had enhanced cell motility and were more adherent to fibronectin. Finally, a real-time quantitative PCR-based microarray was used for genomic analysis of ECM-adhesion-related PMC genes in response to IL-1 and TGF-β treatment. The results showed that IL-1 was more involved in regulating ECM degradation by inducing expression of matrix metalloproteinase (MMP) genes, whereas TGF-β mainly affected genes involved in ECM deposition, including collagens and other ECM components. However, both cytokines were shown to regulate some key genes involved in the development of adhesion, including COL16A1, COL7A1, FN1, ITGA5, and TGFB1. Moreover, IL-1 was shown to reduce ITGA4 and ITGB6 expression affecting adherence of PMC to basement membrane, while TGF-β increased MMP14 and MMP16 expression, which could facilitate invasion of EMT-transformed PMC to the site of tissue repair. In summary, this thesis indicates that LOX plays an important role in peritoneal fibrosis. Secondly, a combination of IL-1 and TGF-β1 treatment demonstrates how these factors can act in concert to orchestrate tissue remodelling during peritoneal repair. Finally, genomic analysis of ECM-adhesion genes increases our understanding of aspects of the pathology of post-operative adhesion and identifies novel potential therapeutic targets to prevent adhesion formation.

Potassium bromate (KBrO3) is a drinking water disinfection by-product and may represent a health hazard if found to be carcinogenic for humans, as was determined in rats (DeAngelo et al., 1998; Hayashi et al., 1986; Kurokawa, 1985; Kurokawa et al., 1985; Kurokawa et al., 1986a; Kurokawa et al., 1982; Kurokawa et al., 1983a; Kurokawa et al., 1987a; Kurokawa et al., 1983b; Kurokawa et al., 1986b; Ohno et al., 1982; Onodera et al., 1985). The purpose of this research was to determine the mechanism of toxicity. The peritoneal mesothelium is a (rat) target organ of potassium bromate carcinogenicity, but has not been studied due to the inherent difficulties of working with this one cell layer-thick tissue. Data from a two-year bioassay were used to more precisely map the location of origin, revealing that these tumors in the male F344 rat originate on the mesorchium of the tunica vaginalis testis, the mesosplenium, or at a point in between. An in vitro cell culture system was developed to study the mechanism of toxicity. It was demonstrated that KBrO3 caused cell cycle arrest and markedly increased the number of apoptotic cells. KBrO3 is a powerful oxidant and glutathione (GSH) is the major cellular antioxidant. Therefore, GSH-related responses were studied revealing mesothelial cells contained substantially less GSH than a human hepatocellular carcinoma cell line (Hep-G2). Studies employing GSH ester or N-acetyl cysteine (a GSH precursor) pre-treatment demonstrated abatement of toxicity in mesothelial, but not Hep-G2, cells. Experiments carried out to determine the chemical or enzymatic nature of the reaction between GSH and KBrO3 revealed differences between the reaction kinetics of the unbuffered and buffered chemical and cell-free/cell lysate reactions, probably due to reaction pH. Both chemical and cellular reactions exhibited a similar first step reaction between GSH and KBrO3; thus, enzyme participation is probably not required. Experiments using diethylmaleate, which depletes GSH by a reaction involving KBrO3, showed that GSH depletion greatly enhanced KBrO3 toxicity, indicating GSH glutathione S-transferase, buthionine sulfoximine (which prevents synthesis of GSH) and was protective. This does not support the hypothesis that the reaction of KBrO3 and GSH itself produces a radical/reactive species that oxidatively damages lipids, proteins and DNA. Rather, depletion of GSH likely precedes oxidative damage. Gene expression studies demonstrated that peritoneal mesothelial cells displayed expression changes in a discrete set of genes, including oxidative stress-responsive genes, after treatment with KBrO3 for four or 12 hours. Mesothelial cells severely damaged by five days KBrO3 treatment recovered from complete cell cycle arrest after four weeks and exhibited explosive growth, focus formation and altered morphology. The redox imbalance created by GSH depletion appears to mediate increased expression of known oxidative stress responsive genes (e.g., HO-1,GADD45, GADD153, QR), activation of transcription factors (AP-1 and NFkB) and down-regulation of cell cycle initiating cyclins (and up-regulation of the CDK inhibitor p21waf1/cip1) in KBrO3-mediated toxicity. These alterations may permit cell survival, as observed after severe toxicity, and may be accompanied by transforming mutations or clastogenic changes. Taken together, these data suggest that mesothelial cells represent a population susceptible to KBrO3-mediated toxicity in vitro, and suggest that tissue susceptibility in vivo plays a role in the nascence of mesotheliomas in the male F344 rat.

2011/2012
Mesenchymal Stem/Stromal Cells (MSCs) are the precursors of various cell types that compose both normal and cancer tissue microenvironments. In order to support the widely diversified parenchymal cells and tissue organization, MSCs are characterized by a large degree of heterogeneity, although available analyses of molecular and transcriptional data do not provide clear evidence. Moreover a wealth of studies has demonstrated a significant role of the microenvironment and MSCs in tumor growth. In the course of the years MSCs were isolated by different groups from different tissues, both healthy and cancerous. The laboratory in which I conducted my PhD project was able to purify MSCs from different healthy tissues (N-MSCs) and, using an adapted protocol, from High-Grade Serous Ovarian Carcinomas (HG-SOC-MSCs). It was possible to shown that these cells do not possess gross chromosomal aberrations and are not tumorigenic in vivo. To better characterize these cells, an integrative bioinformatics analysis was conducted using the deep-CAGE-derived expression profiles obtained from HG-SOC-MSCs, N-MSCs and the FANTOM5 large comprehensive primary cells and tissues dataset. When compared to the other cells and tissues, HG-SOC-MSCs showed a correlation with mesothelial cells and cells hypothesized to have a mesothelial origin, such as smooth muscle cells and fibroblasts. It is known that mesothelial cells in culture can alternate between epithelioid and fibroblastoid morphologies and express high levels of either keratin or vimentin or both depending on their state of growth and the presence of EGF. When cultivated in the absence of EGF, which is known to induce a morphological switch in mesothelial cells, HG-SOC-MSCs switch from a fibroblast-like to an epithelial-like shape. N-MSCs in the same culture conditions, instead, do not change morphology. Moreover, in absence of EGF, HG-SOC-MSCs but not N-MSCs raise the levels of Keratin 7 both in protein and in mRNA. Starting from the list of up-regulated genes in HG-SOC-MSCs compared to N-MSCs a list of mesothelial-related genes was generated. This mesothelial-related gene list was compared to high-throughput gene expression datasets of MSCs derived from other tissues. Such analysis revealed that the mesothelial-related signature is specific to HG-SOC-MSCs. Moreover, Kaplan-Meier survival analysis conducted on a comprehensive SOC microarray dataset showed that patients with higher levels of the mesothelial-related gene signature displayed shorter progression-free survival time. Such correlation was rather specific for HG-SOC given that its performance was either statistically non-significant in the case of lung cancer or correlated with good prognosis in the case of breast cancer. Altogether, the study allowed us to assign a specific identity for MSCs derived from high-grade serous ovarian cancer. We demonstrated a cell-type specific transcriptional activity associated with HG-SOC-MSCs, which identifies them compared to N-MSCs from other districts and position them close to primary mesothelial and mesothelail-derived cells within the FANTOM5 dataset.
XXV Ciclo
1985

The Best M.Phil Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize, 2001-2003.
published_or_final_version
abstract
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Medicine
Master
Master of Philosophy

Background Le perdite aeree prolungate (PAL) sono la principale causa di morbidità dopo interventi di chirurgia polmonare che si traduce in un prolungamento dei tempi di ospedalizzazione e conseguente incremento della spesa sanitaria. Differenti tecniche intraoperatorie e sigillanti chirurgici sono stati sviluppati per cercare di prevenire o ridurre l’incidenza delle perdite aeree post-operatorie. Obiettivi Questo studio vuole valutare la capacità del gel piastrinico ottenuto da cordone ombelicale di riparare un danno pleurico in un modello in vitro ed in un modello animale. Metodi In vitro è stata confrontata la capacità di riparazione del gel piastrinico rispetto alle condizioni standard di cultura di cellule mesoteliali umane dopo scratch assay. Nel modello in vivo, il gel piastrinico derivante da sangue di cordone ombelicale è stato testato sperimentalmente su 53 ratti Wistar, dopo aver effettuato una lesione standardizzata sul polmone sinistro. Dopo sacrificio degli animali a tempistiche prestabilite sono state valutate alterazioni istologiche ed eventuali complicanze. Inoltre, sono stati analizzati cambiamenti nei fattori solubili dell’infiammazione. Risultati In vitro, il gel piastrinico ripara più velocemente il danno delle cellule mesoteliali rispetto ai controlli (24 vs 35 ore). In vivo, la formazione del nuovo tessuto mesoteliale è stata osservata dopo una media di 44±1 ore nel gruppo trattato rispetto a 130±2.5 ore del gruppo di controllo. La guarigione completa dei campioni trattati con gel piastrinico è stata evidenziata dopo 75±1 ore ora rispetto alle 160±6 ore dei casi controllo. Nel 43% dei trattati sono state rilevate aderenze pleuriche mentre solo nel 17% dei controlli. Alcuni fattori dell’infiammazione erano significativamente ridotti negli animali trattati. Conclusioni I risultati ottenuti dal nostro studio sembrano promuovere l’utilizzo del gel piastrinico e dei suoi fattori di crescita nei processi riparativi del tessuto pleurico, inoltre sembra stimolare lo sviluppo di aderenze pleuriche, particolarmente utili nella gestione delle perdite prolungate. Ipotizziamo che il gel piastrinico giochi un duplice ruolo rilasciando citochine e fattori di crescita che promuovono un processo riparativo più rapido e di conseguenza riducono la flogosi.
OBJECTIVES: Prolonged air leak is the major cause of morbidity after pulmonary resection. In this study we tested an innovative approach based on the use of the platelet gel derived from human umbilical cord blood in repairing pleural damage through in vitro and in vivo experimental approaches. METHODS: The in vitro model scratch assay was performed to test the tissue repair capability mediated by platelet gel compared to the standard culture conditions using human primary mesothelial cells. In vivo the animal model consisted of an iatrogenic injury on the left lung surface. Fifty-four Wistar rats were divided into a treated group with cord blood platelet gel (n=31) and a control group (n=23). After the damage, the cord blood platelet gel was placed on the injured area only in treated animals. Rats were sacrificed to evaluate and study histological changes, and possible presence of pleural adhesions and infections. In addition, changes in the soluble inflammatory factor pattern were tested using a multiplex proteome array. RESULTS: In vitro, cord blood platelet gel repaired the damage of mesothelial cells in a shorter time in comparison with the control (24 vs 35 hours, respectively). In vivo, the formation of new mesothelial tissue was already visible at 45+1 hours in treated group vs 130+2.5 hours in control group; complete recovery was obtained respectively 75+1 hours compared to 160+6 hours. There was direct evidence of pleural adhesions in 43% of treated compared to 17% of controls. The gel was not associated with the development of any complications. Interestingly, some crucial soluble factors involved in inflammation were significantly reduced in the treated animals. CONCLUSIONS: Cord blood platelet gel significantly reduces the time to repair the pleural damage. In addition, it positively stimulates the development of pleural adhesions, particularly useful in the management of prolonged air leaks. We hypothesize that platelet gel play at least a double role releasing cytokines and growth factors that support the faster tissue repair and consequently reduce the inflammation site.

Mesothelin is a cell surface glycoprotein highly expressed in mesothelioma, ovarian cancer, pancreatic cancer and some other malignancies. It is a promising candidate for tumour specific therapy and diagnosis, given its limited expression in normal tissues. The purpose of the work presented in this dissertation is to develop and characterize a molecular imaging bioprobe that targets mesothelin. We radiolabelled fab and f(ab´)₂ fragments of the anti-mesothelin antibody mAbK1 with ⁹⁹mTc- tricarbonyl core using a histidine-modified tridentate ligand, while whole mAbK1 was radiolabelled with ⁹⁹mTc using a direct labelling approach. In vivo evaluation of these ⁹⁹mTc labelled radioimmunoconjugates in mesothelin expressing NCI-H226 tumour model, revealed low mesothelin specific tumour uptake. These findings were attributed to low expression of mesothelin on NCI-H226 cells as well as to the low affinity of mAbK1. An anti-mesothelin antibody mAbMB, with higher mesothelin affinity than mAbK1 was labelled with ¹¹¹In and evaluated in A431K5 tumour model which expresses clinically relevant levels of mesothelin. Biodistribution studies and SPECT imaging revealed specific localization of ¹¹¹In-mAbMB in mesothelin expressing A431K5 tumours. An interesting finding with ¹¹¹In-mAbMB was its preferential localization in spleen, which suggests a role of circulating mesothelin antigen in forming immune complexes with ¹¹¹In-mAbMB. In comparison, control studies with ¹¹¹In-mAbK1 revealed low specific uptake into A431K5 tumours. These studies provided evidence that ¹¹¹In-mAbMB is a better choice than ¹¹¹In-mAbK1 for imaging mesothelin expression in tumours. A dual-modality SPECT/MR imaging bioprobe was further developed by conjugating ¹¹¹In-mAbMB with SPIONs (superparamagnetic iron oxide nanoparticles) which demonstrated specific targeting and MR imaging capability in A431K5 tumour bearing mice. The work in this dissertation for the first time demonstrates successful SPECT imaging of mesothelin expressing cancers using radiolabelled antibodies. The radiopharmaceutical ¹¹¹In-mAbMB developed in this work holds promise for clinical use as a radioactive imaging bioprobe. Additionally, bioconjugates of ¹¹¹In-mAbMB and SPIONs are promising as dual-modality SPECT/MRI imaging bioprobes, which may be beneficial in improving the imaging outcomes of these difficult to treat tumours. In conclusion, our studies demonstrate that molecular imaging agents targeting mesothelin have a role to play for the detection and monitoring of mesothelin expressing cancers.

Pancreatic cancer (PDAC) affects 4400 Canadians each year and with 5year survival rates <8%, there is clearly an unmet need for new therapeutic approaches for treating this deadly disease. Herein I report the development of a surgical model of PDAC that recapitulates many of the hallmarks of human disease and has an immune infiltrate consisting of T cells and suppressive regulatory T cells and myeloid derived suppressor cells. This model allows the exploration of new therapeutics that can be used in combination with surgical resection of primary tumours. Furthermore, I propose that the use of neoadjuvant administration of a prime-boost oncolytic vaccine targeting a pancreatic tumour associated antigen (TAA) - mesothelin - could potentiate pancreatic tumour specific immune responses to improve patient prognosis. We demonstrate that immune tolerance to this self antigen can be broken by the complete depletion of circulating Tregs at the time of vaccination, which leads to the activation of a population of CD8+ T cells responsive to mesothelin. We demonstrate that these T cells respond to mesothelin expressing tumour cells ex vivo, and that CD8+ T cells are recruited to the site of tumour challenge. However, despite the generated CD8+ T cell response, oncolytic vaccine strategies targeting mesothelin provide no protection against Pan02 tumours, or against other mesothelin expressing murine tumour lines. I demonstrate that this is not through common tumour escape mechanisms, nor through the upregulation of suppressive immune populations. Any efficacy observed was found to be provided solely by depletion of Tregs, as the depletion of CD8+ T cells did not reduce protection from tumour outgrowth in vaccinated mice. While mesothelin represents a promising target, it is not an ideal target for oncolytic vaccine platforms, potentially due to its nature as a self antigen.

Endometriosis is a benign inflammatory disorder, defined by the presence of endometrial tissue outside the uterus with lesions typically found on the pelvic peritoneum in close association with the peritoneal mesothelium. The prevalence of endometriosis is estimated at 6-10% of women of reproductive age and it is associated with chronic pelvic pain, dysmenorrhoea, dyspareunia and infertility. Surgical excision can provide symptom relief, but symptoms recur in up to 75% of surgical cases and available medical treatments have undesirable side effects. New treatments are limited due to our poor understanding of the aetiology of endometriosis. To date, the majority of research has focused on changes within the ectopic endometrial tissue to explain the development of endometriosis lesions, however, there is increasing evidence that the peritoneal mesothelium plays an important role. According to Sampson’s theory of retrograde menstruation, ectopic endometrial cells must first have to attach to the surface of the peritoneum before undergoing invasion, proliferation, and neoangiogenesis. TGF-β1 is an inflammatory growth factor that regulates a variety of cellular functions including; cell adhesion, cell invasion and angiogenesis. Levels of TGF-β1 are increased in the peritoneal fluid of women with endometriosis compared to controls and research using a mouse model of endometriosis has demonstrated TGF-β1-null mice to develop smaller and fewer endometriosis lesions than their wild-type controls. Together these studies suggest that TGF-β1 plays a major role in the development of peritoneal endometriosis lesions and that targeting this pathway may be of therapeutic potential. However the functional role that TGF-β1 plays in peritoneal endometriosis is still unclear. The overall aim of this thesis was to determine if the peritoneal expression of TGF-β and its target genes are disrupted in women with endometriosis and whether this could contribute to the development of endometriosis lesions. Our first aim was to determine if the peritoneum was a source of TGF-β expression and if reception and/or signalling were altered in women with endometriosis. We found that the peritoneal fluid of women with endometriosis contained increased concentrations of TGF-β1 and that peritoneal mesothelial cells adjacent to endometriosis lesions expressed significantly higher levels of TGF-β1 mRNA. Analysis of TGF-β signalling targets within the peritoneum showed that women with endometriosis express significantly higher levels of TGF-β targeted genes associated with tumourigenesis processes including; EMT, invasion and angiogenesis. We next asked if there are changes in the metabolic phenotype of endometriosis lesions and peritoneum in women with endometriosis, similar to the metabolic changes seen in tumour cells. Endometriosis lesions expressed markers of aerobic glycolysis, including HIF-1α, suggesting that lesions may metabolise in a similar fashion to tumours. Furthermore, peritoneum adjacent to endometriosis lesions expressed significantly higher levels of markers of aerobic glycolysis, including HIF-1α, suggesting that the peritoneum may feed forward high-energy lactate, a by-product of glycolysis, to the endometriosis lesions. These observations were supported by significantly increased lactate concentrations within the peritoneal fluid of women with endometriosis that positively correlated with levels of TGF- β1. TGF-β1 was shown to increase expression of glycolysis markers and lactate expression in peritoneal mesothelial cells in-vitro, suggesting TGF-β may regulate this change. We then determined if TGF-β1 was responsible for the change in peritoneal mesothelial cell metabolism by signalling through the ID-HIF-1α pathway. ID proteins are transcription factors, whose expression is regulated by the TGF-β superfamily. We found expression of ID1 mRNA to be increased in the peritoneum of women with endometriosis and that TGF-β1 significantly increased ID1 but decreased ID2 expression in the peritoneal mesothelial cells in-vitro. ID1 siRNA knockdown decreased glycolysis initiator HIF-1α mRNA and ID2 siRNA knockdown increased HIF-1α mRNA and lactate expression, suggesting TGF-β1 regulates mesothelial cell metabolism, at least in part, through the ID pathway. ID transcription factors are also known to regulate VEGF-A expression, therefore we next determined if TGF-β1 induced ID1 and/or reduced ID2 expression in the peritoneum promoted VEGF-A mRNA and protein expression. VEGF-A, a cytokine essential for angiogenesis, was significantly increased in the peritoneal fluid of women with endometriosis and levels positively correlated with TGF-β1. TGF-β1 increased VEGF-A expression in-vitro and siRNA knockdown of ID1 decreased and siRNA knockdown of ID2 increased VEGF-A mRNA and protein expression in the peritoneal mesothelial cell. Lastly we aimed to determine if TGF-β1 induces EMT in peritoneal mesothelial cells. The peritoneum of women with endometriosis expressed higher levels of EMT markers. Exposure of peritoneal mesothelial cells to TGF-β1 in-vitro induced EMT-like changes, including; changes to cell morphology, gene expression and protein localisation. Peritoneal mesothelial cells were more migratory and invasive suggesting that TGF-β1 induced EMT may disrupt the mesothelial cell monolayer allowing ectopic endometrial cells to invade the peritoneal tissue or for peritoneal mesothelial cells to migrate into endometriosis lesions. In summary, the novel data presented in this thesis provides evidence that the pelvic peritoneum and in particular the peritoneal mesothelial cell may play a critical role in the aetiology of peritoneal endometriosis. Expression of TGF-β1 and its transcriptional target genes are dysregulated in the peritoneum of women with endometriosis. TGF-β regulated ID expression may induce changes in cell metabolism and promote neoangiogenesis, prompting peritoneal endometriosis lesion development. Furthermore other TGF-β1 transcriptional targets, such as those involved in EMT, are also altered in the peritoneum of women with endometriosis and may contribute the development and maintenance of lesion formation. These results point to a central role for TGF-β1 expression and signalling in the aetiology of peritoneal endometriosis. Furthermore it is likely that changes within the expression profile and morphology of the peritoneal mesothelial cells contribute to peritoneal lesion formation. Drugs that target these pathways may provide new therapies for women with endometriosis.

Epithelial Ovarian Cancer (EOC) kills more women annually in the United Kingdom than any other gynaecological cancer. Survival rates for women diagnosed with EOC have not improved over the past 30 years, due to the often advanced stage at presentation, where widespread intra-peritoneal dissemination has occurred. The natural history of the disease remains uncertain but the ovarian surface epithelium (OSE) is a strong candidate for the tissue of origin. The OSE undergoes cyclical damage and repair in women of reproductive age following ovulation, which can be considered an acute inflammatory event. Factors that prevent ovulation (pregnancy, breastfeeding and contraceptive pill use) also protect against the development of EOC. Previously published data show that the OSE is able to upregulate the enzyme 11-beta hydroxysteroid dehydrogenase type 1 (11βHSD1) in response to inflammation, the enzyme responsible for converting inactive cortisone to anti-inflammatory cortisol. This thesis hypothesises that 11βHSD isozymes are deregulated in ovarian cancer; that the peritoneal surface epithelium (PSE) is indistinguishable from the OSE in its response to inflammation and should be considered a potential source of some “ovarian cancers”; and finally that the expression of the tumour suppressor gene OPCML (OPioid binding Cell adhesion Molecule-Like) is altered by inflammation. These hypotheses were examined at three levels. Firstly, primary cultures of EOC were established, and glucocorticoid metabolism and the response to inflammation was compared to normal OSE. Results from these investigations reveal that the11βHSD1 response to IL-1α stimulation is impaired in EOC compared to normal OSE at the mRNA level but there is no significant difference when 11βHSD1 enzyme activity is measured in these tissues. When basal levels of 11βHSD1, 11βHSD2 and COX2 are compared amongst untreated samples of EOC and OSE, there was a significant correlation between 11βHSD1 and COX2 mRNA expression (P<0.001). 11βHSD2 mRNA expression was significantly higher in the EOC specimens compared to OSE (P<0.05). Secondly the response to inflammation was compared in primary cultures of human peritoneal surface epithelial (PSE) cells and OSE. The data suggest that the mRNA response to inflammation was similar in OSE and PSE, but that the 11βHSD1 enzyme activity was reduced in PSE (P<0.05), which may result in differences in tissue healing. Finally, the effect of inflammation on the expression of the ovarian cancer associated tumour suppressor gene (TSG), OPCML (OPioid binding Cell adhesion Molecule-Like) and the other members of the IgLON family, was examined in OSE. These results suggest that OPCML mRNA expression can be induced by IL-1α, an effect that is inhibited by cortisol.

Orientador: João Alexandre Ribeiro Gonçalves Barbosa
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Neste trabalho realizamos estudos estruturais com duas proteínas, a Q4DV70 de Trypanosoma cruzi e a mesotelina de Homo sapiens. O objetivo foi contribuir para a compreensão da função dessas proteínas, as quais possivelmente são importantes para a doença de Chagas e o câncer, respectivamente. A proteína Q4DV70 estava anotada no genoma de T. cruzi como hipotética conservada. Em nosso estudo, a proteína foi pela primeira vez detectada em amostras do parasita. Essa expressão ocorre na fase epimastigota, mas não na fase tripomastigota metacíclica, indicando que a proteína pode ter uma função importante no ciclo de vida do T. cruzi. A estrutura cristalográfica da Q4DV70 foi resolvida por substituição molecular e refinada com dados até 1,5 Å de resolução. Ela apresenta enovelamento tiorredoxina, formado por uma folha ß de 5 fitas cercada por duas hélices a de cada lado. As proteínas que apresentam maior identidade seqüencial e superposição estrutural com Q4DV70 são as tiorredoxinas e PDIs, oxidorredutases de pontes dissulfeto. Porém, as duas cisteínas do sítio ativo dessas proteínas estão substituídas por serinas em Q4DV70, o que impossibilita a função de formação, redução ou isomerização de pontes dissulfeto. Diversas tiorredoxina-like apresentam atividade chaperona independente da função oxidorredutase. Essa função está relacionada a regiões hidrofóbicas na superfície e/ou depende da presença de outro domínio. Q4DV70 é monomérica, composta apenas pelo domínio tiorredoxinalike, e não apresenta regiões hidrofóbicas em sua superfície. Além disso, não demonstrou capacidade de aumentar o enovelamento da GAPDH de T. cruzi. Esses resultados indicam que Q4DV70 não apresenta atividade chaperona. Mesotelina é uma proteína expressa em mesotélio normal e em diversos tipos de câncer, como mesotelioma, câncer de ovário e de pâncreas e leucemia mielóide aguda. Ela é considerada um marcador diagnóstico para esses cânceres e tem sido alvo para o desenvolvimento de drogas anti-tumor. Além disso, ela interage com MUC16, uma proteína presente na superfície de células de câncer de ovário. Apesar da reconhecida importância e uso da mesotelina, pouco se sabe sobre sua estrutura e função. A proteína foi purificada em condições desnaturantes e submetida ao re-enovelamento por diálise. Experimentos de dicroísmo circular, fluorescência e proteólise limitada comprovaram que a mesotelina foi corretamente re-enovelada. Essa amostra foi submetida a ensaios de cristalização, os quais resultaram em cristais que difrataram a baixa resolução. A estrutura de baixa resolução da mesotelina foi calculada a partir de dados de espalhamento de raios X a baixo ângulo e mostra que sua forma é alongada e curvada. O espectro de dicroísmo circular da mesotelina é típico de proteínas ricas em hélices a. Recentemente foi proposto que a estrutura da mesotelina é formada por uma estrutura em super-hélice, composta por repetições do tipo ARM, as quais apresentam 3 hélices a cada uma. Nossos resultados experimentais indicam que esse modelo teórico está correto. Proteólise limitada com quimotripsina resultou em um domínio estável de 20 kDa na região N-terminal da proteína. Esse domínio contém os 64 resíduos descritos como possível sítio de ligação a MUC16 e deve ser formado pelas 4 primeiras repetições do tipo ARM, de acordo com o modelo publicado.
Abstract: In this work we carried out studies with two proteins, Q4DV70 from Trypanosoma cruzi and mesothelin from Homo sapiens. The aim was to help the understanding of the function of these proteins which possibly are important to Chagas disease and cancer, respectively. Q4DV70 protein was annotated in T. cruzi genome as a conserved hypothetical protein. In our studies, the protein was detected for the first time in parasite samples. Its expression occurs in the epimastigote form but not in the metacyclic trypomastigote form indicating that Q4DV70 is important for the pathogen life cycle. Q4DV70 crystal structure was solved by molecular replacement and refined with data to 1.5 Å maximum resolution. It shows a thioredoxin fold, formed by a five stranded ß-sheet flanked by two a-helixes in each side. The proteins more sequentially identical and better structurally superposed to Q4DV70 are thioredoxins and protein disulfide isomerases, which are disulfide oxidoreductases. However, the cysteine residues from CXXC motif of the active site are replaced by serines in Q4DV70, what prevents the function of formation, reduction and isomerization of disulfide bonds. Different thioredoxin-like proteins show chaperone activity independent of oxidoreductase function. This function is related to hydrophobic regions on the surface and/or is dependent of another domain. Q4DV70 is monomeric, composed only by thioredoxin-like domain and does not show hydrophobic regions on its surface. Moreover the protein does not increase the refolding of GAPDH from T. cruzi. These results indicate that Q4DV70 does not present chaperone activity. Mesothelin is a protein expressed in normal mesothelium and in different types of cancer, such as mesothelioma, ovarian cancer, acute myeloid leukemia and cancer of pancreas. It is considered a diagnostic marker for these cancers and it has been used in the development of antitumor drugs. Besides that, it binds to MUC16, a protein present on the surface of ovarian cancer cells. In spite of its recognized significance and use in cancer, little is known about its structure and function. The protein was purified under denaturing conditions and submitted to refolding by dialysis. Circular dichroism, fluorescence and limited proteolysis experiments confirmed that mesothelin was correctly refolded. This sample was submitted to crystallization trials that resulted in crystals which diffracted at low resolution. The low resolution structure of mesothelin was calculated from small angle X-ray scattering data and it shows an elongated and curved shape. The circular dichroism spectrum for mesothelin is typical of proteins that are rich in a-helixes. Recently it was proposed that mesothelin has a superhelix structure made by ARM repeats which are composed by 3 a-helixes each one. Our experimental results indicate that this theoretical model is correct. Limited proteolysis with chymotrypsin resulted in a stable domain of 20 kDa in the Nterminal region of the protein. This domain has the 64 residues described as the possible binding site for MUC16 and should be composed by the first four ARM repeats according to the model.
Doutorado
Bioquimica
Doutor em Biologia Funcional e Molecular

L'ensemencement cellulaire des protheses vasculaires vise a ameliorer l'hemocompatibilite des protheses de faible diametre. Les cellules mesotheliales (cms) disponibles en grande quantite a partir de graisse epiploique et dotees de proprietes antithrombotiques comparables a celles des cellules endotheliales (ces) representent une alternative a l'utilisation des ces. Cependant les cms expriment constitutivement in vitro une activite procoagulante liee au facteur tissulaire. Cette expression pourrait constituer un obstacle a leur utilisation. Pour la diminuer, l'effet de deux agents pharmacologiques : l'heparine et l'ilomedine a ete etudie. L'ilomedine, seulement, induit une diminution de l'activite procoagulante des cms mais ne l'abolit pas completement. Dans ces conditions on ne peut pas envisager l'utilisation directe de ces cellules pour le recouvrement des protheses. La modification genetique des cms a ete suggeree pour ameliorer leurs proprietes anticoagulantes avant l'ensemencement sur protheses. La mise en place d'une telle technologie necessite des etudes in vitro et sa validation dans un modele animal. Afin de pouvoir transferer in vivo, les resultats obtenus in vitro, nous nous sommes donnes comme objectif de disposer d'un modele experimental de grande taille, ayant une physiologie proche de celle de l'espece humaine : le porc, modele experimental en pathologie vasculaire et recemment dans le transfert de gene. Dans un premier temps, une technique d'isolement de cms porcines a partir d'epiploon porcin a ete mise au point. Les cellules epiploiques porcines montrent des caracteristiques phenotypiques et fonctionnelles de cms et qui sont communes avec celles des cms humaines. Ces cellules porcines ont pu etre modifiees genetiquement par des vecteurs non viraux. Ces premiers resultats constituent une premiere approche dans l'elaboration d'un ensemencement par des cms genetiquement modifiees.

BACKGROUND: Cytology has a crucial role for diagnosing pleural and abdominal effusions. A prompt accurate diagnosis has both prognostic and therapeutic significance. However, cell morphology alone is not always sufficient to formulate such a diagnosis. In human medicine, immunocytochemistry of effusion cytology has now standardized procedures that provide reliable insights into various diagnostic dilemmas. OBJECTIVE: To describe the method of immunocytochemistry in effusion cytology and to estimate the value of a panel of markers in identifying cells in canine and feline effusions. MATERIALS AND METHODS: Human, feline and canine mesothelial cells were isolated in culture. Western‐blot (WB) analysis was used to ascertain antibody cross‐reactivity for all the markers, with the exception of HBME‐1. Forty‐four cytospined or smeared effusion specimens from dogs and cats with a cytological diagnosis of reactive effusion or malignancy of non‐hematopoietic origin were stained with a standard panel of Vimentin, Cytokeratin (CK) AE1/AE3, CK 5/6 and HBME‐1 as mesothelial cell markers; desmin as mesothelial cell malignancy marker; and CK7/CK20 as a marker of metastasis. Malignancy was confirmed by histologic evaluation; non‐malignant conditions were confirmed by follow‐up. Sensitivities, specificities and predictive values were calculated. RESULTS: The WB analysis confirmed the specific crossreactivity of the human antibodies for canine and feline proteins in mesothelial tissue. No significant differences were found between canine and feline results. Vimentin/cytokeratin coexpression had a sensitivity of 79% and a specificity of 92%, HBME‐1 had 89% sensitivity and 23% specificity, and CK5/6 had 26% sensitivity and 100% specificity for mesothelial cells. Desmin had only 20% specificity for benign mesothelial cells, while CK7‐/CK20+ had a specificity of 79% and sensitivity of 30% for metastatic cells on effusions. CONCLUSION: Immunocytochemistry can be applied in effusion samples, and valuable results can be obtained. The most useful marker, with the highest overall accuracy for the identification of mesothelial cells in effusion, is the Vim/CK coexpression, being CK5/6 the more specific and HBME‐1 the more sensitive antibody. Desmin is not useful for discriminating between benign and malignant mesothelial cells. The coordinate expression of CK7‐/CK20+ has not proved to be useful on the identification of metastatic cells on effusions.

Au cours de la dialyse peritoneale, les cellules mesotheliales peritoneales sont directement exposees aux solutes de dialyse hyperosmolaires et aux cytokines liberees au cours des infections peritoneales. Dans ces conditions, la balance entre leurs capacites de synthese et de degradation de la matrice extracellulaire (mec), qui sont importantes, peut etre considerablement modifiee et aboutir a l'accumulation de matrice et donc a la perte des capacites de filtration du peritoine. Notre etude a porte plus particulierement sur les metalloproteases et les activateurs du plasminogene, et leurs inhibiteurs parce qu'ils interviennent non seulement dans les processus de remodelage de la mec mais aussi dans les phenomenes de migration cellulaire et de differenciation. Tres peu d'etudes sont disponibles sur les proteases synthetisees par les cellules mesotheliales peritoneales et leur regulation par les liquides de dialyse et par le tgf, cytokine pro-fibrogene par excellence, qui joue un role majeur au cours des processus de reparation tissulaire et de fibrose. Dans une lignee differenciee que nous avons etablie et dans des cultures primaires de cellules mesotheliales peritoneales, nous avons montre que l'hyperosmolalite, equivalente a celle des solutes de dialyse peritoneale, inhibe considerablement l'expression et l'activite de la mmp9, alors qu'elle stimule celles du tpa, principal activateur du plasminogene produit par les cellules mesotheliales, grace a la presence d'un element de reponse a l'hyperosmolalite localise dans le promoteur du gene du tpa. Par contre, le tgf1 stimule l'expression et l'activite du pai-1 par un effet sur la transcription du gene, et de facon surprenante stimule egalement l'activite et l'expression de la mmp9 dans les cultures en phase exponentielle de croissance et dans les cultures exposees a l'hyperosmolalite. Ces resultats suggerent que le tgf1 libere au cours des infections peritoneales favorisent la fibrose en permettant la degradation du collagene de type iv et son remplacement par des collagenes interstitiels, et que l'hyperosmolalite pourrait favoriser l'accumulation de collagenes de type iv et une desorganisation de la membrane basale peritoneale. Enfin, nous avons montre que les cellules mesotheliales peritoneales humaines produisent les proteines sp-a et sp-d identiques a celles du surfactant alveolaire et leurs arnm specifiques. L'etude de la regulation des proteines sp-a et sp-d dans le peritoine au cours de la dialyse peritoneale pourrait ouvrir des perspectives therapeutiques interessantes.

Utilistions d'anticorps monoclonaux est une partie prometteuse de la thérapie du cancer. À ce jour, il existe plus de 30 anticorps monoclonaux approuvés pour la thérapie contre le cancer. Plus de 350 anticorps se situent également dans différentes phases du développement clinique. La mésothéline est l'une des cibles les plus prometteuses pour l'immunothérapie. La mésothéline est présente à des niveaux relativement faibles dans les cellules mésothéliales de la plèvre, du péritonéum et du péricarde normaux, mais est fortement exprimée dans un certain nombre de cancers différents, y compris les mésothéliomes, le cancer de l'estomac, les carcinomes à cellules squameuses, le cancer de la prostate, le cancer du pancréas, le cancer du poumon et le cancer de l'ovaire. La mésothéline est une glycoprotéine liée au glycosylphosphatidylinositol (GPI) synthétisée sous la forme d'un précurseur de 69 kDa et transformée de façon protéolytique en une forme sécrétée à 30 kDa (anciennement appelée Facteur de potentialisation des mégacaryocytes (MPF)) et une forme liée à la membrane de 40 kDa. Par ailleurs, il peut être clivé par une protéase et peut produire une forme de mésothéline soluble. Il a été déjà montré que cette forme soluble de mésothéline agit comme un ligand et neutralise les anticorps thérapeutiques ciblant la mésothéline. Par conséquent, les anticorps ne pouvaient pas atteindre les cellules cancéreuses et reste inefficaces. Dans notre travail, nous avons décidé de développer un anticorps discriminant spécifique à la forme associée à la membrane pour surmonter l'antagonisme produit par les formes solubles de mésothéline. Pour ce but, nous avons utilisé une nouvelle méthode d'immunisation de souris, que nous avons d'abord toléré la souris avec une mésothéline soluble et ensuite ré-immunisée avec des cellules exprimant la mésothéline. En utilisant la technologie de phage display, nous avons obtenu près de 150 clones de ciblant mésothéline dans 34 familles de VH-CDR3 parmi lesquelles nous avons identifié seulement 2 familles qui se lient à la mésothéline membranaire avec une affinité élevée et ne reconnaissent aucune autre forme soluble de mésothéline. Ici, nous proposons qu'ils puissent être des bons candidats pour être utilisés pour la thérapie contre le cancer de qui permet de passer à travers la barrière de mésothéline soluble. Pour démontrer leur efficacité pour une utilisation thérapeutique, nous avons construit une CAR avec le sc-Fv d'un anticorps discriminant de la forme membranaire
Antibody based immune treatment is a promising component of cancer therapy. To date there are more than 30 approved monoclonal antibodies for cancer therapy. More than 350 antibodies are also in different phases of clinical development. Mesothelin is one of the most promising targets for immunotherapy. It is present at relatively low levels in mesothelial cells of the pleura, peritoneum and pericardium of healthy individuals, but is highly expressed in a number of different cancers, including mesotheliomas, stomach cancers, squamous cell carcinomas, as well as prostate, pancreatic, lung, and ovarian cancers. Mesothelin is a glycosylphosphatidylinositol (GPI)-linked glycoprotein synthesized as a 69 kDa precursor and proteolytically processed into a 30 kDa NH2-terminal secreted form (formerly referred to as Megakaryocyte Potentiating Factor (MPF)) and a 40 kDa membrane-bound form. Besides that it can be cleaved by a protease leading to the production of a soluble, shedded, form of mesothelin. It has already been shown that this soluble form of mesothelin acts as a ligand and neutralizes the mesothelin targeting therapeutic antibodies. Therefore antibodies could not reach cancer cells and remained inefficient. In our work we decided to develop discriminating antibodies specific to a membrane associated form so as to overcome the antagonism produced by soluble forms of mesothelin. To this aim we used a novel method of mouse immunization, in which we first tolerized the mouse with soluble mesothelin before immunization with mesothelin expressing cells. By using phage display technology we obtained nearly 150 mesothelin recognizing clones in 34 VH-CDR3 families, among which we identified only 2 families that bind membrane mesothelin with high affinity and do not recognize any other soluble form of mesothelin. Here we suggest that this Fab can be effective candidates to be used for mesothelin expressing cancer therapy being allowed to pass through the soluble mesothelin barrier. To show their efficacy for therapeutic use we constructed a CAR with the sc-Fv of a membrane-form discriminating antibody

Dans le present travail, la cytotoxicite et la genotoxicite de differentes particules naturelles ou synthetiques ayant des caracteristiques de forme, de chimie et de granulometrie differentes ont ete determinees en utilisant des cellules mesotheliales pleurales de rat (cmpr). Les resultats obtenus en appliquant des tests de cytotoxicite et les tests de detection d'anomalies anaphasiques et telophasiques nous suggerent que les dimensions des particules sont des parametres plus importants pour expliquer la toxicite des particules, que leur composition chimique. Les criteres definis par stanton (longueur> 8 m et diametre < 0,25 m) pour selectionner les fibres les plus toxiques constituent un parametre tres pertinent pour predire un eventuel effet genotoxique dans le test de detection d'anaphases anormales. Par ailleurs, en considerant ce parametre nous avons pu mettre en evidence que les fibres les plus actives pour produire des anomalies anaphasiques sont egalement les plus cancerogenes par inoculation intrapleurale chez le rat. Bien que les caracteristiques granulometriques ne refletent pas totalement le potentiel toxique, nous avons constate que la teneur en fer des echantillons n'apparaissait pas etre un parametre pertinent pour evaluer la toxicite des fibres, ce qui n'exclut pas que d'autres modes d'expression de la teneur en fer pourraient rendre compte de la toxicite

Le trastuzumab est le traitement de référence des cancers du sein « HER2 amplifié ». Outre les limitations inhérentes à toute IgG, cet anticorps est inefficace pour traiter les tumeurs exprimant que modérément (cancers hormonaux) ou faiblement (triple négatif) HER2. L'objectif de ces travaux de thèse est de concevoir de nouveaux anticorps bispécifiques destinés au traitement de cancers du sein échappant à l'action du trastuzumab. Nous nous sommes appuyés sur des formats innovants, basés sur l'utilisation d'anticorps simple domaine de lama (sdAb) comme unité de reconnaissance antigénique. Deux anticorps bispécifiques Fab-like (bsFab) ont été développés, l'un dirigé contre HER2 (HER2bsFab) et l'autre ciblant la mésothéline (MesobsFab), un antigène surexprimé dans 30 à 70% des cancers du sein « triple négatif ». En liant spécifiquement et de façon efficace FcγRIII, ces deux bsFabs ne compètent pas avec les IgG endogènes, ne fixent pas FcγRIIB et activent fortement les NKs. In vitro, HER2bsFab induit de fortes sécrétions de cytokines pro-inflammatoires et de puissantes ADCCs contre des lignées de cancer du sein indépendamment du niveau d'expression d'HER2 et du polymorphisme FcγRIIIA-158. In vivo, HER2bsFab montre une nette supériorité comparé au trastuzumab contre des tumeurs ne surexprimant que modérément HER2. HER2bsFab et MesobsFab induisent in vitro de fortes cytotoxicités contre deux lignées de cancer du sein « triple négatif » et des résultats préliminaires réalisés chez la souris semblent confirmer ces observations. A termes, l'utilisation de ces anticorps permettrait d'étendre la proportion de patientes traitables de façon efficace par immunothérapie
Trastuzumab is established as standard of care for the treatment of HER2high breast cancers. However, in addition to Fc-related limitations inherent to IgG antibodies, trastuzumab is inefficient to treat low- (triple-negative) or moderate-HER2-overexpressing (hormone-receptor-positive) breast cancers. Based on the unique structural and functional properties of llama single domain antibodies (sdAbs), we report the design of two Fab-like bispecific antibodies targeted to HER2 (HER2bsFab) and mesothelin (MesobsFab), an antigen overexpressed in several human tumors, including triple-negative breast cancers. The two bsFabs display a unique, specific and high affinity for FcγRIII. As a consequence, they do not bind the FcγRIIB inhibitor receptor and bypass competition with endogenous IgGs. HER2bsFab mediated ADCC at picomolar concentration against HER2high as well as HER2moderate cell lines. In vivo HER2bsFab potently inhibited HER2high tumor growth and more importantly, exhibited a net superiority over trastuzumab at inhibiting HER2moderate tumor growth. Moreover, FcγRIIIA-engagement by HER2bsFab was independent of FcγRIIIA-158 polymorphism and induced a stronger NK cells activation in response to target cell recognition. Such findings led us to investigating the efficacy of bsFabs in a context of low-HER2-overexpression displays by triple-negative breast cancers. In vitro characterization showed that both HER2bsFab and MesobsFab trigger efficient lysis of two different triple-negative breast cancer cell lines. Altogether, these findings would enable the treatment of a broader population of patients than that eligible with current HER2-targeted therapies

Le cancer colorectal (CCR) est l'un des cancers les plus fréquents au monde avec les cancers du poumon et de la prostate. Une estimation précoce du pronostic pourrait aider à réduire la mortalité de ce cancer et l'implémentation de nouveaux biomarqueurs est devenu indispensable. Dans cette étude nous avons évalué deux molécules pour mieux caractériser et apprécier le pronostic du CCR : l'angiopoiétine-2 (Ang2) et la Mésotheline (Msln). Nous avons d'abord analysé des données transcriptomiques associés à des données de survie et collectés à partir de bases de données publiques. 1617 transcriptomes avaient été produites par des puces ADN et ont été téléchargés de la base GEO (NCBI Gene Expression Omnibus) 573 autres transcriptomes produits par RNAseq ont été extraits de la base de données du TCGA (The Cancer Genome Atlas). Nous avons ensuite réalisé des dosages plasmatiques par méthode ELISA chez 20 donneurs sains et 51 patients atteints de CRC métastatiques suivis dans le cadre d'une étude locale. Les analyses de survie pour Ang2 dans les transcriptomes ont montré qu'une expression intratumoral élevée d'Ang2 était associée à une baisse de la survie globale (OS, "Overall survival") et de la survie sans rechute (RFS, "relapse free survival") y compris chez les patients en stade localisé. Comme attendu, l'expression intratumoral d'Ang2 était associée à l'infiltration stromale et l'angiogenèse mais les analyses multivariées ont montré qu'Ang2 était bien un facteur indépendant de la survie (OS) après ajustement avec toutes les variables disponibles (incluant la classification CMS, "Consensus Molecular Subtypes"). Les dosages plasmatiques d'Ang2 chez les patients atteints de CRC métastatique ont montré que les taux d'Ang2 étaient plus élevés chez les patients que chez les donneurs et qu'un taux élevé était associé à une PFS plus basse. Une expression de Msln élevée dans les transcriptomes était associée à une survie réduite (OS et RFS). C'était également un facteur de risque indépendant de la survie (OS et RFS). L'analyse des voies de signalisations a montré que l'expression de Mésotheline était associée à la voie des éxosomes. Les dosages sériques de Msln ont également montré que les taux sont plus élevés chez les patients que chez les donneurs sains. Enfin, les patients avec des valeurs plus élevées de Msln avaient une survie OS plus basse. Au total, ces résultats suggèrent qu'Ang2 et Msln sont tous deux des biomarqueurs pronostiques pour le CRC. Nous planifions maintenant de stratifier le pronostic en fonction d'une utilisation combinée de ces deux marqueurs
Colorectal cancer (CRC) is the third most commonly diagnosed cancer after lung cancer, and prostate cancer. The early prognostic estimation could reduce the mortality and the implementation of new biomarkers is getting indispensable. In this study we investigated two molecules for better characterization and prognosis of CRC: Angiopietin2 (Ang2) and Mesothelin (Msln). We first analyzed transcriptomic and clinicopathological data of primary tumors collected from online repositories. 1617 transcriptomes had been produced by microarrays and were downloaded from NCBI Gene Expression Omnibus (GEO) and additionally 573 RNAseq transcriptomic data were collected from The Cancer Genome Atlas (TCGA) repository. Plasma ELISA assays for Ang2 and Msln were then performed in 20 healthy donors and in 51 CRC patients from a local cohort in order to validate those proteins as potential biomarkers. Ang2 survival analyses performed on transcriptomic data showed that high intratumoral Ang2 expression was associated with poor overall survival (OS) and relapse-free survival (RFS) in CRC patients with localized stages. As expected, Ang2 expression was associated with stroma infiltration and angiogenesis but multivariate analyses showed that Ang2 was an independent factor for OS after adjusting it with all available variables (including Consensus Molecular Subtypes of CRC). The plasma measurement of Ang2 showed that the metastatic CRC (mCRC) patients had higher levels than the healthy donors and the progression free survival (PFS) was lower in patients with higher Ang2. High intratumoral MSLN expression in CRC transcriptomes was associated with poor OS and RFS. It was also an independent factor for OS and RFS. Pathway analyses revealed that Msln expression was associated with tumor exosome pathway and cell invasion. ELISA measurements of Msln levels showed that the patients with colorectal cancer had significantly higher Msln levels than healthy donors. Furthermore, the CRC patients with increased levels of Msln had poor OS than the patients with low Msln levels. Taken together, these results suggest that both Ang2 and Msln are independent prognostic biomarkers for CRC. We now aim to stratify risk using both Ang2 and Msln to improve the treatment of CRC patients

碩士
臺北醫學大學
醫學技術學系
94
In Taiwan, there are at present more than forty thousand patients with end-stage renal disease (ESRD) under regular dialysis therapy. Kidney disease is the seventh of ten leading causes of mortality in our country.The incidence rate of ESRD in Taiwan has gained the second place in the world. Continuous ambulatory peritoneal dialysis (CAPD) is a convenient therqpy as one kind of renal replacement therapy which interferes less of daily activity in ESRD patients. Peritoneal fibrosis (PF) is one of the most common complications in peritoneal dialysis (PD). It has been found that bio-incompatible PD solutions bearing characteristics of high glucose (HG), hyperosmolality, and low pH value may leads to the development of PF. The glucose concentration of PD solutions in Taiwan is 1.5％(83.8mM), 2.5％(138mM), or 4.25％(236mM). After long-term exposure of HG condition, human peritoneal mesothelial cells (HPMCs) will result in changes of peritoneal structure and function. Histological evaluations had demonstrated that HPMCs apoptosis and accumulation of extracellular matrix (ECM) are main pathogenesis of PF. However, the mechanisms of HG-induced apoptosis of HPMCs and the role of mitochondria as well as reactive oxygen species (ROS) in PF remains undetermined. This research is aims to evaluate effects of HG in apoptosis of HPMCs. We also investigated the efficacy of mitochondria oxidative phosphorylation and ROS generation in HPMCs under HG condition. It was found that HG induced HPMC apoptosis, cytochrome c release, PARP cleavage, activations of caspase-9, caspase-3, and an up-regulated gene expression of type I collagen with 138mM and 236mM glucose treatment. It was also demonstrated that these detrimental effects of HG in RMC caould be significantly debased apoptosis 50％ by 1mM l-N-acetylcystein (L-NAC), debased apoptosis 60％ by 1μM rotenone or debased apoptosis 12％by 1μM (CCCP) carbonyl cyanide m-chlorophenylhydrazone supplemented. Induced apoptosis and ECM accumulation were also revealed in rat mesangial cell cuture with 35mM glucose treatment. Our work may have great clinical implications in management of diabetic nephropathy and provide pharmacological information for prevention of PF in long-term PD patients.

碩士
國立陽明大學
生理學研究所
96
Background. Myofibroblasts are known to be important in the pathogenesis of peritoneal fibrosis by promoting cytokine release, extracellular matrix formation and angiogenesis. Fas-mediated apoptosis plays a vital role in the regulation of myofibroblast clearance, while decoy receptor 3 (DcR3) is a soluble receptor that competitively binds to Fas ligand (FasL). In the present study, we sought to investigate the protective effects of DcR3 in the Fas-mediated apoptosis of human peritoneal mesothelial cells (HPMCs) , in both its native and transdifferentiated states. Methods. HPMCs and human peritoneal fibroblasts (HPFs) were isolated from human omentum tissue, and transdifferented HPMCs (tPMCs) were initiated by induction with 3 ng/ml of transforming growth factor β1 (TGF-β1) for 7 days. Morphological differences and the expression of e-cadherin, vimentin, cytokeratins, and α-smooth muscle actin were observed. Production of DcR3 was induced by TNFα and subsequently measured using ELISA and reverse transcription PCR of DcR3 mRNA. Fas receptor expression was characterized by immunofluorescent cytometry and RT-PCR. Apoptosis was induced by sensitizing the cells with cycloheximide (CHX) and then treatment with FasL 100 ng/ml, and incremental doses of human recombinant DcR3-Fc (rhDcR3-Fc) were added to evaluate their effects on FasL-induced apoptosis. Apoptosis was assessed by FACS measurement of TUNEL, PI staining and immunoblotting for the active form of caspase 3. Results. HPMCs and HPFs exhibit a negligible constitutive production of DcR3 and low production even after induction with TNFα, while tPMCs show a constitutive expression of DcR3 that was significantly increased after induction with TNFα. Fas receptor expression was significantly down-regulated in tPMCs. The percentage of apoptotic tPMCs were not significantly increased with FasL treatment alone, but pretreatment with CHX sensitized cells to FasL-induced apoptosis. HPMCs exhibited a dramatic increase in apoptosis percentage after induction with CHX/FasL, while apoptosis in tPMCs and HPFs were less pronounced. The addition of rhDcR3-Fc inhibits FasL-induced cell death in all three cell lines at a concentration of 100ng/ml. Conclusion. tPMCs up-regulate DcR3 expression and down-regulate Fas receptor expression. tPMCs are also more resistant to FasL-mediated apoptosis, and the presence of DcR3 in culture medium protects HPMCs, tPMCs and HPFs cells against FasL-induced apoptosis. tPMCs have previously been implicated as a protagonist in peritoneal fibrosis, and their higher DcR3 secretion and lower Fas expression may play an important role in the balance between healing and fibrosis after peritoneal inflammation.

博士
國立臺灣大學
臨床醫學研究所
87
Pleural mesothelial cells (PMCs), which cover the surface of the parietal and visceral pleura, play important roles to reconstruct and restore the normal pleural function after pleural injury. Although infectious, inflammatory and neoplastic diseases frequently involved the pH change of pleural effusion, the regulatory mechanisms have not yet completely illustrated in a variety of pleural disorders. We hypothesized the pH of pleural effusion might be regulated by the transmembrane transporters in the pleural lining cells, i.e., pleural mesothelial cells. We established the methods to isolate and identify the cultured pleural mesothelial cells from pleural effusions and pleural tissues. Morphologically, the cells formed a homogeneous cell population, and polygonal when confluent. Cultured PMCs co-expressed cytokeratin and vimentin, which is a characteristic of epithelial and mesodermal origin, respectively. Then we provided a model to study intracellular pH (pHi) regulation in PMCs, pleural effusion formation, and pleural injury and carcinogenesis. Responding extrinsic or intrinsic stimuli, PMCs can produce a broad spectrum of extracellular matrix, including at least three collagen types (I, III, IV), elastin, fibronectin and laminin. The extracellular matrix could be the ligands, which interact with integrins, a family of cell adhesion molecules, in PMCs. Integrins are heterodimers, * and * subu-nits, which mediate cell- to- cell or extracellular connections. Interactions of ligands and integrins mediated cell morphological change, cellular attachment and detachment, activation Na-H exchanger, then induced intracellular alkalization. By using flow cytometry, we found the a2, a3, a5, b1, b3, and avb3 subunits were uniformly expressed at higher (>70%) positive cells for integrin staining on cultured human PMCs. The expression a1 subunit was intermediate (30-70%), and a4 and a6 subunits were low (<30%). The immunofluorescent stain also showed similar findings. The expression of integrins on Met-5A cells showed that a3, a5, a6, and b1 subunits were high, a1 subunit, intermediate and a2, a4, b3 and avb3 subunits, low. The patterns of immnunohistochemical staining of integrin expression on pleural tissues were generally and consistently similar to the results of the flow cytometry in the cultured human PMCs, except b3. To further investigate the regulation of pHi in PMCs, we studied the intrinsic buffering power of H+ ions (bi) and the pHi regulatory systems by using a pH-sensitive fluorescent probe, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) with microspectrofluorimetry. We found: (1) that at the resting pHi, the bi was low and increased as the pHi decreased; (2) that the pHi recovery was largely inhibited either with Na-free medium or nominally HCO3 free medium containing ethyl-isopropyl amiloride (EIPA); (3) a 4-4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-sensitive, Na/HCO3-dependent, but Cl-independent acid extrusion mechanism in CO2/HCO3 buffer; and (4) that in the same buffer, a DIDS-sensitive but Na-independent alkalosis was induced by intracellular Cl depletion. We therefore conclude that at least three membrane pHi regulators are involved in regulating the pHi in PMCs, these being the EIPA-sensitive Na-H exchanger; a novel electroneutral, DIDS-sensitive Na-HCO3 cotransporter; and the DIDS-sensitive Cl-HCO3 exchanger. Furthermore, under physiologic conditions, the Na-HCO3 cotransporter plays a more important role in extrusion of excess intracellular H+ ions than does the Na-H exchanger. EGF (epidermal growth factor) has been shown to stimulate cell proliferation in various cell types, including rat mesothelial cells. EGF also stimulates the Na-H exchanger, leading to enhanced cell proliferation. In human PMCs, the intracellular signaling mechanism mediating the EGF-induced stimulation of the Na-H exchanger has not yet been identified. Using a pH-sensitive fluorescent probe, BCECF with microspectrofluorimetry, to measure changes in the intracellular pH (pHi), we have found that: (1) EGF and 12-O-tetradecanoyl-phorbol-13-acetate (TPA, a phorbol ester) both stimulate the ethyl-isopropyl amiloride (EIPA)-sensitive Na+-H+ exchanger; (2) the TPA-induced alkalosis can be blocked by protein kinase C (PKC) inhibitors (chelerythrine and staurosporine) or by PKC down-regulation, indicating that PKC activation is involved in the stimulation of the Na+-H+ exchanger. However, the TPA-induced alkalosis is not blocked by tyrosine kinase inhibitors; (3) the stimulatory effect of EGF on the Na-H exchanger acts via stimulation of receptor tyrosine kinase activity, since it is inhibited by tyrosine kinase inhibitors (genistein, lavendustin A and herbimycin A), but, in addition, involves PKC activation, since the EGF-induced alkalosis is blocked by PKC inhibitors. (4) cell proliferation is both significantly enhanced by addition of EGF and the changes in pHi resulting from the change of extracellular pH (pHo). Taken together our observations indicate that the stimulatory effect on the Na-H exchanger with addition of EGF is mainly caused by PKC activation. The proliferative ability induced by EGF in human PMCs, however, is at least partly controlled by the changes in pHi which are mainly regulated by the Na- H exchanger. Based on these studies, we established a model to investigate the cellular function and physiology of human PMCs. The expression of integrins was first reported in PMCs. We also illustrated the regulation of pHi, through ion transporters and intrinsic buffering capacity. Further, we found the EGF-stimulated Na-H exchanger in PMCs via PKC pathway. This information provides new modalities for treatment of pleural injury and pleural carcinogenesis. We will investigate further the relationship of the effects and the mechanisms of fibrinolytics and pleurodesis agents with the integrins and ion transporters in PMCs.

博士
國立陽明大學
臨床醫學研究所
91
Background: Peritoneal dialysis (PD) is a widely used renal replacement therapy modality for end-stage renal disease patient. Patients receiving PD have higher technical failure rate than that of hemodialysis patients. Repeated peritonitis and ultrafiltration failure after long-term use of bio-incompatible peritoneal dialysate are two main reasons of technical failure. Peritonitis in PD patients is characterized by prolonged repair and was believed to cause peritoneum damage due to chronic inflammation. However, peritoneal mesothelial cells have good proliferating ability. Therefore, what is the reason of prolonged repair of peritoneum in PD peritonitis? We try to investigate the significance of human peritoneal mesothelial cells (HPMC) apoptosis during peritonitis and the role of nitric oxide, Fas-Fas ligand system in the process of peritoneal mesothelial cells apoptosis. Methods: We use cytokines (Tumor necrosis factor-a (TNF-α) 、Interleukin-1b (IL-1b)、Interferon-g (IFN-γ) 及lipopolysaccharide (LPS)) singly or in various combinations to stimulate HPMC and scrutinize the expression of nitric oxide synthase type II (iNOS) by western blot, reverse transcriptase polymerase chain reaction and Griess method. HPMC apoptosis was investigated by propidium iodide staining and TUNEL method. We also studied the expression of Fas in HPMC by flow cytometry and use anti-Fas antibody (clone CH11) to stimulate cytokine-primed HPMC. Apoptosis of HPMC was demonstrated by propidium iodide, TUNEL method and M30 cytodeath antibody staining. The quantity of apoptosis was studied by flow cytometry. The activities of caspase-3, caspase-8 and Bcl-2 were investigated by western blot. The apoptotic HPMC were co-incubated with adhered macrophages to see if macrophages could engulf apoptotic HPMCs. The peritoneal dialysate effluents of PD peritonitis patients were studied for the presence of apoptotic HPMCs. Results: Our results showed that iNOS is inducible after concomitant stimulation of TNF-a (5ng/ml) and IFN-g (5ng/ml). However, endogenous production of nitric oxide did not lead to apoptosis of HPMC. On the other hand, pre-incubation of HPMC with TNF-a (5ng/ml) and then stimulation with anti-Fas antibody (clone CH11) cause apoptosis of HPMC. Activation of both caspase-8 and caspase-3 were noted during the apoptotic process. The addition of caspase inhibitor could prevent the occurrence of apoptotic process. The apoptotic HPMC could be engulfed by macrophages. We found increased mesothelial cell apoptosis in peritoneal dialysate effluent during recovery phase of peritonitis. Conclusions: Our results reveal that bio-incompatible peritoneal dialysate probably aggravate the magnitude of HPMC apoptosis or affect the removal of apoptotic HPMC by macrophages during peritonitis. Failure to clear apoptotic HPMC might prolong peritoneal inflammation and therefore hamper the repair of peritoneum. The apoptosis of HPMC is related to the regulation of inflammation and subsequent repair of peritoneum during peritonitis.

La disseminazione tumorale sistemica è considerato uno dei passaggi cruciali della carcinosi peritoneale, complicanza che nel carcinoma dello stomaco e del colon-retto si manifesta con elevata prevalenza anche dopo il trattamento chirurgico di resezione del tumore (Sadeghi et al. 2000, Jayne et al. 2002). I meccanismi di disseminazione peritoneale prevedono l’intervento di una sequenza di eventi tra cui si annoverano il distacco di sottopopolazioni di cellule neoplastiche dalla massa del tumore primario, il loro passaggio nel fluido peritoneale, la susseguente colonizzazione del mesotelio e la formazione di una nuova massa tumorale. Uno dei passaggi critici che condiziona lo sviluppo della recidiva peritoneale consiste nell’adesione ai foglietti mesoteliali delle cellule tumorali esfoliate dal carcinoma. In questo processo risultano coinvolti diversi meccanismi molecolari imperniati soprattutto sul ruolo e sulle caratteristiche delle cellule tumorali (Harada et al. 2001, Kajiyama et al. 2008, Saito et al. 2010) mentre il contributo della controparte mesoteliale è stato, fino ad oggi, meno studiato, nonostante sembri anch’esso accreditato di un ruolo primario (Casey et al. 2003, Takatsuki et al. 2004, Alkhamesi et al. 2005). L’analisi dell’adesione tumorale al mesotelio è basata sull’impiego di modelli sperimentali sviluppati in vitro; essi, in genere, prevedono l’utilizzo di monostrati mesoteliali derivati da colture primarie su cui viene testata la capacità di adesione di linee cellulari tumorali in presenza o meno di agenti promotori o bloccanti (Jayne et al. 1999, Heyman et al. 2008, Cabourne et al. 2010). Le colture primarie mesoteliali peritoneali comunemente utilizzate per allestire questi sistemi in vitro vengono ottenute a partire da frammenti bioptici di omento, mentre un approccio meno utilizzato prevede l’impiego di campioni ottenuti da lavaggio peritoneale (Ivarsson et al. 1998). Tra le numerose molecole espresse dal mesotelio e potenzialmente coinvolte nel processo di adesione delle cellule tumorali, ICAM-1 (Intercellular Adhesion Molecule 1) sembra svolgere un ruolo chiave. L’espressione di ICAM-1 sulle cellule mesoteliali aumenta come risultato dello stress ossidativo e della senescenza e la modulazione positiva della sua espressione è in grado di promuovere l’adesione delle cellule neoplastiche al mesotelio peritoneale dei pazienti affetti da carcinoma ovarico, gastrico e del colon-retto. (Ksiazek et al. 2008, 2009, 2010). La prima parte delle attività del Dottor Danilo Ranieri, svolta con il supporto dell’Unità di Chirurgia A dell’Azienda Ospedaliera Sant’Andrea, ha avuto come obiettivo quello di definire il contributo del mesotelio nel processo di adesione delle cellule tumorali attraverso il tentativo di riprodurre in vitro nel modo più fedele possibile le condizioni presenti in vivo. A tale scopo si è deciso di utilizzare come materiale biologico di partenza il liquido di lavaggio peritoneale prelevato durante le procedure operatorie volte al trattamento chirurgico di pazienti con carcinoma gastrico o del colon-retto. Questo approccio è stato giustificato dalla peculiare composizione cellulare del liquido: da un lato la ricchezza di elementi mesoteliali di sfaldamento ha rappresentato una valida fonte per allestire colture primarie mesoteliali destinate alla messa a punto di modelli in vitro per studiare l’adesione di linee cellulari tumorali, dall’altro la presenza eventuale di cellule tumorali disseminate ha permesso una migliore definizione del comportamento biologico della neoplasia attraverso la loro identificazione e la loro caratterizzazione morfologica e molecolare. Nella sequenza metodologica sperimentale la prima fase è stata rivolta all’ottimizzazione di modelli di cocoltura in vitro su cui testare l’adesione di linee cellulari tumorali su un monostrato di mesotelio. Il monostrato mesoteliale è stato ricreato utilizzando una linea cellulare immortalizzata di mesotelioma pleurico (MeT-5A); su di esso sono state successivamente seminate cellule tumorali di linee di carcinoma gastrico (AGS) e di colon (Caco2). Per identificare le cellule utilizzate in cocoltura è stata condotta un’analisi di immunofluorescenza su specifiche proteine caratteristicamente espresse dalle cellule epiteliali (EpCAM/CD326; molecola di adesione pan epiteliale) e dal mesotelio (vimentina). Per analizzare le modalità di crescita delle linee cellulari tumorali sul monostrato di Met-5A è stata utilizzata la microscopia in contrasto di fase. Le cellule AGS hanno mostrato la tendenza a crescere come elementi isolati mentre le Caco2 hanno mostrato la tendenza a crescere in isole compatte. Queste osservazioni sono state supportate da un’analisi della proliferazione delle cellule tumorali adese al mesotelio. Utilizzando il marcatore ki67 in microscopia a fluorescenza si è visto che, diversamente dalla linea AGS, le Caco2 presentavano cellule ciclanti solo ai margini dell’isola, comportamento biologico atteso in base alla loro tendenza a differenziare in vitro (Visco et al. 2001). Per una valutazione quantitativa dell’adesione delle cellule AGS e Caco2 al monostrato di mesotelio si è deciso di utilizzare il marcatore lipofilico di membrana DiI, colorante in grado di marcare le cellule tumorali in cocoltura senza influenzare le loro proprietà di adesione. Le cellule sono state marcate in vivo, seminate sul monostrato mesoteliale e lasciate aderire per diversi intervalli di tempo. Le due linee tumorali hanno mostrato analoghe percentuali di adesione dopo la prima ora di cocoltura, mentre a tempi più lunghi le Caco2 hanno mostrato percentuali di adesione maggiori rispetto alle AGS. Questa differenza è stata attribuita essenzialmente alle diverse capacità adesive mostrate dalle due linee, visto che un’analisi della proliferazione a 48 ore non ha mostrato differenze significative tra AGS e Caco2. Una volta ottimizzato il test di adesione in vitro, il passaggio successivo ha previsto l’allestimento di colture primarie mesoteliali da lavaggi peritoneali di pazienti neoplastici. Per eliminare l’eventuale presenza di cellule neoplastiche derivate dallo sfaldamento della massa tumorale, il liquido di lavaggio è stato sottoposto ad una deplezione immunomagnetica della componente cellulare epiteliale EpCAM positiva. La frazione cellulare privata della componente epiteliale è stata seminata in appropriato mezzo di coltura per ottenere le colture primarie di mesotelio peritoneale (HPMCs). Le colture ottenute (8 da pazienti con carcinoma del colon, 4 da pazienti con carcinoma gastrico, 2 da pazienti con malattie non neoplastiche) sono state caratterizzate con tecniche di immunofluorescenza utilizzando un pannello di marcatori caratteristicamente espressi dal mesotelio, tra cui vimentina, pan-citocheratine e calretinina. La disponibilità di colture primarie ottenute da lavaggio peritoneale ha permesso di ripetere i test di adesione precedentemente ottimizzati su una serie di monostrati mesoteliali; sono stati utilizzati tre monostrati differenti rappresentativi di altrettanti microambienti: uno derivato da un paziente con carcinoma gastrico, uno da paziente con carcinoma del colon, uno da un paziente con patologia non neoplastica. L’analisi quantitativa dell’adesione delle cellule DiI+ ha confermato una maggiore capacità adesiva delle cellule Caco2 rispetto alle cellule AGS, indipendente dal microambiente, neoplastico o meno, del lavaggio peritoneale di origine. Oltre a ciò, le AGS hanno mostrato una maggiore tendenza ad aderire al monostrato mesoteliale ottenuto dal paziente con carcinoma del colon, lasciando ipotizzare la presenza di peculiari caratteristiche biologiche appartenenti al monostrato stesso. Tra le caratteristiche biologiche del mesotelio in grado di promuovere l’adesione delle cellule tumorali è stato ampiamente documentato il ruolo della senescenza il cui grado appare, inoltre, correlato con l’espressione di molecole di adesione come ICAM-1 (Ksiazek et al. 2009, 2010). Queste caratteristiche sono state valutate anche nei modelli di cocoltura precedentemente utilizzati: un’analisi quantitativa dell’espressione di ICAM-1 sulle tre colture primarie in esame ha indicato, al primo passaggio in vitro, una maggiore espressione di ICAM-1 nel monostrato mesoteliale proveniente dal paziente affetto da carcinoma del colon, lo stesso che aveva manifestato un maggior grado di permissività all’adesione per la linea tumorale AGS. Per documentare ulteriormente il ruolo giocato dalla senescenza nel processo di adesione si è provveduto ad indurre in vitro, in questa stessa coltura primaria, un maggior livello di senescenza sfruttando il principio secondo cui è possibile raggiungere questo obiettivo nel corso di passaggi di coltura successivi e ravvicinati nel tempo (Ksiazek et al. 2009). Come atteso, la coltura primaria al passaggio P4 ,oltre a mostrare una maggiore espressione di ICAM-1 rispetto alla coltura al passaggio P2, è risultata più permissiva all’adesione della linea tumorale Caco2. La progressiva inibizione dose-dipendente dell’adesione ottenuta attraverso il blocco di ICAM-1, ha dimostrato che l’aumentata capacità delle cellule neoplastiche di interagire con il monostrato mesoteliale senescente è direttamente correlata con l’espressione di questa molecola di adesione. L’insieme dei risultati ottenuti dal Dottor Ranieri in questa parte di progetto ha permesso di dimostrare che le colture primarie ottenute dai campioni di lavaggio peritoneale sono uno strumento pratico e riproducibile per analizzare in vitro i meccanismi mesoteliali coinvolti nel processo di adesione delle cellule tumorali e di documentare che l’adesione delle cellule neoplastiche al monostrato mesoteliale non è dipendente dal microambiente mesoteliale di origine della coltura o dal possibile stato di attivazione delle cellule mesoteliali associato ai diversi tipi di carcinoma, ma piuttosto dai livelli di espressione di ICAM-1 direttamente correlati al grado di senescenza cellulare. La seconda parte delle attività di ricerca del Dottor Danilo Ranieri è stata rivolta all’ottimizzazione di metodiche di rilevazione e caratterizzazione morfologica e molecolare delle cellule disseminate tumorali (FPTCs, free peritoneal tumor cells) nei lavaggi peritoneali e alla valutazione dell’impatto prognostico della loro presenza nei pazienti studiati (27 affetti da carcinoma gastrico e 48 affetti da tumore del colon-retto). I carcinomi dello stomaco e del colon-retto, tumori tra i più diffusi, possono dare entrambi origine a metastasi per via ematica, linfatica e a disseminazione peritoneale. Quest’ultima, più frequente nel tumore gastrico rispetto a quello del colon, è dovuta in entrambi i casi allo sfaldamento delle cellule dal tumore primario (Khair et al. 2007, Pantel et al. 2008). Lo studio del lavaggio peritoneale con metodiche citologiche tradizionali viene utilizzato per rilevare la presenza di cellule tumorali libere nella cavità peritoneale. La positività citologica costitusce da molto tempo un’indicazione prognostica sfavorevole (Fujiwara et al. 2007, Lee et al. 2009, Katsuragi et al. 2007) anche se la carcinosi peritoneale può svilupparsi frequentemente in pazienti con lavaggi peritoneali negativi. La scarsa sensibilità delle metodiche citologiche tradizionali è stata oggi superata dall’impiego di metodiche di diagnostica molecolare; l’analisi con real-time qRT-PCR basata sulla ricerca nel lavaggio peritoneale di tipici marcatori tumorali ed epiteliali (antigene carcinoembrionario, citocheratine CK19 e CK20) è stata impiegata con successo nei pazienti con carcinoma gastrico e la positività è risultata un valido indicatore di prognosi sfavorevole. L’approccio sperimentale multiplo disegnato dal Dottor Ranieri ha previsto l’analisi del liquido peritoneale in toto (Real Time qRT-PCR) o dopo arricchimento immunomagnetico della componente epiteliale. L’applicazione delle tecniche di arricchimento immunomagnetico è stata associata all’analisi in immunofluorescenza e, su casi selezionati, ad una valutazione ultrastrutturale condotta con microscopia elettronica a trasmissione; i risultati sono stati confrontati con quelli ottenuti dall’analisi citologica convenzionale e dall’analisi molecolare con real time qRT-PCR. Sono stati definiti positivi per FPTCs i liquidi di lavaggio che all’immunofluorescenza presentavano cellule caratterizzate dalla doppia positività per il marcatore epiteliale CD326/EpCAM e per il marcatore tumorale CEA o che all’analisi molecolare mostravano livelli di mRNA per CEA e/o CK20 superiori al cut-off. I liquidi di lavaggio provenienti dai pazienti con carcinoma dello stomaco hanno mostrato una positività globale in immunofluorescenza e qRT-PCR pari al 15% e al 78%. Lo studio citologico è risultato positivo solo su 4 pazienti (15%) con carcinosi peritoneale massiva. L’analisi statistica dei dati ha mostrato che la positività in immunofluorescenza è risultata associata al grading, all’invasione locale della parete gastrica, agli stadi avanzati di malattia e alla concomitante positività citologica. L’analisi molecolare con real time qRT-PCR ha mostrato una prevalenza di positività per FPTCs molto superiore a quella ottenuta con l’immunofluorescenza; il 70% dei campioni è risultato positivo al CEA, il 41% a CK20, il 36% all’associazione CEA/CK20. La positività riscontrata all’analisi molecolare non è risultata associata al grado di invasione locale o allo stadio della malattia ma solo al grading istopatologico. L’analisi delle curve di sopravvivenza e di recidiva e l’analisi multivariata hanno dimostrato il ruolo prognostico sfavorevole della positività per FPTCs ottenuta sia in immunofluorescenza che in real time qRT-PCR. Nei liquidi di lavaggio provenienti dai pazienti con carcinoma del colon-retto la positività globale in immunofluorescenza e qRT-PCR è risultata pari al 17% e al 42%. La citologia è risultata negativa in tutti i pazienti. Contrariamente a quanto osservato nei carcinomi gastrici, non è stata documentata associazione tra positività in immunofluorescenza e grading istologico, grado di invasione locale o stadio della neoplasia. L’analisi molecolare in real time qRT-PCR ha documentato una maggiore positività per il CEA (42%) a fronte di una ridotta positività per CK20 (10%). Come osservato nei pazienti con carcinoma gastrico, l’analisi delle curve di sopravvivenza e di recidiva e l’analisi multivariata hanno documentato il ruolo prognostico sfavorevole della positività per FPTCs ottenuta in qRT-PCR. L’insieme dei risultati ottenuti dal Dottor Ranieri in questa parte di progetto ha permesso di dimostrare che l’associazione di tecniche di immunoarricchimento epiteliale con metodiche di analisi di immunofluorescenza e real time qRT-PCR costituisce un valido sistema per identificare e caratterizzare le FPTCs nei liquidi di lavaggio peritoneale; l’analisi molecolare in real time qRT-PCR dei livelli di mRNA di CEA e CK20 in tali campioni può, peraltro, essere un utile strumento prognostico non solo per i pazienti affetti da carcinoma gastrico ma anche per quelli affetti da carcinoma del colon-retto. Le informazioni che possono essere ottenute da questo tipo di approccio contribuiscono complessivamente ad una migliore definizione del comportamento biologico delle neoplasie epiteliali del tratto gastrointestinale.

碩士
高雄醫學大學
醫學研究所
96
Introduction and aims: Chronic peritoneal dialysis is one of the major therapies for patients with end stage renal disorders. Dialysates with bio-incompatibility may lead to peritoneal mesothelial cell injury, fibrosis and subsequent poor dialysis efficacy. The aim of this study investigates the toxic effects of conventional glucose-based dialysates in primary human peritoneal mesothelial cells (HPMCs) and the possible roles of amtioxidants in HPMC during conventional dialysate exposure. Methods：HPMCs were collected from peritoneal effluent of patients receiving peritoneal dialysis (PD). Conventional 1.5% dextrose peritoneal dialysate was utilized. HPMCs were exposed to conventional peritoneal dialysate with 1.5% dextrose for different time course and then the survival rate of HPMCs was evaluated by MTT assay. Curcumin（CUR）and N-acetylcysteine (NAC) were utilized as antioxidants. ROS accumulation, such as intracellular hydrogen peroxide, and superoxide anion in HPMCs were detected with flow cytometry by using intracellular probes, H2DCFDA and dHE, respectively,. Protein expression of HSP72 and HSP27 were detected by Western blots analysis. Antioxidant enzymes, such as catalase and superoxide dimutase (SOD), and glutathione were detected by ELSA reader. Result：Present study shows that conventional 1.5% dextrose dialysate exposure resulted in significant decrease of cell survival in a time dependent manner. NAC treatment contributes to decrease the conventional dialysate-induced cell death. However, curcumin can not offer the protective effects. Moreover, HSP72 is induced in HPMCs after dialysate exposure. NAC treatment decreases HSP72 induction. The intensity of DCF green fluorescence and the EB red fluorescence are increased, and shifting to right showed in histogram after conventional dialysate exposure. The accumulation of intracellular hydrogen peroxide and superoxide anion are in an exposure time-dependent manner. However, NAC treatment contributes to decrease dialysate induced-ROS accumulation. Activity of SOD and catalase is not different in every group. However, the reduced glutathione is significant decrease after dialysate exposure and NAC treatment contributes to preserve the expression of reduced glutathione in HPMCs. Conclusion: ROS accumulation during convention peritoneal dialysate exposure is an important cause of leading cell damage. NAC treatment contributes to protect the HPMCs from conventional dialysates-induced cellular damage by preserving the reduced glutathione to decrease ROS accumulation and oxidative stresses. HSP72 could be used as a stress marker.

碩士
國防醫學院
藥理學研究所
96
Introduction Intracellular pH (pHi) changes were found to influence many cellular functions. To date, the pHi regulators include Na+/H+ exchanger (NHE), Na+/HCO3- symporter (NHS), lactate-/H+ symporter (LHS), Cl-/OH- exchanger (CHE) and Cl-/HCO3- exchanger (AE) in the mammalian cells. Then, peritoneal dialysis (PD) is one method of treating chronic uremic patients. Available 4 commercial peritoneal dialysis fluids (PDFs), Dianeal, Extraneal, Nutrineal and Physioneal, have various but dramatic different on pH values (pH is 5.2, 5.2, 6.3 and 7.4 respectively). Objects The aims of the study used the human peritoneal mesothelial cells (HPMCs) to: (1.) investigate the underlying mechanisms for its effects on pHi regulation (NHE and NHS) and NHE isoforms; (2.) explore the effects of commercial PDFs and different pHo solutions on pHi; (3.) investigate the LHS, the effects of 40 mM lactate and different buffered solutions on LHS activity. Materials and Methods 1.HPMCs were cultured directly from peritoneal tissue and identified by immunocytochemistry. 2.The measurement of pHi was detected by microspectrofluorimetry method with BCECF-AM. We recorded fluorescence ratio (R490/440), which represented the change of pHi. 3.Immunocyto/histochemistry were used to identify NHE-1, NHE-2 and NHE-3 in HPMCs or peritoneal biopsy tissues. Results 1.We have successfully cultured human peritoneal mesothelial primary cells from tissue and set-up a reproduceable model for routine use. 2.In HEPES-buffered solution, removal of extracellular Na+ or adding HOE 694 (a specific NHE inhibitor) alone can totally inhibit the recovery from NH4Cl-induced intracellular acidosis, which prove the existence of NHE. 3.In bicarbonate-buffered solution, either HOE 694 or DIDS (a specific NHS inhibitor) alone only partially inhibited the recovery from NH4Cl-induced intracellular acidosis, while removal of Na+ or adding together HOE 694 and DIDS totally inhibited the recovery. This demonstrate the existence of NHS. 4.Both in HEPES- and bicarbonate-buffered solution, all 4 different PDFs (pH 5.2, 5.2, 6.3, 7.4, respectively) significantly changed HPMCs pHi. 5.Both in HEPES- and bicarbonate-buffered solution, we demonstrated that, in the HPMCs, pHi is increased linearly as pHo rising from 5.2 to 8.0. 6.In sodium citrate solution, CHC (a LHS inhibitor) inhibited pHi recovery from lactate-induced intracellular acidosis, which proves the existence of LHS. Lactate-induced intracellular acidosis kinetice was not affected by HOE 694 or 40 mM lactate, either in bicarbonate- or HEPES-buffered solutions. Conclusions 1.We have demonstrated for the first time that, two Na+-dependent acid-extruders (NHE and NHS) and LHS are functionally existed in the HPMCs. NHE-1 and NHE-3 were identified in HPMCs. 2.Commercial PDFs used in clinics significantly affect HPMCs pHi, which suggest the space for the improvement or developing new PDFs. 3.We have demonstrated that pHi is functional linearly as pHo. In bicarbonate- buffered solution (pH 5.2~7.4), it was best buffered solution for HPMCs.

碩士
高雄醫學大學
醫學研究所碩士班
93
Traditional peritoneal dialysis solutions are bioincompatible resulting from high glucose concentration, high osmolality, low pH, and glucose degradation products（GDP）. Nutrineal○R is 1.1％ amino acid-based peritoneal dialysis solution and is designed to replace daily losses of amino acids and proteins during peritoneal dialysis. Its pH value is 6.5, closer to physiological value. And there is no GDPs in Nutrineal○R . It may be more biocompatible. L-Methionine, 85 mg/dl in Nutrineal®, is one of the components. However, the absorption of methionine may increase the synthesis of a well-recognized independent risk factor for cardiovascular diseases, homocysteine (Hcy). Poor appetite was reported after use of Nutrineal®. In this study, we observed the changes of serum biochemistry, serum homocysteine and leptin, Kt/V and nPCR, CA125 and PICP of peritoneal effluent, and the response of human peritoneal mesothelial cell after treating with various peritoneal dialysis solution. Methods: Seventeen adults CAPD patients (11M/6F) who have received CAPD for at least 6 months were enrolled. They all received daily four-exchange CAPD treatment and Nutrineal® was used as once daily substitute, given at the first or second exchange. Blood samples and peritoneal effluents were collected every month. Serum albumin, total protein, BUN, creatinine, homocysteine, and leptin were measured. Kt/V , nPCR, and body mass index (BMI) were calculated. And we checked the CA125 and PICP of peritoneal effluent. Human peritoneal mesothelial cells were cultured from peritoneal effluent and then were treated with various peritoneal dialysis solutions. LDH, heat shock protein 70, and CA125 were measured. Results: Serum BUN and homocysteine increased significantly（p=0.007, p=0.003）. Other serum data did not change. Body weight and BMI increased significantly（p = 0.0344, p = 0.037）; but there was significant change in serum leptin. Peritoneal effluent CA125 increased significantly（p=0.045） and PICP did not change. There were a similar pattern in increase of LDH, heat shock protein 70, and CA125 measured from cultured mesothelial cell treated by various solutions. The increase by Nutrineal○R was lower; the increase by 4.25％ Dianeal○R and Extraneal○R were higher. Conclusion: Nutrineal○R is more biocompatible to peritoneal membrane, but there was a disadvantage in increases of BUN and homocysteine after use of Nutrineal○R .Body weight and BMI were increased after use of Nutrineal○R . CA125 from human peritoneal mesothelial cells was increased after stimulated by various solutions. Further studies will be needed.

碩士
國立臺灣大學
臨床醫學研究所
91
The patients undertaking peritoneal dialysis are inclined to have peritonitis and further, denudation of the mesothelial cell monolayer. The recovery from the said requires regeneration of the mesothelial cells (including adhesion, migration and proliferation) to keep the integrity of the peritoneum. This is highly related to the degree of achievement of prevention in peritoneal fibrosis and the maintenance of peritoneal function. It’s known that the occurrence of peritonitis would stimulate the production of prostaglandin (PG), however, the role it plays is not yet clear. Based on some reports, PG can promote migration of the mesangial cells and the endothelial cells though there is no answer that is there similar effect on the mesothelial cells. Moreover, PG also helps the proliferation of gastric epithelial cells through the increasing production of hepatocyte growth factor (HGF). But the PG’s effect on the mesothelial cells is not yet studied. PG also works to prevent the production of extracellular matrix in the mesangial cells. Is there similar effect on the mesothelial cells still needs to clarified. Hence, our study is designed to ask (1) whether PG can work to help migration, adhesion and proliferation in the mesothelial cell as well. And if the promotion of proliferation is proved, this effect is related to the incremental of HGF or not; (2) PG can also function in prohibiting the expression of collagen gene of the mesothelial cell or not; and (3) the clinical use of NSAID or COX-2 inhibitor does harm to the peritoneal repair. Our study evaluated adhesion of the mesothelial cell by trypsin assay first. Secondly, migration and proliferation of the cells would be evaluated with cell culture techniques by establishing a simulation model of damaging mesothelial cell in vitro. And the expression of collagen gene is measured by Northern blot analysis. Test the mesothelial cells with various dosage of PGE2、PGI2、IL-1β、indomethacin and COX-2 inhibitor (NS398) to verify their influences on the mesothelial cell’s adhesion, migration, proliferation and expression of collagen gene. And further check there is IL-1β → PG → HGF Pathway existing in the mesothelial cells or not by the enzymeimmunoassay analysis. The results showed both PGE2 and PGI2 can promote the mesothelial cell’s adhesion, as well as the IL-1β. PGE2 does not stimulate migration of mesothelial cells. However, PGI2 has the opposite trend on migration though there is no statistic significance. IL-1β can increase the secretion of PGE2 which peaks at the 16 hour of treatment and decades at the following hours. Nevertheless, both PGE2 and PGI2 have no effect on the secretion of HGF. The Northern blot analysis reveals PGE2 and PGI2 can suppress the expression of collagen gene, and COX-2 inhibitor and NSAID can block the suppression effect of PG in the stimulation of IL-1β. This study proves part of our hypothesis that both PGE2 and PGI2 can promote adhesion and suppress the collagen gene expression of the mesothelial cells. Therefore, we know that the clinical use of NSAID and COX-2 inhibitor may be harmful to the integrity of peritoneum, especially during the course of peritonitis when COX-2 is highly activated and the repair of peritoneum needs the function of PG.