Gene expression fingerprint of uterine serous papillary carcinoma: identification of novel molecular markers for uterine serous cancer diagnosis and therapy

Uterine serous papillary cancer (USPC) represents a rare but highly aggressive variant of endometrial cancer, the most common gynecologic tumour in women. We used oligonucleotide microarrays that interrogate the expression of some 10 000 known genes to profile 10 highly purified primary USPC cultures and five normal endometrial cells (NEC). We report that unsupervised analysis of mRNA fingerprints readily distinguished USPC from normal endometrial epithelial cells and identified 139 and 390 genes that exhibited >5-fold upregulation and downregulation, respectively, in primary USPC when compared to NEC. Many of the genes upregulated in USPC were found to represent adhesion molecules, secreted proteins and oncogenes, such as L1 cell adhesion molecule, claudin-3 and claudin-4, kallikrein 6 (protease M) and kallikrein 10 (NES1), interleukin-6 and c-erbB2. Downregulated genes in USPC included SEMACAP3, ras homolog gene family, member I (ARHI), and differentially downregulated in ovarian carcinoma gene 1. Quantitative RT–PCR was used to validate differences in gene expression between USPC and NEC for several of these genes. Owing to its potential as a novel therapeutic marker, expression of the high-affinity epithelial receptor for Clostridium perfringens enterotoxin (CPE) claudin-4 was further validated through immunohistochemical analysis of formalin-fixed paraffin-embedded specimens from which the primary USPC cultures were obtained, as well as an independent set of archival USPC specimens. Finally, the sensitivity of primary USPC to the administration of scalar doses of CPE in vitro was also demonstrated. Our results highlight the novel molecular features of USPC and provide a foundation for the development of new type-specific therapies against this highly aggressive variant of endometrial cancer

Packed bed bioreactor for the isolation and expansion of placental-derived mesenchymal stromal cells

Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm2) with murine-derived green fluorescent proteinexpressing MSCs, and then scaled-up to a 2800 cm2 format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs

Guanylated polymethacrylates : a class of potent antimicrobial polymers with low hemolytic activity

We have synthesized a series of copolymers containing both positively charged (amine, guanidine) and hydrophobic side chains (amphiphilic antimicrobial peptide mimics). To investigate the structure–activity relationships of these polymers, low polydispersity polymethacrylates of varying but uniform molecular weight and composition were synthesized, using a reversible addition–fragmentation chain transfer (RAFT) approach. In a facile second reaction, pendant amine groups were converted to guanidines, allowing for direct comparison of cation structure on activity and toxicity. The guanidine copolymers were much more active against Staphylococcus epidermidis and Candida albicans compared to the amine analogues. Activity against Staphylococcus epidermidis in the presence of fetal bovine serum was only maintained for guanidine copolymers. Selectivity for bacterial over mammalian cells was assessed using hemolytic and hemagglutination toxicity assays. Guanidine copolymers of low to moderate molecular weight and hydrophobicity had high antimicrobial activity with low toxicity. Optimum properties appear to be a balance between charge density, hydrophobic character, and polymer chain length. In conclusion, a suite of guanidine copolymers has been identified that represent a new class of antimicrobial polymers with high potency and low toxicity.

Antimicrobial Polymethacrylates Synthesized as Mimics of Tryptophan-Rich Cationic Peptides

This study describes a facile and high yielding route to two series of polymethacrylates inspired by the naturally occurring, tryptophan-rich cationic antimicrobial polymers. Appropriate optimization of indole content within each gave rise to polymers with high potency against <i>Staphylococcus epidermidis</i> (e.g., PGI-3 minimum inhibitory concentration (MIC) = 12 μg/mL) and the methicillin-resistant strain of <i>Staphylococcus aureus</i> (e.g., PGI-3 MIC = 47 μg/mL) with minimal toxicity toward human red blood cells. Future work will be directed toward understanding the cooperative roles that the cationic and indole pendant groups have for the mechanism of these polymers

Guanylated Polymethacrylates: A Class of Potent Antimicrobial Polymers with Low Hemolytic Activity

We have synthesized a series of copolymers containing both positively charged (amine, guanidine) and hydrophobic side chains (amphiphilic antimicrobial peptide mimics). To investigate the structure–activity relationships of these polymers, low polydispersity polymethacrylates of varying but uniform molecular weight and composition were synthesized, using a reversible addition–fragmentation chain transfer (RAFT) approach. In a facile second reaction, pendant amine groups were converted to guanidines, allowing for direct comparison of cation structure on activity and toxicity. The guanidine copolymers were much more active against Staphylococcus epidermidis and Candida albicans compared to the amine analogues. Activity against Staphylococcus epidermidis in the presence of fetal bovine serum was only maintained for guanidine copolymers. Selectivity for bacterial over mammalian cells was assessed using hemolytic and hemagglutination toxicity assays. Guanidine copolymers of low to moderate molecular weight and hydrophobicity had high antimicrobial activity with low toxicity. Optimum properties appear to be a balance between charge density, hydrophobic character, and polymer chain length. In conclusion, a suite of guanidine copolymers has been identified that represent a new class of antimicrobial polymers with high potency and low toxicity

Characterisation of plasma modified surface and GFP-mMSCs expansion in our packed bed bioreactor in static and perfusion conditions.

<p>(A) Surface composition of the air plasma treated polystyrene scaffold determined by XPS. (B) GFP-mMSC attachment after one and half hours, initially seeded at 3000 cells/cm² (n = 4). (C) The growth after 3 days of culture of GFP-mMSC seeded at 1000 cells/cm² (n = 6). (D) Growth of GFP-mMSC in the bioreactor (BR) under static conditions (n = 4) and (E) under 5 mL/day perfusion (n = 4). (F) Cell harvest recovery and viablity from the bioreactor. (G, H, I and J) Fluorescent microscopy showed that the GFP-mMSC attached to the scaffold (scale bar is 500 μm). (K) IVIS imaging of the fluorescent intensity of PI stained GFP-mMSC in the bioreactor under static and (L) 5 mL/day perfusion conditions. IVIS images are a red (low) / yellow (high) heat map of fluorescent intensity.</p

Pre-isolated passage four pMSC expanded in our bioreactor in static and perfusion conditions.

<p>(A) pMSC expansion in the small-scale 160 cm² bioreactor (BR) in static (n = 4) and (B) 5 mL/day perfusion (n = 4), with the 2D controls (2D). (C) IVIS imaging of PI stained pMSC under perfusion conditions. IVIS images are a red (low) / yellow (high) heat map of fluorescent intensity. (D) Two week tri-lineage mesodermal differentiation induction of bioreactor expanded pMSC and 2D controls down the adipogenic (Oil Red O, 10x, scale bar is 100 μm), osteogenic (Alizarin Red, 5x, scale bar 500 μm) and chondrogenic (Alcian Blue, 10x, scale bar 500 μm) lineages. Quantification of (E) triglycerides (n = 4), (F) ALP activity (n = 4) and (G) GAG production.</p