Commercialisation of regenerative human tissue : Regulation and reform in Australia and England, Wales and Northern Ireland

The commercialisation of therapeutic products containing regenerative human tissue is regulated by the common law, statute and ethical guidelines in Australia and England, Wales and Northern Ireland. This article examines the regulatory regimes in these jurisdictions and considers whether reform is required to both support scientific research and ensure conformity with modern social views on medical research and the use of human tissue. The authors consider the crucial role of informed consent in striking the balance between the interests of researchers and the interests of the public.

Genetic polymorphisms in the human tissue kallikrein (KLK) locus and their implication in various malignant and non-malignant diseases

The Kallikrein (KLK) gene locus encodes a family of serine proteases and is the largest contiguous cluster of protease-encoding genes attributed an evolutionary age of 330 million years. The KLK locus has been implicated as a high susceptibility risk loci in numerous cancer studies through the last decade. The KLK3 gene already has established clinical relevance as a biomarker in prostate cancer prognosis through its encoded protein, prostate-specific antigen. Data mined through genome-wide association studies (GWAS) and next-generation sequencing point to many important candidate single nucleotide polymorphisms (SNPs) in KLK3 and other KLK genes. SNPs in the KLK locus have been found to be associated with several diseases including cancer, hypertension, cardiovascular disease and atopic dermatitis. Moreover, introducing a model incorporating SNPs to improve the efficiency of prostate-specific antigen in detecting malignant states of prostate cancer has been recently suggested. Establishing the functional relevance of these newly-discovered SNPs, and their interactions with each other, through in silico investigations followed by experimental validation, can accelerate the discovery of diagnostic and prognostic biomarkers. In this review, we discuss the various genetic association studies on the KLK loci identified either through candidate gene association studies or at the GWAS and post-GWAS front to aid researchers in streamlining their search for the most significant, relevant and therapeutically promising candidate KLK gene and/or SNP for future investigations.

The human tissue kallikrein and kallikrein-related peptidase family

The human kallikrein-related peptidases are a subgroup of trypsin and chymotrypsin-like serine peptidases that are characterized by their homology to tissue kallikrein or kallikrein 1 (KLK1) encoded by the KLK1 gene (reviewed in[1-4]). The human KLK locus spans an approximately 320 kb region on chromosome 19q13.3-13.4 and contains fifteen genes encoding KLK1 and fourteen other kallikrein-related peptidases, KLK2-KLK15, which have been named contiguously in the locus in the order of their discovery [5-8] (Figure 606.1). It is the largest contiguous cluster of serine protease encoding genes in the human genome which has evolved from gene duplication of KLK1 and then subsequent reduplication of the newly evolved KLK genes [2]. The high conservation noted for KLK1-KLK3 (62-77%) reflects the proposed duplication of the KLK1 gene that produced the KLK2 gene which further generated the KLK3 gene. In contrast, the newer KLK4-KLK15 proteases share much less similarity, from 24-66%, although strong homology between KLK4 and KLK5, KLK9 and KLK11, and KLK10 and KLK12 suggests these genes are duplications of each other [2]...

High and low mammographic density human breast tissues maintain histological differential in murine tissue engineering chambers

Mammographic density (MD) is the area of breast tissue that appears radiologically white on mammography. Although high MD is a strong risk factor for breast cancer, independent of BRCA1/2 mutation status, the molecular basis of high MD and its associated breast cancer risk is poorly understood. MD studies will benefit from an animal model, where hormonal, gene and drug perturbations on MD can be measured in a preclinical context. High and low MD tissues were selectively sampled by stereotactic biopsy from operative specimens of high-risk women undergoing prophylactic mastectomy. The high and low MD tissues were transferred into separate vascularised biochambers in the groins of SCID mice. Chamber material was harvested after 6 weeks for histological analyses and immunohistochemistry for cytokeratins, vimentin and a human-specific mitochondrial antigen. Within-individual analysis was performed in replicate mice, eliminating confounding by age, body mass index and process-related factors, and comparisons were made to the parental human tissue. Maintenance of differential MD post-propagation was assessed radiographically. Immunohistochemical staining confirmed the preservation of human glandular and stromal components in the murine biochambers, with maintenance of radiographic MD differential. Propagated high MD regions had higher stromal (p = 0.0002) and lower adipose (p = 0.0006) composition, reflecting the findings in the original human breast tissue, although glands appeared small and non-complex in both high and low MD groups. No significant differences were observed in glandular area (p = 0.4) or count (p = 0.4) between high and low MD biochamber tissues. Human mammary glandular and stromal tissues were viably maintained in murine biochambers, with preservation of differential radiographic density and histological features. Our study provides a murine model for future studies into the biomolecular basis of MD as a risk factor for breast cancer.

Molecular architecture of the human sinus node: insights into the function of the cardiac pacemaker.

BACKGROUND: Although we know much about the molecular makeup of the sinus node (SN) in small mammals, little is known about it in humans. The aims of the present study were to investigate the expression of ion channels in the human SN and to use the data to predict electrical activity. METHODS AND RESULTS: Quantitative polymerase chain reaction, in situ hybridization, and immunofluorescence were used to analyze 6 human tissue samples. Messenger RNA (mRNA) for 120 ion channels (and some related proteins) was measured in the SN, a novel paranodal area, and the right atrium (RA). The results showed, for example, that in the SN compared with the RA, there was a lower expression of Na(v)1.5, K(v)4.3, K(v)1.5, ERG, K(ir)2.1, K(ir)6.2, RyR2, SERCA2a, Cx40, and Cx43 mRNAs but a higher expression of Ca(v)1.3, Ca(v)3.1, HCN1, and HCN4 mRNAs. The expression pattern of many ion channels in the paranodal area was intermediate between that of the SN and RA; however, compared with the SN and RA, the paranodal area showed greater expression of K(v)4.2, K(ir)6.1, TASK1, SK2, and MiRP2. Expression of ion channel proteins was in agreement with expression of the corresponding mRNAs. The levels of mRNA in the SN, as a percentage of those in the RA, were used to estimate conductances of key ionic currents as a percentage of those in a mathematical model of human atrial action potential. The resulting SN model successfully produced pacemaking. CONCLUSIONS: Ion channels show a complex and heterogeneous pattern of expression in the SN, paranodal area, and RA in humans, and the expression pattern is appropriate to explain pacemaking.

Ryanodine receptor phosphorylation in human heart failure

Heart failure is a complex disorder, characterized by activation of the sympathetic nervous system, leading to dysregulated Ca2+ homeostasis in cardiac myocytes and tissue remodeling. In a variety of diseases, cardiac malfunction is associated with aberrant fluxes of Ca2+ across both the surface membrane and the internal Ca2+ store, the sarcoplasmic reticulum (SR). One prominent hypothesis residues is that in heart failure, the activity of the ryanodine receptor (RyR2) Ca2+ release channel in the SR is increased due to excess phosphorylation and that this contributes to excess SR Ca2+ leak in diastole, reduced SR Ca2+ load and decreased contractility (Huke & Bers, 2008). There is controversy over which serine residues in RyR2 are hyperphosphorylated in animal models of heart failure and whether this is via the CaMKII or the PKA-linked signaling pathway. S2808, S2814 and S2030 in RyR2 have been variously claimed to be hyperphosphorylated. Our aim was to examine the degree of phosphorylation of these residues in RyR2 from failing human hearts. The use of human tissue was approved by the Human Research Ethics Committee, The Prince Charles Hospital, EC28114. Left ventricular tissue samples were obtained from an explanted heart of a patient with endstage heart failure (Emery Dreifuss Muscular Dystrophy with cardiomyopathy) and non-failing tissue was from a patient with cystic fibrosis undergoing heart-lung transplantation with no history of heart disease. SR vesicles were prepared as described by Laver et al. (1995) and examined with SDS-Page and Western Blot. Transferred proteins were probed with antibodies to detect total protein phosphorylation, phosphorylation of RyR2 serine residues S2808, S2814, S2030 and for the key proteins calsequestrin, triadin, junctin and FKBP12.6. To avoid membrane stripping artifact, each membrane was exposed to one phosphorylation-specific antibody and signal densities quantified using Bio-Rad Quantity One software. We found no distinguishable difference between failing and healthy hearts in the protein expression levels of RyR2, triadin, junctin or calsequestrin. We found an expected upregulation of total RyR2 phosphorylation in the failing heart sample, compared to a matched amount of RyR2 (quantified using densiometry) in healthy heart. Probing with antibodies detecting only the phosphorylated form of the specific RyR2 residues showed that the increase in total RyR2 phosphorylation in the failing heart was due to hyperphosphorylation of S2808 and S2814. We found that S2030 phosphorylation levels were unchanged in human heart failure. Interestingly, we found that S2030 has a basal level of phosphorylation in the healthy human heart, different from the absence of basal phosphorylation recently reported in rodent heart (Huke & Bers, 2008). Finally, preliminary results indicate that less FKBP 12.6 is associated with RyR2 in the failing heart, possibly as a consequence of PKA activation. In conclusion, residues S2808 and S2814 are hyperphosphorylated in human heart failure, presumably due to upregulation of the CaMKII and/or PKA signaling pathway as a result of chronic activation of the sympathetic nervous system. Such changes in RyR2 phosphorylation are believed to contribute to the leaky RyR2 phenotype associated with heart failure, which increases the incidence of arrhythmia and contributes to the severely impaired contractile performance of the failing heart. Huke S & Bers DM. (2008). Ryanodine receptor phosphorylation at serine 2030, 2808 and 2814 in rat cardiomyocytes. Biochemical and Biophysical Research Communications 376, 80-85. Laver DR, Roden LD, Ahern GP, Eager KR, Junankar PR & Dulhunty AF. (1995). Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle. Journal of Membrane Biology 147, 7-22. Proceedings

Pten and Ndufb8 aberrations in cervical cancer tissue

Cervical cancer is one of the world's major health issues. Despite many studies in this field, the carcinogenetic events of malignant conversion in cervical tumours have not been significantly characterised. The first aim of this project was to investigate the mutation status of the tumour suppressor gene- Phosphatase and Tension Homolog (PTEN)- in cervical cancer tissue. The second aim of this study was the analysis in the same cervical cancer tissue for aberrations in the mitochondrial electron transport chain subunit gene NDUFB8, which is localised to the same chromosomal contig as PTEN. The third aim was the evaluation of the potential therapeutic anti-cancer drug 2,4-Thiazolidinediones (TZDs) and its affect in regulating the PTEN protein in a cervical cancer cell line (HeLa). To approach the aims, paraffin-embedded cancerous cervical tissue and non-cancerous cervical tissue were obtained. DNA recovered from those tissues was then used to investigate the putative genomic changes regarding the NDUFB8 gene utilising SYBR Green I Real-Time PCR. The PTEN gene was studied via Dual-Labelled probe Real-Time PCR. To investigate the protein expression change of the PTEN protein, HeLa cells were firstly treated with different concentrations of 2,4-Thiazolidinediones and the level of PTEN protein expression was then observed utilising standard protein assays. Results indicated that there were putative copy-number changes between the cancerous cervical tissue and non-cancerous cervical tissue, with regard to the PTEN locus. This implies a potential gain of the PTEN gene in cancerous cervical tissue. With regards to normal cervical tissue versus cancerous cervical tissue no significant melting temperature differences were observed with the SYBR Green I Real-Time PCR in respect to the NDUFB8 gene. A putative up-regulation of PTEN protein was observed in TZD treated HeLa cells. © 2008 Springer Science+Business Media, LLC.

Expression and prognostic significance of a panel of tissue hypoxia markers in head-and-neck squamous cell carcinomas

Purpose: To investigate the expression pattern of hypoxia-induced proteins identified as being involved in malignant progression of head-and-neck squamous cell carcinoma (HNSCC) and to determine their relationship to tumor pO 2 and prognosis. Methods and Materials: We performed immunohistochemical staining of hypoxia-induced proteins (carbonic anhydrase IX [CA IX], BNIP3L, connective tissue growth factor, osteopontin, ephrin A1, hypoxia inducible gene-2, dihydrofolate reductase, galectin-1, IκB kinase β, and lysyl oxidase) on tumor tissue arrays of 101 HNSCC patients with pretreatment pO 2 measurements. Analysis of variance and Fisher's exact tests were used to evaluate the relationship between marker expression, tumor pO 2, and CA IX staining. Cox proportional hazard model and log-rank tests were used to determine the relationship between markers and prognosis. Results: Osteopontin expression correlated with tumor pO 2 (Eppendorf measurements) (p = 0.04). However, there was a strong correlation between lysyl oxidase, ephrin A1, and galectin-1 and CA IX staining. These markers also predicted for cancer-specific survival and overall survival on univariate analysis. A hypoxia score of 0-5 was assigned to each patient, on the basis of the presence of strong staining for these markers, whereby a higher score signifies increased marker expression. On multivariate analysis, increasing hypoxia score was an independent prognostic factor for cancer-specific survival (p = 0.015) and was borderline significant for overall survival (p = 0.057) when adjusted for other independent predictors of outcomes (hemoglobin and age). Conclusions: We identified a panel of hypoxia-related tissue markers that correlates with treatment outcomes in HNSCC. Validation of these markers will be needed to determine their utility in identifying patients for hypoxia-targeted therapy. © 2007 Elsevier Inc. All rights reserved.

Identification of abundant alkyl ether glycerophospholipids in the human lens by tandem mass spectrometry techniques

Previous studies have shown that the human lens contains glycerophospholipids with ether linkages. These lipids differ from conventional glycerophospholipids in that the sn-1 substituent is attached to the glycerol backbone via an 1-O-alkyl or an 1-O-alk-1'-enyl ether rather than an ester bond. The present investigation employed a combination of collision-induced dissociation (CID) and ozone-induced dissociation (OzID) to unambiguously distinguish such 1-O-alkyl and 1-O-alk-1'-enyl ethers. Using these methodologies the human lens was found to contain several abundant 1-O-alkyl glycerophos-phoethanolamines, including GPEtn(16:0e/9Z-18:1), GPEtn(11Z-18:1e/9Z-18:1), and GPEtn(18:0e/9Z-18:1), as well as a related series of unusual 1-O-alkyl glycerophosphoserines, including GPSer(16:0e/9Z-18:1), GPSer(11Z-18:1e/9Z-18:1), GPSer(18:0e/9Z-18:1) that to our knowledge have not previously been observed in human tissue. Isomeric 1-O-alk-1'-enyl ethers were absent or in low abundance. Examination of the double bond position within the phospholipids using OzID revealed that several positional isomers were present, including sites of unsaturation at the n-9, n-7, and even n-5 positions. Tandem CID/OzID experiments revealed a preference for double bonds in the n-7 position of 1-O-ether linked chains, while n-9 double bonds predominated in the ester-linked fatty acids [e.g., GPEtn(11Z-18:1e/9Z-18:1) and GPSer(11Z-18:1e/9Z-18:1)]. Different combinations of these double bond positional isomers within chains at the sn-1 and sn-2 positions point to a remarkable molecular diversity of ether-lipids within the human lens.

Association of CYP1B1 codon 432 mutant allele in head and neck squamous cell cancer is reflected by somatic mutations of p53 in tumor tissue

Tobacco use is causally associated with head and neck squamous cell cancer (HNSCC). Here, we present the results of a case-control study that investigated the effects that the genetic variants of the cytochrome (CYP)1A1, CYP1B1, glutathione-S-transferase (GST)M1, GSTT1, and GSTP1 genes have on modifying the risk of smoking-related HNSCC. Allelisms of the CYP1A1, GSTT1, GSTM1, and GSTT1 genes alone were not associated with an increased risk. CYP1B1 codon 432 polymorphism was found to be a putative susceptibility factor in smoking-related HNSCC. The frequency of CYP1B1 polymorphism was significantly higher (P < 0.001) in the group of smoking cases when compared with smoking controls. Additionally, an odds ratio (OR) of 4.53 (2.62-7.98) was discovered when investigating smoking and nonsmoking cases for the susceptible genotype CYP1B1*2/*2, when compared with the presence of the genotype wild type. In combination with polymorphic variants of the GST genes, a synergistic-effect OR was observed. The calculated OR for the combined genotype CYP1B1*2/*2 and GSTM1*2/*2 was 12.8 (4.09-49.7). The calculated OR for the combined genotype was 13.4 (2.92-97.7) for CYP1B1*2/*2 and GSTT1*2/*2, and 24.1 (9.36-70.5) for the combination of CYP1B1*2/*2 and GSTT1-expressors. The impact of the polymorphic variants of the CYP1B1 gene on HNSCC risk is reflected by the strong association with the frequency of somatic mutations of the p53 gene. Smokers with susceptible genotype CYP1B1*2/*2 were 20 times more likely to show evidence of p53 mutations than were those with CYP1B1 wild type. Combined genotype analysis of CYP1B1 and GSTM1 or GSTT1 revealed interactive effects on the occurrence of p53 gene mutations. The results of the present study indicate that polymorphic variants of CYP1B1 relate significantly to the individual susceptibility of smokers to HNSCC.

Expression profiling in spondyloarthropathy synovial biopsies highlights changes in expression of inflammatory genes in conjunction with tissue remodelling genes

Background: In the spondyloarthropathies, the underlying molecular and cellular pathways driving disease are poorly understood. By undertaking a study in knee synovial biopsies from spondyloarthropathy (SpA) and ankylosing spondylitis (AS) patients we aimed to elucidate dysregulated genes and pathways. Methods RNA was extracted from six SpA, two AS, three osteoarthritis (OA) and four normal control knee synovial biopsies. Whole genome expression profiling was undertaken using the Illumina DASL system, which assays 24000 cDNA probes. Differentially expressed candidate genes were then validated using quantitative PCR and immunohistochemistry. Results: Four hundred and sixteen differentially expressed genes were identified that clearly delineated between AS/SpA and control groups. Pathway analysis showed altered gene-expression in oxidoreductase activity, B-cell associated, matrix catabolic, and metabolic pathways. Altered «myogene» profiling was also identified. The inflammatory mediator, MMP3, was strongly upregulated (5-fold) in AS/SpA samples and the Wnt pathway inhibitors DKK3 (2.7-fold) and Kremen1 (1.5-fold) were downregulated. Conclusions: Altered expression profiling in SpA and AS samples demonstrates that disease pathogenesis is associated with both systemic inflammation as well as local tissue alterations that may underlie tissue damaging modelling and remodelling outcomes. This supports the hypothesis that initial systemic inflammation in spondyloarthropathies transfers to and persists in the local joint environment, and might subsequently mediate changes in genes directly involved in the destructive tissue remodelling.

Diverse populations of T cells with NK cell receptors accumulate in the human intestine in health and in colorectal cancer

T cells expressing NK cell receptors (NKR) display rapid MHC-unrestricted cytotoxicity and potent cytokine secretion and are thought to play roles in immunity against tumors. We have quantified and characterized NKR+ T cells freshly isolated from epithelial and lamina propria layers of duodenum and colon from 16 individuals with no evidence of gastrointestinal disease and from tumor and uninvolved tissue from 19 patients with colorectal cancer. NKR+ T cell subpopulations were differentially distributed in different intestinal compartments, and CD161+ T cells accounted for over one half of T cells at all locations tested. Most intestinal CD161+ T cells expressed alpha beta TCR and either CD4 or CD8. Significant proportions expressed HLA-DR,CD69 and Fas ligand. Upon stimulation in vitro, CD161+ T cells produced IFN-gamma and TNF-alpha but not IL-4. NKT cells expressing the Valpha24Vbeta11 TCR, which recognizes CD1d,were virtually absent from the intestine, but colonic cells produced IFN-gamma in response to the NKT cell agonist ligand alpha-galactosylceramide. NKR+ T cells were not expanded in colonic tumors compared to adjacent uninvolved tissue. The predominance, heterogeneity and differential distribution of NKR+ T cells at different intestinal locations suggests that they are central to intestinal immunity.

LRP5 regulates human body fat distribution by modulating adipose progenitor biology in a dose- and depot-specific fashion

Summary Common variants in WNT pathway genes have been associated with bone mass and fat distribution, the latter predicting diabetes and cardiovascular disease risk. Rare mutations in the WNT co-receptors LRP5 and LRP6 are similarly associated with bone and cardiometabolic disorders. We investigated the role of LRP5 in human adipose tissue. Subjects with gain-of-function LRP5 mutations and high bone mass had enhanced lower-body fat accumulation. Reciprocally, a low bone mineral density-associated common LRP5 allele correlated with increased abdominal adiposity. Ex vivo LRP5 expression was higher in abdominal versus gluteal adipocyte progenitors. Equivalent knockdown of LRP5 in both progenitor types dose-dependently impaired β-catenin signaling and led to distinct biological outcomes: diminished gluteal and enhanced abdominal adipogenesis. These data highlight how depot differences in WNT/β-catenin pathway activity modulate human fat distribution via effects on adipocyte progenitor biology. They also identify LRP5 as a potential pharmacologic target for the treatment of cardiometabolic disorders. © 2015 The Authors.

Examination of pencil beam predictions of small-field dose using dosimetry film and Monte Carlo simulations

The iPlan treatment planning sys-tem uses a pencil beam algorithm, with density cor-rections, to predict the doses delivered by very small (stereotactic) radiotherapy fields. This study tests the accuracy of dose predictions made by iPlan, for small-field treatments delivered to a planar solid wa-ter phantom and to heterogeneous human tissue using the BrainLAB m3 micro-multileaf collimator.

Development of a 3D culture system to study the skeletal metastasis of prostate cancer

In the cancer research field, most in vitro studies still rely on two-dimensional (2D) cultures. However, the trend is rapidly shifting towards using a three-dimensional (3D) culture system. This is because 3D models better recapitulate the microenvironment of cells, and therefore, yield cellular and molecular responses that more accurately describe the pathophysiology of cancer. By adopting technology platforms established by the tissue engineering discipline, it is now possible to grow cancer cells in extracellular matrix (ECM)-like environments and dictate the biophysical and biochemical properties of the matrix. In addition, 3D models can be modified to recapitulate different stages of cancer progression for instance from the initial development of tumor to metastasis. Inevitably, to recapitulate a heterotypic condition, comprising more than one cell type, it requires a more complex 3D model. To date, 3D models that are available for studying the prostate cancer (CaP)-bone interactions are still lacking. Therefore, the aim of this study is to establish a co-culture model that allows investigation of direct and indirect CaP-bone interactions. Prior to that, 3D polyethylene glycol (PEG)-based hydrogel cultures for CaP cells were first developed and growth conditions were optimised. Characterization of the 3D hydrogel cultures show that LNCaP cells form a multicellular mass that resembles avascular tumor. In comparison to 2D cultures, besides the difference in cell morphology, the response of LNCaP cells to the androgen analogue (R1881) stimulation is different compared to the cells in 2D cultures. This discrepancy between 2D and 3D cultures is likely associated with the cell-cell contact, density and ligand-receptor interactions. Following the 3D monoculture study, a 3D direct co-culture model of CaP cells and the human tissue engineered bone (hTEBC) construct was developed. Interactions between the CaP cells and human osteoblasts (hOBs) resulted in elevation of Matrix Metalloproteinase 9 (MMP9) for PC-3 cells and Prostate Specific Antigen (PSA) for LNCaP cells. To further investigate the paracrine interaction of CaP cells and (hOBs), a 3D indirect co-culture model was developed, where LNCaP cells embedded within PEG hydrogels were co-cultured with hTEBC. It was found that the cellular changes observed reflect the early event of CaP colonizing the bone site. In the absence of androgens, interestingly, up-regulation of PSA and other kallikreins is also detected in the co-culture compared to the LNCaP monoculture. This non androgenic stimulation could be triggered by the soluble factors secreted by the hOB such as Interleukin-6. There are also decrease in alkaline phosphatase (ALP) activity and down-regulation of genes of the hOB when co-cultured with LNCaP cells that have not been previously described. These genes include transforming growth factor β1 (TGFβ1), osteocalcin and Vimentin. However, no changes to epithelial markers (e.g E-cadherin, Cytokeratin 8) were observed in both cell types from the co-culture. Some of these intriguing changes observed in the co-cultures that had not been previously described have enriched the basic knowledge of the CaP cell-bone interaction. From this study, we have shown evidence of the feasibility and versatility of our established 3D models. These models can be adapted to test various hypotheses for studies pertaining to underlying mechanisms of bone metastasis and could provide a vehicle for anticancer drug screening purposes in the future.