Examining the potential for porcine-derived islet cells to harbour viral pathogens

With an onus on safety in the potential use of porcine islet cells as a treatment for diabetes, the use of animals lacking exogenous pathogens is clearly important and multilevel screening strategies have been presented on testing animals and the product. In this study, we wished to investigate whether islet cells indeed harboured the same viral pathogens of concern in the source animal. PMBC and islet cells from both adult and neonatal source animals were directly compared and tested for PCMV, PLHV, PCV2, PPV and HEV using both molecular and serological assays. Adult PBMC were found positive for all viruses with the exception of PCV2 and HEV. Neonatal PBMC were only found positive for PCMV and HEV. All animals were found negative for HEV antibodies. Interestingly, islet cells were negative for all viruses tested regardless of status in the animal-derived PBMC. Given that other laboratories have demonstrated the lack of virus detection during the culture of islets, this study also demonstrates that the hygiene status of the herd may not reflect the status of the product. This is important for establishing guidelines for any risk evaluation and mitigation process utilised during product manufacture

Assessment of porcine endogenous retrovirus transmission across an alginate barrier used for the encapsulation of porcine islets

BACKGROUND: Subcutaneous implantation of a macroencapsulated patch containing human allogenic islets has been successfully used to alleviate type 1 diabetes mellitus (T1DM) in a human recipient without the need for immunosuppression. The use of encapsulated porcine islets to treat T1DM has also been reported. Although no evidence of pathogen transfer using this technology has been reported to date, we deemed it appropriate to determine if the encapsulation technology would prevent the release of virus, in particular, the porcine endogenous retrovirus (PERV). METHODS: HEK293 (human epithelial kidney) and swine testis (ST) cells were co-cultured with macroencapsulated pig islets embedded in an alginate patch, macroencapsulated PK15 (swine kidney epithelial) cells embedded in an alginate patch and free PK15 cells. Cells and supernatant were harvested at weekly time points from the cultures for up to 60 days and screened for evidence of PERV release using qRT-PCR to detect PERV RNA and SG-PERT to detect reverse transcriptase (RT). RESULTS: No PERV virus, or evidence of PERV replication, was detected in the culture medium of HEK293 or pig cells cultured with encapsulated porcine islets. Increased PERV activity relative to the background was not detected in ST cells cultured with encapsulated PK15 cells. However, PERV was detected in 1 of the 3 experimental replicates of HEK293 cells cultured with encapsulated PK15 cells. Both HEK293 and ST cells cultured with free PK15 cells showed an increase in RT detection. CONCLUSIONS: With the exception of 1 replicate, there does not appear to be evidence of transmission of replication competent PERV from the encapsulated islet cells or the positive control PK15 cells across the alginate barrier. The detection of PERV would suggest the alginate barrier of this replicate may have become compromised, emphasizing the importance of quality control when producing encapsulated islet patches

The Role of p53 in Cell Transformation by BPV-4

Bovine papillomavirus-type 4 is associated with benign papillomas which can progress to carcinoma in cattle grazing on bracken fern. Bracken is known to contain immunosuppressants and cocarcinogens which can act as cofactors and these are known to be required for progression in vivo. One of the best characterised of these cofactors is the mutagen quercetin. BPV-4 has been shown to be capable of partially transforming primary bovine fibroblasts (PalF) cells in vitro in the presence of an activated ras and when these cells are additionally treated with quercetin they become tumourigenic in nude mice. The transforming functions of BPV-4 are encoded by the E7 and the E8 ORF's. BPV-4 is unusual in that it does not possess an E6 ORF. E6 has been shown to be one of the major transforming oncoproteins of the human papillomaviruses and is known to bind and degrade the tumour suppressor protein p53. This abrogation of the p53 protein is known to contribute to tumour progression by the high risk papillomaviruses such as HPV-16 and 18. As BPV-4 does not encode E6 functions, this would suggest that if p53 dysfunction is important in BPV-4 cell transformation, it may occur by alteration of the cellular gene. In order to look at possible p53 mutations in alimentary cancer of cattle, a 14kb genomic clone containing wild type p53 sequence was isolated from a bovine liver genomic DNA library and sequenced. This clone was used to localise bovine p53 to chromosome 19q15 which is known to be syntenic with human chromosome 17 and mouse chromosome 11 both of which harbour the p53 gene. Immunocytochemical analysis of BPV-4 derived lesions demonstrated that elevated levels of p53 protein were detectable in papillomas but not in carcinomas. BPV-4 E7 was found to be present at all stages of papillomatosis as was BPV-4 viral DNA as detected by in-situ hybridisation. Neither viral DNA nor BPV-4 E7 were detected in normal palatine tissue or in carcinomas which is coincident with the loss of viral DNA previously reported for tumours in vivo. The elevated levels of p53 in some of the papillomas were shown to be a result of the presence of mutations in p53 exon 7 as determined by SSCP-PCR and sequencing. Mutant p53 was also detected in two metastases of upper alimentary canal tumours. The addition of mutant p53 to PalF cells in vitro in the presence of BPV-4, ras and HPV-16E6 caused these cells to become tumourigenic in nude mice. In these tumorigenic PalF cell lines, mRNA expression of the BPV-4 viral oncoprotein E8 was shown to be elevated. Cotransfected mutant p53 mRNA expression was also detectable. This would suggest that p53 mutation plays a role in tumourigenic progression by BPV-4 both in vivo and in vitro. In this study we demonstrate that the lack of an E6 in BPV-4 may be compensated by the presence of a mutated p53 gene in BPV-4 induced carcinomas. These p53 mutations may arise due to cofactors present in bracken fern, and p53 mutations were detected in BPV-4 and ras transfected PalF cells that had been treated with quercetin. However, p53 mutations were detected in transfected PalF cells which had not been exposed to quercetin treatment. We also demonstrate that the additional functions provided by HPV-16 E6 (16E6) in vitro are at least in part, independent of the p53 binding and degradation function. The data suggests that an exogenous 16E6 can supplement in vitro other functions of BPV-4 which are responsible for progression in vivo. HPV-16 E6 was also demonstrated to possess a function confering anchorage independence, which mapped to the cysteines present in the zinc finger motifs of the protein. This study has demonstrated BPV-4 lacks more than one function that can be provided by an exogeneous E6 oncoprotein and that these functions may be independent of p53. In addition, p53 mutations were shown to be present in BPV-4 induced papillomas and carcinomas

Cellular xenotransplantation of animal cells into people: benefits and risk

The main benefit of xenotransplantation is its potential to overcome the worldwide organ shortage experienced in allotransplantation. Allogeneic transplantation is the only successful therapy for several life-threatening diseases, with cell, tissue or organ donation only partially meeting the demand and many patients dying while waiting for treatment. With supply falling short of demand, it is foreseen that the use of porcine material may at some stage overcome the existing gap between organ availability and clinical need. Recently, pig islet cells have been utilised in clinical trials, with safety being demonstrated. Indeed, pig-derived cells present several advantages: i) porcine cells have a stable function and differentiation pattern and are not tumorigenic; ii) pig cells have been shown to meet the physiological needs in large animal models; iii) the source of pig cells can be scaled up to meet demands in a highly standardised manner, and with respect to animal welfare regulations; iv) ‘designated-pathogen-free’ (DPF) pig lines can be produced, which could result in a higher safety profile than allotransplantation itself; v) the risk of zoonosis, which was raised years ago as the major hurdle, has been recently circumvented and is actually viewed as a controlled risk; and vi) immune risks are being circumvented via the use of genetically modified donor animals and encapsulation of porcine cells, particularly for the treatment of diabetes. Overall, the benefit appears to outweigh potential risks with respect to cellular xenotransplantation and this is discussed further in this review

Real-time PCR-based methods for detection of Hepatitis E virus in pork products: a critical review

Standard methods for detection of hepatitis A virus and norovirus in at-risk foodstuffs are available, but currently there is no standard method for detection of hepatitis E virus (HEV) in pork products or other foods that can be contaminated with the virus. Detection assays for HEV are mainly based on nucleic acid amplification, particularly the reverse transcription polymerase chain reaction (RTPCR) in real-time format. RTPCR-based methods can be sensitive and specific, but they require a suite of controls to verify that they have performed correctly. There have been several RTPCR methods developed to detect HEV in pork products, varying in details of sample preparation and RTPCR target sequences. This review critically discusses published HEV detection methods, with emphasis on those that have been successfully used in subsequent studies and surveys. RTPCR assays have been used both qualitatively and quantitatively, although in the latter case the data acquired are only reliable if appropriate assay calibration has been performed. One particular RTPCR assay appears to be ideal for incorporation in a standard method, as it has been demonstrated to be highly specific and sensitive, and an appropriate control and calibration standard is available. The review focuses on the detection of HEV in pork products and similar foodstuffs (e.g., boar). The information may be useful to inform standardisation activities

Characterization of porcine endogenous retrovirus expression in neonatal and adult pig pancreatic islets

BACKGROUND: Pig islets represent an alternative to the current modes of treatment for patients with diabetes. However, the concerns over pathogen transmission including that of PERV limit their immediate, widespread usage in humans. It has been previously demonstrated that PERV copy number and particularly expression levels can vary considerably between individuals and within different tissues of a single animal. In general, expression levels have been found to be particularly low in the pancreas compared to other porcine tissues suggesting a reduced risk associated with the use of this tissue. Data regarding this crucial aspect, however, remain limited and little is known about PERV status of islets themselves, which represent the final product to be transplanted. In addition, comparative analysis of the PERV status of neonatal piglets with adults is important as they are increasingly considered as potential islet donors for xenotransplantation. METHODS: Tissue samples from 51 neonatal piglets (age between 14 and 21 days) and 29 adult pigs were collected from Belgian landrace pigs used for pancreas procurement and islet isolation. Tissue biopsies were used to extract DNA for PERV copy number quantification by qPCR and RNA for PERV expression by qRT-PCR. RESULTS: As expected, PERV expression demonstrated great variation and was significantly lower in pancreas compared to other tissues. More importantly, PERV RNA expression was found to be specifically enriched in pancreatic islets reaching values similar to those found in other tissues such as liver and kidney. Interestingly, this expression was not coupled with the detection of reverse transcriptase in islet cultures or indeed detection of PERV virus. Lung, spleen, and lymph node consistently showed the highest levels of PERV expression. Comparison of PERV in neonatal and adult pigs showed that copy number did not vary significantly from birth to adulthood. PERV expression on the other hand was significantly lower in neonatal pig islets compared to adult islets and did not increase over the period of culture. CONCLUSION: Our study confirms the low level of PERV expression in whole pancreas in a large population of both neonatal and adult pigs (n=80). The level of PERV expression was however higher in the endocrine tissue than in the exocrine cells. There was no correlation between PERV status in donor PBMCs and islet cells, and no evidence of active replication in in vitro regardless of PERV expression in islet cells. Moreover, neonatal pig islets were found to have significantly lower PERV expression compared to adult islets. Neonatal islets have been suggested as the best choice for xenotransplantation in terms of economic and procurement considerations; the PERV status reported here would also potentially support their use

Uptake of synthetic low density lipoprotein by leukemic stem cells — a potential stem cell targeted drug delivery strategy

Chronic Myeloid Leukemia (CML) stem/progenitor cells, which over-express Bcr-Abl, respond to imatinib by a reversible block in proliferation without significant apoptosis. As a result, patients are unlikely to be cured owing to the persistence of leukemic quiescent stem cells (QSC) capable of initiating relapse. Previously, we have reported that intracellular levels of imatinib in primary primitive CML cells (CD34<sup>+</sup>38<sup>lo/−</sup>), are significantly lower than in CML progenitor cells (total CD34<sup>+</sup>) and leukemic cell lines. The aim of this study was to determine if potentially sub-therapeutic intracellular drug concentrations in persistent leukemic QSC may be overcome by targeted drug delivery using synthetic Low Density Lipoprotein (sLDL) particles. As a first step towards this goal, however, the extent of uptake of sLDL by leukemic cell lines and CML patient stem/progenitor cells was investigated. Results with non-drug loaded particles have shown an increased and preferential uptake of sLDL by Bcr-Abl positive cell lines in comparison to Bcr-Abl negative. Furthermore, CML CD34<sup>+</sup> and primitive CD34<sup>+</sup>38<sup>lo/−</sup> cells accumulated significantly higher levels of sLDL when compared with non-CML CD34<sup>+</sup> cells. Thus, drug-loading the sLDL nanoparticles could potentially enhance intracellular drug concentrations in primitive CML cells and thus aid their eradication