Graft dysfunction in compassionate use of genetically engineered pig-to-human cardiac xenotransplantation: a case report.

BACKGROUND: A genetically engineered pig cardiac xenotransplantation was done on Jan 7, 2022, in a non-ambulatory male patient, aged 57 years, with end-stage heart failure, and on veno-arterial extracorporeal membrane oxygenation support, who was ineligible for an allograft. This report details our current understanding of factors important to the xenotransplantation outcome. METHODS: Physiological and biochemical parameters critical for the care of all heart transplant recipients were collected in extensive clinical monitoring in an intensive care unit. To ascertain the cause of xenograft dysfunction, we did extensive immunological and histopathological studies, including electron microscopy and quantification of porcine cytomegalovirus or porcine roseolovirus (PCMV/PRV) in the xenograft, recipient cells, and tissue by DNA PCR and RNA transcription. We performed intravenous immunoglobulin (IVIG) binding to donor cells and single-cell RNA sequencing of peripheral blood mononuclear cells. FINDINGS: After successful xenotransplantation, the graft functioned well on echocardiography and sustained cardiovascular and other organ systems functions until postoperative day 47 when diastolic heart failure occurred. At postoperative day 50, the endomyocardial biopsy revealed damaged capillaries with interstitial oedema, red cell extravasation, rare thrombotic microangiopathy, and complement deposition. Increased anti-pig xenoantibodies, mainly IgG, were detected after IVIG administration for hypogammaglobulinaemia and during the first plasma exchange. Endomyocardial biopsy on postoperative day 56 showed fibrotic changes consistent with progressive myocardial stiffness. Microbial cell-free DNA testing indicated increasing titres of PCMV/PRV cell-free DNA. Post-mortem single-cell RNA sequencing showed overlapping causes. INTERPRETATION: Hyperacute rejection was avoided. We identified potential mediators of the observed endothelial injury. First, widespread endothelial injury indicates antibody-mediated rejection. Second, IVIG bound strongly to donor endothelium, possibly causing immune activation. Finally, reactivation and replication of latent PCMV/PRV in the xenograft possibly initiated a damaging inflammatory response. The findings point to specific measures to improve xenotransplant outcomes in the future. FUNDING: The University of Maryland School of Medicine, and the University of Maryland Medical Center.

Immunological response in cynomolgus macaques to porcine α-1,3 galactosyltransferase knockout viable skin xenotransplants-A pre-clinical study.

BACKGROUND: Allogeneic skin recovered from human deceased donors (HDD) has been a mainstay interim treatment for severe burns, but unfortunately risk of infectious disease and availability limitations exist. Genetically engineered ɑ-1,3 galactosyltransferase knockout (GalT-KO) porcine source animals for viable skin xenotransplants may provide a promising clinical alternative. METHODS: Four cynomolgus macaque recipients received full-thickness surgical wounds to model the defects arising from excision of full-thickness burn injury and were treated with biologically active skin xenotransplants derived from GalT-KO, Designated Pathogen Free (DPF) miniature swine. Evaluations were conducted for safety, tolerability, and recipient immunological response. RESULTS: All skin xenotransplants demonstrated prolonged survival, vascularity, and persistent dermal adhesion until the study endpoint at post-operative day 30. No adverse outcomes were observed during the study. Varying levels of epidermolysis coincided with histologic detection of CD4+ and CD8+ T cells, and other cellular infiltrates in the epidermis. Recipient sera IgM and IgG demonstrated significant antibody immune response to non-α-1,3-galactose porcine xenoantigens. Separately, specific wound healing mediators were quantified. Neither porcine cell migration nor PERV were detected in circulation or any visceral organs. CONCLUSIONS: These results provide a detailed analysis of vital skin xenotransplants utilizing a non-human primate model to predict the anticipated immunological response of human patients. The lack of adverse rejection even in the presence of elevated Ig indicates this is a prospective therapeutic option. The findings reported here directly supported regulatory clearance for a first-in-man, Phase I xenotransplantation clinical trial.

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.

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.

Hepatitis E virus (HEV) in Scotland: evidence of recent increase in viral circulation in humans.

BackgroundPrevious studies showed low levels of circulating hepatitis E virus (HEV) in Scotland. We aimed to reassess current Scottish HEV epidemiology. Methods: Blood donor samples from five Scottish blood centres, the minipools for routine HEV screening and liver transplant recipients were tested for HEV antibodies and RNA to determine seroprevalence and viraemia. Blood donor data were compared with results from previous studies covering 2004-08. Notified laboratory-confirmed hepatitis E cases (2009-16) were extracted from national surveillance data. Viraemic samples from blood donors (2016) and chronic hepatitis E transplant patients (2014-16) were sequenced. Results: Anti-HEV IgG seroprevalence varied geographically and was highest in Edinburgh where it increased from 4.5% in 2004-08) to 9.3% in 2014-15 (p = 0.001). It was most marked in donors < 35 years. HEV RNA was found in 1:2,481 donors, compared with 1:14,520 in 2011. Notified laboratory-confirmed cases increased by a factor of 15 between 2011 and 2016, from 13 to 206. In 2011-13, 1 of 329 transplant recipients tested positive for acute HEV, compared with six cases of chronic infection during 2014-16. Of 10 sequenced viraemic donors eight and all six patients were infected with genotype 3 clade 1 virus, common in European pigs. Conclusions: The seroprevalence, number of viraemic donors and numbers of notified laboratory-confirmed cases of HEV in Scotland have all recently increased. The causes of this change are unknown, but need further investigation. Clinicians in Scotland, particularly those caring for immunocompromised patients, should have a low threshold for testing for HEV.

Detection of viral hepatitis E in clinical liver biopsies.

AIMS: To determine the relative utility of in-situ testing for hepatitis E virus (HEV) RNA and paraffin-section polymerase chain reaction (PCR) to diagnose HEV infection in paraffin-embedded clinical liver biopsies, and to correlate with clinicopathological characteristics. METHODS AND RESULTS: We evaluated in-situ and quantitative PCR (qPCR)-based approaches to identifying HEV in clinical liver biopsies from infected patients from multiple centres, correlating with clinical setting (immunocompetent, allograft or immunosuppressed native liver) and histological findings. Thirty-six biopsies from 29 patients had histological data, 27 and 23 of which had satisfactory material for in-situ RNA testing and tissue qPCR, respectively. Both approaches specifically identified HEV infection, but tissue qPCR was significantly more sensitive than RNAscope in-situ testing (P = 0.035). In immunocompetent but not immunosuppressed patients the tissue qPCR yield correlated with the severity of lobular hepatitis (rho = 0.94, P < 0.001). qPCR viral yield was comparably high in allografts and immunosuppressed native livers and significantly greater than with native liver infection. Immunosuppressed patients showed reduced severity of hepatitis and cholestatic changes, compared with immunocompetent patients. Indeed, HEV-infected liver allografts could show minimal hepatitis for many months. In individual cases each technique was useful when serum was not available to identify chronic infection retrospectively (in biopsies taken 4-31 months before diagnosis), to identify persistent/residual infection when contemporary serum PCR was negative and to identify cleared infection. CONCLUSIONS: qPCR is more effective than in-situ RNA testing to identify HEV infection in paraffin-embedded liver biopsies and has diagnostic utility in selected settings.

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.

Selective blockade of CD28-mediated T cell costimulation protects rhesus monkeys against acute fatal experimental autoimmune encephalomyelitis.

Costimulatory and coinhibitory receptor-ligand pairs on T cells and APC control the immune response. We have investigated whether selective blockade of CD28-CD80/86 costimulatory interactions, which preserves the coinhibitory CTLA4-CD80/86 interactions and the function of regulatory T (Treg) cells, abrogates the induction of experimental autoimmune encephalomyelitis (EAE) in rhesus monkeys. EAE was induced by intracutaneous immunization with recombinant human myelin oligodendrocyte glycoprotein (rhMOG) in CFA on day 0. FR104 is a monovalent, PEGylated-humanized Fab' Ab fragment against human CD28, cross-reactive with rhesus monkey CD28. FR104 or placebo was administered on days 0, 7, 14, and 21. FR104 levels remained high until the end of the study (day 42). Placebo-treated animals all developed clinical EAE between days 12 and 27. FR104-treated animals did not develop clinical EAE and were sacrificed at the end of the study resulting in a significantly prolonged survival. FR104 treatment diminished T and B cell responses against rhMOG, significantly reduced CNS inflammation and prevented demyelination. The inflammatory profile in the cerebrospinal fluid and brain material was also strongly reduced. Recrudescence of latent virus was investigated in blood, spleen, and brain. No differences between groups were observed for the ß-herpesvirus CMV and the polyomaviruses SV40 and SA12. Cross-sectional measurement of lymphocryptovirus, the rhesus monkey EBV, demonstrated elevated levels in the blood of FR104-treated animals. Blocking rhesus monkey CD28 with FR104 mitigated autoreactive T and B cell activation and prevented CNS pathology in the rhMOG/CFA EAE model in rhesus monkeys.

Expression of recipient cytomegalovirus in immunosuppressed and xenotransplanted Macaca fascicularis may be related to more severe gastrointestinal lesions.

BACKGROUND: Xenotransplantation is a potential answer to the current organ shortage, but the risk of infections related to overimmunosuppression is an important parameter that may predict the recipient's long-term survival. Cytomegalovirus (CMV) in xenotransplanted and immunosuppressed primates is a well-known cause of disease particularly affecting the gastrointestinal (GI) tract and a zoonotic concern. METHODS: Post-mortem sera and tissues from 45 immunosuppressed and xenografted Macaca fascicularis were evaluated for CMV using antisera specific for the immediate early 1 (IE1), anti-RhCMV, and QPCR for virus. RESULTS: Serological analysis showed 100% positivity for the presence of CMV antibodies following the application of a specific test designed for RhCMV. Five of 45 primates showed typical lesions of CMV infection in the GI tract, including neutrophilic enteritis and inclusion bodies. Molecular analysis confirmed the presence of recipient's CMV in the tissues with CMV histopathology. Porcine CMV from the donor animals was not found in any of the CMV-specific IHC-positive recipients. CONCLUSION: The presence of active CMV infection in animals intended for xenograft experiments can lead to severe gastrointestinal lesions that could impact the overall aims of the study. In such cases, the animals should be investigated using appropriate (non-human primate-specific) diagnostic tools prior to use and treated aggressively with state-of-the-art antiviral therapy.

Long-term IgG response to porcine Neu5Gc antigens without transmission of PERV in burn patients treated with porcine skin xenografts.

Acellular materials of xenogenic origin are used worldwide as xenografts, and phase I trials of viable pig pancreatic islets are currently being performed. However, limited information is available on transmission of porcine endogenous retrovirus (PERV) after xenotransplantation and on the long-term immune response of recipients to xenoantigens. We analyzed the blood of burn patients who had received living pig-skin dressings for up to 8 wk for the presence of PERV as well as for the level and nature of their long term (maximum, 34 y) immune response against pig Ags. Although no evidence of PERV genomic material or anti-PERV Ab response was found, we observed a moderate increase in anti-αGal Abs and a high and sustained anti-non-αGal IgG response in those patients. Abs against the nonhuman sialic acid Neu5Gc constituted the anti-non-αGal response with the recognition pattern on a sialoglycan array differing from that of burn patients treated without pig skin. These data suggest that anti-Neu5Gc Abs represent a barrier for long-term acceptance of porcine xenografts. Because anti-Neu5Gc Abs can promote chronic inflammation, the long-term safety of living and acellular pig tissue implants in recipients warrants further evaluation.

Hepatitis E seroprevalence in recipients of renal transplants or haemodialysis in southwest England: a case-control study.

Locally acquired HEV infection is increasingly recognized in developed countries. Anti-HEV IgG seroprevalence has been shown to be high in haemodialysis patients in a number of previous studies, employing assays of uncertain sensitivity. The aim of this study was to investigate anti-HEV IgG seroprevalence in recipients of haemodialysis and renal transplants compared to a control group using a validated, highly sensitive assay. Eighty-eight patients with functioning renal transplants and 76 receiving chronic haemodialysis were tested for HEV RNA and anti-HEV IgG and IgM. Six hundred seventy controls were tested for anti-HEV IgG. Anti-HEV IgG was positive in 28/76 (36.8%) of haemodialysis and 16/88 (18.2%) of transplant patients. HEV RNA was not found in any patient. 126/670 (18.8%) of control subjects were anti-HEV IgG positive. After adjusting for age and sex, there was a significantly higher anti-HEV IgG seroprevalence amongst haemodialysis patients compared to controls (OR = 1.97, 95% CI = 1.16-3.31, P = 0.01) or transplant recipients (OR = 2.63, 95% CI = 1.18-6.07, P = 0.02). Patients with a functioning transplant showed no difference in anti-HEV IgG seroprevalence compared to controls. The duration of haemodialysis or receipt of blood products were not significant risk factors for HEV IgG positivity. Patients receiving haemodialysis have a higher seroprevalence of anti-HEV IgG than both age- and sex-matched controls and a cohort of renal transplant patients. None of the haemodialysis patients had evidence of chronic infection. The reason haemodialysis patients have a high seroprevalence remains uncertain and merits further study.

Suggestions for the diagnosis and elimination of hepatitis E virus in pigs used for xenotransplantation.

The hepatitis E virus (HEV) is considered a zoonotic pathogen. In xenotransplantation, given the high prevalence of HEV infection in pigs, the risk of zoonotic transmission from a porcine source is considered high. Currently no clear data are available on how to diagnose and eliminate HEV in herds used for medical purposes and the importance of viral infection at the stage of harvest. In this study, several groups of animals currently used for medical purposes were found RNA positive in both serum and faeces for HEV genotype 3. In addition, viraemia was found in animals up to 3.6 yr of age, which is much longer than originally expected. Herd transmission rates appeared to be significantly lower in animals kept under minimal barrier conditions, compared with those observed for commercial animals, and as expected, segregation of animals at an early age prevented spread of infection. This study makes suggestions to ensure appropriate detection and eradication of HEV from a donor herd to be used for xenotransplantation purposes.

Co-induction of cyclooxygenase-2 [correction of cyclooxyenase-2] and early growth response gene (Egr-1) in spinal cord in a clinical model of persistent inflammation and hyperalgesia.

BACKGROUND: This study characterised the effects of persistent peripheral inflammation of the foot on pain and spinal cord expression of cyclooxygenase-1 and -2 (COX-1 and COX-2) and early growth response gene 1 (Egr-1), known markers of neuronal plasticity, in a clinical model of naturally-occurring inflammatory disease and hyperalgesia in sheep ('footrot'), before and after routine treatment (parenteral treatment with antibiotics and antiseptic footbathing). The temporal pattern of expression of COX-1, COX-2 and Egr-1 mRNA and protein were analysed using real-time PCR and Western blotting. RESULTS: Animals affected with persistent peripheral inflammation displayed significant hyperalgesia and lameness (a proxy indicator of spontaneous pain) restricted to the inflamed limb. Hyperalgesia and lameness were significantly attenuated 1 day after treatment, and resolved further by day 7 and day 3, respectively. COX-2 but not COX-1, protein expression was up-regulated in spinal cord from lame animals on day 0, before treatment. Following treatment and attenuation of pain behaviours, levels of COX-2 returned to control levels. Significant induction of Egr-1 mRNA and protein were observed in spinal cord from lame animals. Three days after treatment, levels of Egr-1 mRNA returned to control levels, however, Egr-1 protein remained elevated. CONCLUSION: Elevated levels of spinal COX-2 and Egr-1 protein correlate with the presence of pain and hyperalgesia, and may underlie persistent pain, although a direct causal link has still to be established. Understanding the temporal pattern of expression of key mediators in clinical pain states may lead to better strategies to manage pain.

Activation of metabotropic glutamate receptor 7 in spinal cord inhibits pain and hyperalgesia in a novel formalin model in sheep.

This study set out to characterize the contribution of group III metabotropic glutamate receptor 7 activation to nociceptive behaviour and mechanical hypersensitivity in a novel formalin test in sheep. The mGlu receptor 7 allosteric agonist, N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082; 2-20 mM), the nonselective group III mGlu receptor agonist L-(+)-2-amino-4-phosphonobutyric acid (0.2-20 mM) and drug vehicle were injected intrathecally into naive subjects (n=7 per group), or 5 min preformalin (3%; 0.2 ml)/saline injection (intradermal), into the lower forelimb of adult female sheep (n=5-7 per group). Forelimb withdrawal thresholds to noxious mechanical stimulation and pain behaviours (time spent nonweight bearing or flinching) were assessed for up to 180 min. Formalin induced a characteristic biphasic pain-behaviour response and mechanical hyperalgesia between 1-5 and 30-120 min postinjection. Treatment with AMN082, but not L-(+)-2-amino-4-phosphonobutyric acid significantly inhibited both early and late phase formalin-induced hyperalgesia and pain behaviours. AMN082 also induced a rapid but short lasting analgesia in naive subjects. These data suggest that enhancing endogenous metabotropic glutamate receptor 7 activity in spinal cord, using the novel allosteric modulator, AMN082, blocks pain and hyperalgesia, and may be of therapeutic benefit for the treatment of inflammatory pain.

Assessment of the potential risk of infection associated with Clostridium difficile from porcine xenografts.

There are numerous concerns over the potential for transfer of pathogens between species during clinical xenotransplantation, and although current clinical application is limited, porcine xenografts have been previously used to treat patients with severe burns. Donor animals providing the xenografts are sourced from a healthy commercial herd, however, as pigs are a known source of zoonotic agents, a number of diseases are required to be excluded from pigs used for xenotransplantation purposes. Many studies have indicated the relevance of viral zoonoses, however, little has been done with regard to the potential for transfer of pathogens related to health care associated infections. Clostridium difficile is a major cause of neonatal enteritis in pigs and an important feature of this organism is that pigs can be asymptomatic carriers. This study has examined the incidence of C. difficile PCR ribotypes present in healthy donor pigs to determine if pig faeces, and in particular, contamination of skin with faecal matter, is a potential route for the transfer of C. difficile. Animals were found to have human ribotype 017 present in the faecal matter, however, no C. difficile was isolated from skin samples taken from the same animals. In addition, due to the risk factors associated with C. difficile infection, the antimicrobial susceptibility of the C. difficile isolates has been determined.

First Report of the Presence of Hepatitis E Virus in Scottish-Harvested Shellfish Purchased at Retail Level.

Shellfish samples (n = 310) purchased from local supermarkets were analysed for the presence of hepatitis E virus (HEV) by nested RT-PCR and real-time qRT-PCR. Overall, 2.9% of samples tested positive for the presence of HEV. Phylogenetic analysis of HEV sequences revealed all as being genotype 3 HEV. This is the first report of the detection of HEV in commercially sold shellfish in Scotland. These findings may encourage further research that will help address the gaps in the knowledge in respect to foodborne transmission of HEV in Scotland and the rest of the United Kingdom.

Hepatitis E virus in patients with acute severe liver injury.

AIM: To examine the incidence of hepatitis E (HepE) in individuals with acute liver injury severe enough to warrant treatment at a transplant unit. METHODS: Hepatitis E virus (HEV) is an emerging pathogen in developed countries causing severe illness, particularly in immunocompromised patients or those with underlying chronic liver disease. HepE infection is often under diagnosed, as clinicians can be reluctant to test patients who have not travelled to regions traditionally considered hyperendemic for HepE. There are few data regarding the significance of HEV in patients with very severe acute liver injury in developed countries. Eighty patients with acute severe liver injury attending the Scottish Liver Transplant unit were tested for HEV and anti-HEV IgG and IgM. Severe acute liver injury was defined as a sudden deterioration in liver function confirmed by abnormal liver function tests and coagulopathy or presence of hepatic encephalopathy. Eighty percent of these patients were diagnosed with paracetomol overdose. No patients had a history of chronic or decompensated chronic liver disease at time of sampling. IgG positive samples were quantified against the World Health Organization anti-HEV IgG standard. Samples were screened for HEV viral RNA by quantitative reverse transcription polymerase chain reaction. RESULTS: Four cases of hepatitis E were identified. Three of the four cases were only diagnosed on retrospective testing and were initially erroneously ascribed to drug-induced liver injury and decompensated chronic liver disease, with the cause of the decompensation uncertain. One case was caused by HEV genotype 1 in a traveller returning from Asia, the other three were autochthonous and diagnosed on retrospective testing. In two of these cases (where RNA was detected) HEV was found to be genotype 3, the most prevalent genotype in developed countries. Three patients survived, two of whom had been misdiagnosed as having drug induced liver injury. The fourth patient died from sepsis and liver failure precipitated as a result of hepatitis E infection and previously undiagnosed cirrhosis. Histopathology data to date is limited to mainly that seen for endemic HepE. All patients, with the exception of patient 1, demonstrated characteristics of HepE infection, as seen in previously described locally acquired cases. CONCLUSION: In patients with acute severe liver injury, HEV testing should be part of the initial diagnostic investigation algorithm irrespective of suspected initial diagnosis, age or travel history.

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+38(lo/⁻)), are significantly lower than in CML progenitor cells (total CD34+) 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+ and primitive CD34+38(lo/⁻) cells accumulated significantly higher levels of sLDL when compared with non-CML CD34+ cells. Thus, drug-loading the sLDL nanoparticles could potentially enhance intracellular drug concentrations in primitive CML cells and thus aid their eradication.