Influence of human fulminant hepatic failure sera on endogenous retroviral expression in pig hepatocytes

Abstract

A porcine endogenous retrovirus (PERV) has been shown to infect human embryonic kidney 293 (HEK293) cells in vitro. The PERV proviral sequence exists in the genome of all porcine cells, including hepatocytes used in a bioartificial liver (BAL). We examined the possibility of PERV infection in HEK293 cells during exposure to supernatant from cultured pig hepatocytes. Pig hepatocytes were cultured in media supplemented with serum from patients in fulminant hepatic failure (FHF) to simulate conditions of an extracorporeal BAL. Pig hepatocytes were cultured in serum-free media for 24 hours and then exposed to fresh medium containing serum from a patient with FHF (22 patients tested). Twenty-four hours later, supernatant was collected and analyzed by polymerase chain reaction (PCR), with and without reverse transcriptase. Primers targeting the pol gene of PERV were used for PCR. Products of amplification were detected by an enzyme-linked immunosorbent assay-based technique using an internal capture probe also targeting the pol gene. Levels of PERV sequences were estimated by serial dilution. All positive samples were tested for infectivity in HEK293 cells. Porcine kidney 15 cell supernatant and fresh culture media were studied as positive and negative controls, respectively. Pig hepatocytes were also studied in the absence of FHF sera and in the presence of mitogenic stimulation with phytohemagglutinin (PHA) and phorbol 12-myristate-13-acetate (PMA). PERV DNA and PERV RNA were detected in all supernatants of cultured pig hepatocytes. The level of PERV RNA in the supernatant of pig hepatocytes was not altered by exposure to human FHF serum or stimulation with PHA and PMA. In addition, PERV RNA was undetectable in the supernatant of HEK293 cells for up to 50 days after exposure to pig hepatocyte supernatant (with or without FHF sera). These findings show that production of PERV by cultured pig hepatocytes was unaffected by exposure to growth factors and cytokines present in human FHF sera.

Prospects for Extracorporeal Liver Support

“…One of their major advantages, apart from easy availability, is that they can be satisfactorily cryopreserved, thus simplifying the issues of cell storage and transport to the treatment centre. 23 24 Concerns remain however regarding immune reactions to foreign antigens, 25 as well as xenozoonosis in the form of cross species infection with porcine endogenous retrovirus (which has been demonstrated in vitro [26][27][28][29][30][31] although never in vivo [32][33][34] ).…”

Microbiological safety of a novel bio-artificial liver support system based on porcine hepatocytes: a experimental study

“…Nyberg et al . [43] reported that HEK293 cells could not be infected by the supernatant of in vitro cultured porcine hepatocytes, and that human plasma from patients with fulminant hepatic failure would not affect its infectivity. In other trials, no HEK293 cells were found to be infected in an in vitro infection experiment of another two extracorporeal BAL systems, the Academic Medical Center (AMC)-BAL and the HepatAssist system [18,28].…”

No evidence of PERV infection in healthcare workers exposed to transgenic porcine liver extracorporeal support

“…These patients have shown no evidence of PERV infection (PERV DNA or PERV seroreactivity), despite immunosuppression with tacrolimus‐based regimens (both patients) and anti‐CD3 monoclonal antibody therapy for the treatment of steroid‐resistant rejection (one patient) [21]. The negative results for PERV transmission to the study subjects would be predicted by the previous in vitro analyses for expression of infectious PERV in hepatocytes used under similar conditions [25,26].…”