Porcine endogenous retrovirus does not infect human cells using a bioartificial liver model system

July 7, 2017 | Autor: Valentina Adami | Categoria: Endogenous Retroviruses, Cell line, Humans, Animals, Swine, Model System, Swine Diseases, Model System, Swine Diseases
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Porcine Endogenous Retrovirus Does Not Infect Human Cells Using a Bioartificial Liver Model System E. Falasca, V. Adami, G. Astori, A. Donini, F. Biffoni, A. Degrassi, G.A. Botta, and C. Pipan

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RGAN transplantation could save or greatly improve the quality of life of many people. However, there is a shortage of suitable donated human organs, so attention has turned to the possibility of transplanting nonhuman organs. Pigs have been suggested as potential source of cells, tissues, and organs for xenotransplants or bioartificial supports. The possibility of infection of recipients with xenogeneic agents and risk of transmission to the general population are of concern; consequently, the use of porcine tissues for xenotransplantation involves potential microbiological hazard, and the risk of porcine endogenous retrovirus (PERV) transmission to humans has been reported as a critical issue.1,2 Recently, several reports published data about this topic either showing infection of human cells in vitro or lack of transmission and infection in patients exposed to pig’s tissue.3,4 The risks can be reduced or eliminated by using specific, pathogen-free animal colonies, but this approach will not reduce the risk of porcine endogenous retrovirus infection because the genome of these viruses is in the germline of every pig. The consideration that retroviruses results in lifelong infection and that PERV from cell lines and porcine lymphocytes can infect human cells in vitro have prompted the US Food and Drug Administration to put porcine xenograft trials on hold until previously exposed patients are assessed for PERV infection and until prospective monitoring of xenograft recipients has been established.5 The bioartificial hepatic support (BHS) is an extracorporal therapy that uses porcine hepatocytes as a source of hepatic function. A reasonable first step toward estimating the potential of PERV transmission during BHS therapy is to determine the rate of PERV transfer across hollow fiber membranes.6 We therefore tested transmission of PERV to human cells across BHS hollow fiber membranes. MATERIALS AND METHODS Bioartificial Hepatic Support BHS consists of a hollow fibers cartridge, with a porosity of 600 nm, and was loaded with cryopreserved hepatocytes. We performed an in vitro analysis of recirculated human plasma in this device. Six hundred milliliters of the same plasma was recirculated for 3 hours in three different experiments at a flow rate of 200 mL/min, 0041-1345/01/$–see front matter PII S0041-1345(00)02677-4

resembling the bioartificial liver support procedure experimentally used in vivo. Plasma samples were collected and stored at ⫺80°C before recirculation (T0) and at the end of the procedure (T1).

Porcine Hepatocytes Cryopreserved porcine hepatocytes were previously isolated by enzymatic digestion from a pathogen-free pig’s liver and loaded in the extraluminal space of hollow fiber bioreactor, as previously described.7

Cell Line Cultures Infectivity assays were performed by co-culture of plasma samples with human embryonic kidney 293 cell line (HK 293), susceptible to PERV infection and with MRC-5 fetal diploid fibroblast cell line (MRC-5), not susceptible to PERV infection. As positive control, pig kidney 15 cell line (PK15) was used. Cell line cultures reached confluence and were grown in static culture at 37°C using RPMI medium supplemented with 10% fetal calf serum and 1 mmol/L sodium pyruvate.

Coculture Assay Plasma samples at T0 and T1 were co-cultured with the cell lines HK 293 and MRC-5 for 6 and 24 hours of contact, and kept in culture for 4 weeks, followed by PCR analysis of PERV DNA in cells and RT-PCR analysis of PERV RNA in cells free supernatants. Pig kidney cell line was used as PCR and RT-PCR PERV positive control.

PCR Analysis PCR analysis was a modification of the protocol described by Patience et al.1 and detection of PERV DNA and RNA was From Consorzio Fenice (E.F., V.A., G.A., A.D., A.De., C.P.); the Institute of Microbiology (E.F., G.A.B., C.P.); the Department of Experimental and Clinical Pathology and Medicine (V.A., A.De.), and the Department of Surgery (A.D.), University of Udine, Udine, Italy; and the Immuno Transfusional Center, Udine General Hospital (F.B.), and the Department of Surgery (A.D.), University of Ferrara, Ferrara, Italy. Supported in part by the Consiglio Nazionale delle Ricerche (CNR) Contributo di Ricerca n. 97.04090.CT04, from the Associazione Italiana per la ricerca sul Cancro (AIRC) and from Fondazione CA.RI.FE. Address reprint requests to Elisabetta Falasca, Institute of Microbiology, University Hospital Udine, c/o Pad Petracco, P. le Santa Maria della Misericordia, 33100 Udine, Italy. E-mail: [email protected] © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

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Transplantation Proceedings, 33, 1780–1781 (2001)

PORCINE ENDOGENOUS RETROVIRUS developed based on polymerase chain reaction (PCR) and RTPCR, respectively. DNAs and RNAs were extracted from 106 cells (MRC5, HK 293) and plasma (T0 and T1). Ten microliters of extract were used as templated for PCR and RT-PCR with PERV protease specific primers.1 As positive control, in each PCR and RT-PCR amplification included DNA or RNA, respectively, extracted from 106 PK 15 cells.

RESULTS

Plasma samples, collected at T0 and T1, were negative for both PERV DNA and RNA. HK293 and MRC-5 cells co-cultured with plasma for 6 and 24 hours were negative, for both PERV DNA and RNA, after 4 weeks of culture. Porcine hepatocytes, loaded in the extraluminal space of hollow fiber bioreactor, were positive for PERV DNA and RNA by PCR and RT-PCR. CONCLUSIONS

From our results, PERV positive porcine hepatocytes do not transfer PERV RNA to human plasma recirculating in

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bioartificial liver support system. Furthermore co-culture of plasma after circulation above described in the apparatus does not result in transmission of PERV to human sensitive cell line or production of viral RNA. Although these observations cannot exclude the possibility of viral transmission from animal tissues to humans, the use of porcine hepatocytes in a bioartificial liver device, do not promote the transmission of viral infection in human cell line. REFERENCES 1. Patience C, Takeuchi Y, Weiss RA: Nat Med 33:282, 1997 2. Heneine W, Tibell A, Switzer WM, et al: Lancet 352:695, 1998 3. Falasca E, Donini A, Baccarani U, et al: Cell Transplant 8:177, 1999 4. Martin U, Kiessig V, Blusch JH, et al: Lancet 352:692, 1998 5. Stoye J: Lancet 352:666, 1998 6. Nyberg SL, Hibbs JR, Hardin JA, et al: Transplantation 67:1251, 1999 7. Corno V, Donini A, Vianello V, et al: Transplant Proc 30:2469, 1998

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