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TRANSPLANTATION
From the Transplantation Biology Research Center,
Massachusetts General Hospital/Harvard Medical School, Boston, MA;
Biotransplant, Inc., Charlestown, MA; the Department of Pathology,
Massachusetts General Hospital, Boston, MA; and the Department of
Infectious Diseases, Massachusetts General Hospital, Boston, MA.
Posttransplantation lymphoproliferative disease (PTLD) is a major
complication of current clinical transplantation regimens. The lack of
a reproducible large-animal model of PTLD has limited progress in
understanding the pathogenesis of and in developing therapy for this
clinically important disease. This study found a high incidence of PTLD
in miniature swine undergoing allogeneic hematopoietic stem cell
transplantation and characterized this disease in swine. Two days
before allogeneic peripheral blood stem cell transplantation, miniature
swine were conditioned with thymic irradiation and in vivo T-cell
depletion. Animals received cyclosporine daily beginning 1 day before
transplantation and continuing for 30 to 60 days. Flow cytometry and
histologic examination were performed to determine the cell type
involved in lymphoproliferation. Polymerase chain reaction was
developed to detect and determine the level of porcine gammaherpesvirus
in involved lymph node tissue. PTLD in swine is morphologically and
histologically similar to that observed in human allograft recipients.
Nine of 21 animals developed a B-cell lymphoproliferation involving
peripheral blood (9 of 9), tonsils, and lymph nodes (7 of 9) from 21 to
48 days after transplantation. Six of 9 animals died of PTLD and 3 of 9 recovered after reduction of immunosuppression. A novel porcine gammaherpesvirus was identified in involved tissues. Miniature swine
provide a genetically defined large-animal model of PTLD with many
characteristics similar to human PTLD. The availability of this
reproducible large-animal model of PTLD may facilitate the development
and testing of diagnostic and therapeutic approaches for
prevention or treatment of PTLD in the clinical setting.
(Blood. 2001;97:1467-1473) Our laboratory has been successful in developing
protocols for establishing mixed chimerism and tolerance across major
histocompatibility complex (MHC) barriers in miniature swine without
the use of whole-body irradiation (WBI).1 In one of our
protocols developed for this purpose we have observed a high incidence
of posttransplantation lymphoproliferative disorder (PTLD). Because of
the importance of PTLD clinically, we have attempted to characterize
this phenomenon in the miniature swine model.
The development of lymphoid neoplasms in allograft recipients receiving
immunosuppressive therapy has been recognized as a major complication
of solid organ and bone marrow transplantation for over 30 years.2,3 PTLD and acquired immunodeficiency syndrome
(AIDS)-associated B-cell lymphoma are serious and often lethal
complications of immunosuppression. The majority of neoplasms involved
in PTLD, including those lacking surface immunoglobulin expression, are
of B-cell origin.4,5 A strong correlation has been
reported between B-cell neoplasms developing in immunosuppressed patients and the presence of the B-lymphotropic gammaherpesvirus Epstein-Barr virus (EBV).6 In humans, PTLD is thought to
represent a spectrum of EBV-driven lymphoid proliferations ranging in
histologic appearance from a reactive polymorphic expansion of
EBV-infected lymphocytes to monoclonal B-cell lymphomas.7
Studies of patients who developed PTLD have implicated several risk
factors, including T-cell depletion and the degree of
immunosuppression; however, the pathogenesis of PTLD is not completely
understood.8,9 The lack of a reproducible large-animal
model of PTLD has limited progress in understanding the pathogenesis of
and in developing therapy for this clinically important disease.
Animals
Thymic irradiation
Recipient T-cell depletion T-cell depletion was achieved using the antiswine CD3 immunotoxin, pCD3-CRM9,14 made by conjugating the diphtheria toxin-binding site mutant, CRM9, to the antiporcine CD3 monoclonal antibody (mAb), 898H2-6-15.15 On day 2, 0.05 mg/kg pCD3-CRM9 was administered intravenously to recipient animals.
Cyclosporin A treatment Cyclosporine A (CyA; Neoral; Novartis, East Hanover, NJ) was administered orally at approximately 15 to 30 mg/kg per day in divided doses from day1 to day 30 with or without tapering to day 60. Serum
CyA levels were monitored daily and dosage of CyA was adjusted to
attempt to maintain a level of 400 to 800 mg/dL over the first
30 days.
Peripheral blood stem cell collection A stem cell mobilizing regimen was administered to donors of peripheral blood stem cells (PBSCs). This regimen consisted of daily treatments with porcine stem cell factor (pSCF; 100 µg/kg) in combination with porcine interleukin 3 (pIL-3; 100 µg/kg), both from Biotransplant (Boston, MA), administered subcutaneously. Collection of PBSCs was achieved by leukapheresis (Cobe Spectra Apheresis System, Lakewood, CO) beginning on day 5 of cytokine therapy and continuing daily until sufficient numbers of cells were collected. PBSCs, either fresh or frozen and thawed, were adjusted to a concentration of 2.0 × 108/mL, and the appropriate volume was infused via catheter over 15 to 20 minutes. PBSCs were administered beginning on day 0, and on 2 to 3 successive days, for a total of 100 to 200 × 108/kg.Antibodies and flow cytometry Antibodies used for flow cytometry were as follows: CD1 76-7-4 Balb/c IgG2aK16; CD2 MSA-4; CD3 898H2-6-15 C3H/HEJ IgG2aK15; CD5 BB6-9G12 IgG117; CD16 G7; class II DR 40D; CD21 BB6-11C9; anti-IgM 5C9; anti- light-chain K139 3E1;
pig monocyte/granulocyte-specific SWC3a 74-22-15 (Balb/c,
IgG1K)16; and PAA 1038H-10-9 (B10.PD1, IgMK).12 Flow cytometry was performed using a Becton
Dickinson FACScan (San Jose, CA). Staining of whole blood and lymph
node cell suspensions was performed as previously
described.13 Data were analyzed using Winlist list mode
analysis software (Verity Software House, Topsham, ME).
Detection of cytoplasmic immunoglobulin by flow cytometry was done using Fix & Perm cell permeabilization kit (Caltag Laboratories, Burlingame, CA) according to the manufacturer's instructions. Cytoplasmic staining for immunoglobulin was done using fluorescein isothiocyanate (FITC) goat antiswine IgM (Kirkegaard and Perry, Gaithersburg, MD) and FITC K139 3E1 mouse antiswine light-chain mAb. Histology Lymph node tissues were obtained by sequential biopsies and at autopsy. Representative sections were fixed in formalin and snap frozen. Formalin-fixed tissue sections were stained with hematoxylin and eosin.Polymerase chain reaction to identify novel porcine gammaherpesviruses Sample preparation. Genomic DNA from the lymph nodes of miniature swine that were observed to have PTLD was extracted using the Qiamp Blood Kit (Qiagen, Santa Clara, CA). Then, 100 ng of the DNA pool was added to each 50-µL polymerase chain reaction (PCR) containing the following reagents: 25 mM KCl, 10 mM Tris-HCl (pH 8.3), 3.5 mM MgCl2 (Stratagene, La Jolla, CA), 0.2 mM dNTP, and 2.5 U Amplitaq Gold (PerkinElmer, Norwalk, CT) along with 20 pM of each primer. The following primer pair designed to amplify the glycoprotein B (gpB) gene of known gammaherpesviruses yielded a 627-bp product: QLIVF4 5'-CAR ITI CAR TWT GCM TAY GAC-3'; FREYNR4 5'-GTA RTA RTT RTA YTC YCT RAA-3'; (R = A + G, Y = C + T, M = A + C, W = A + T, I = Inosine). The PCRs were cycled 9 minutes at 95°C followed by 30 cycles of 94°C for 30 seconds, 45°C for 1 minute, and 72°C for 1 minute. The program concluded with a 5-minute incubation at 72°C. The PCR products were purified using Microspin G-50 columns (Amersham Pharmacia Biotech, NJ), ligated into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA), and used to transform competent TOP10F' Escherichia coli. Colonies were grown up and plasmid DNA extracted using the Wizard miniprep kit (Promega, Madison, WI). The plasmids were sent to Lark Technologies (Houston, TX) for automated sequencing.PCR to detect relative level of porcine gammaherpesvirus DNA in miniature swine lymph node Genomic DNA from peripheral blood mononuclear cells (PBMCs) was extracted using the Qiamp Blood Kit (Qiagen, Valencia, CA) or from tissue samples using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN) following the manufacturers' instructions. Sequence information from the PCR product derived from degenerate herpesvirus primers was used to design specific primers to the porcine gammaherpesvirus (pGHV) gpB gene: TEF-16 (forward primer) 5'-CAC AAG CGT CAT GAG CAT G-3'; TER-13 (reverse primer) 5'-TAA GCC TCT TCT CGT CCC TG-3'.The final PCR mixture consisted of 25 mM KCl, 10mM Tris-HCl, pH 8.3, 1.5 mM MgCl2 (Stratagene), 0.4 mM dNTP, and 2.5 U Amplitaq Gold (PerkinElmer), 15 pmol of each primer and 50 ng genomic DNA. Cycling conditions were as follows: 1 cycle of 9 minutes at 95°C followed by 25 cycles of 96°C for 10 seconds, 59°C for 30 seconds, and 72°C for 30 seconds. The program concluded with a 5-minute incubation at 72°C. Plasmid DNA containing the fragment of the pGHV gpB gene was quantitated on an agarose gel. Known amounts of the plasmid were added with 50 ng pGHV-negative pig DNA to a PCR tube containing the PCR reagents as detailed above. Thus, the standards were amplified simultaneously with the test samples.
Lymphoproliferation in miniature swine Twenty-one miniature swine were conditioned with thymic irradiation and in vivo T-cell depletion on day2, followed by a short course (either 30 days or 60 days with tapering from day 30) of
CyA starting on day 1. Eight animals were treated with the 30-day CyA
protocol and 13 were treated with the 60-day CyA protocol, although not
all animals completed their CyA course (see below). Conditioned
recipients received PBSC transplants on days 0 to 2 with a high dose
(1-2 × 1010/kg) of donor PBSC mobilized for 5 to 7 days
with pIL-3 and pSCF as shown in Figure 1.
This regimen was successful in establishing long-term mixed chimerism
and donor-specific tolerance across both minor and major
histocompatibility barriers in miniature swine, as previously
reported.1 However, as summarized in Table 1, a high percentage (43%, 9 of 21) of
animals treated with this protocol developed a lymphoproliferative
disorder, which was transient in some animals (3 of 9) and fatal in
others (6 of 9).
Transient lymphoproliferation was observed in 2 animals receiving PBSC transplants, one MHC-matched (no. 12757) and one MHC-mismatched (no. 13100), with a 30-day course of CyA. The peripheral blood white blood cell (WBC) count rapidly rose in these animals reaching a maximum on days 34 and 35 (Table 1). The rise in WBCs was coincident with a drastic decline in the degree of lymphocyte chimerism in the peripheral blood, as previously reported.1 Immediately following cessation of CyA on day 30, both animals developed a high fever (106°F) with loss of appetite and lethargy, and a slight decline in platelet count and blood hemoglobin level (data not shown). Blood cultures were consistently negative. The blood counts returned to normal, and both animals appeared clinically well with no evidence of PTLD by day 40. The animal that received MHC-matched PBSC remained healthy, with stable chimerism for over 550 days, whereas the animal that received MHC-mismatched PBSC developed signs of skin and intestinal graft-versus-host disease (GVHD) beginning on day 43 and was killed on day 73 due to prolonged, persistent diarrhea associated with chronic GVHD.1 A third animal (no. 13 318) treated with the 30-day CyA protocol died of complications due to PTLD on day 32 (Table 1). In an attempt to avoid GVHD in an additional series of animals receiving transplants across an MHC barrier, we extended CyA administration to 60 days, with tapering from day 30 to 60. Six of 13 (46%) animals treated with this extended CyA protocol developed PTLD (Table 1). Clinical signs of PTLD in miniature swine included lethargy, anorexia, rapid rise in WBC and palpable lymph nodes on physical examination. Five of these animals died of respiratory failure in 24 to 48 days (median, 35 days). Autopsy examination revealed massive enlargement of tonsils and lymph nodes throughout the body with involvement of the gastrointestinal tract and spleen. The enlarged pulmonary hilar lymph nodes and palatine tonsils resulted in airway obstruction and respiratory failure in these animals. Histologic examination of lymph node tissue showed typical polymorphous changes of PTLD with a mixture of immunoblasts, plasmacytoid cells, and plasma cells (Table 1). The sixth animal (no. 13 629) was killed because of complications of GVHD, which developed after CyA was stopped in an effort to halt the progression of PTLD (see below). Although PTLD was more apparent when we began treating animals using the 60-day CyA protocol, the prevalence of PTLD was similar among animals treated with either CyA protocol. Of 8 animals intentionally treated with the 30-day CyA protocol, 2 (no. 12 757 and no. 13 100) developed transient PTLD that resolved following CyA cessation, and one (no. 13 318) died of complications due to PTLD on day 32 (Table 1). Because 2 of the animals in the 60-day CyA protocol (no. 13 271 and no. 13 813) developed PTLD and died prior to day 30 (Table 1), the prevalence of PTLD among the 10 animals given CyA for no more than 30 days (5 of 10) was comparable to the prevalence of PTLD among the 11 animals receiving a course of CyA beyond 30 days (4 of 11). Immunophenotyping of peripheral blood and lymph node cells Flow cytometry and immunohistochemistry were performed to determine the immunophenotype of the cells proliferating in the peripheral blood and lymph nodes of animals with PTLD (Table 2). Donor cells can be distinguished from the host by flow cytometry using a mAb that recognizes a PAA present on all swine leukocytes.12 Eight of 9 cases of PTLD were of host origin. In all cases examined, the abnormal cells were class II+, CD3 , CD2dim, and
CD16 (Table 2). These cells were negative for the
available swine peripheral B-cell marker, CD21 (data not shown). Flow
cytometric analysis of immunoglobulin µ heavy-chain and light-chain staining was performed on lymph node cell suspensions from
7 of the 9 PTLD animals. Five of 7 examined were positive for surface
immunoglobulin. Cytoplasmic staining was performed on the 2 samples
that were negative for surface immunoglobulin. Both were positive for
cytoplasmic µ and one of the 2 was also positive for cytoplasmic light chain. Based on this analysis, all 7 cases examined were
determined to be of B-cell origin (Table 2). The variable pattern of
light-chain staining of involved lymph node cells in 6 of the 7 cases examined suggested that the lesions were most likely either
polyclonal or oligoclonal (data not shown). In one case (no. 13 271),
more than 95% of the lymph node consisted of PTLD cells and they were all surface and cytoplasmic light-chain negative (Table 2), suggesting a possible monoclonal outgrowth. This was a very aggressive case with rapid onset at 21 days (Table 1). Interestingly, this is the
only case so far in which we have been successful in growing out a
continuous cell line in culture.
Resolution of PTLD following CyA cessation One of the 6 animals that developed PTLD on the 60-day CyA protocol survived the disease (no. 13 629, Table 1). In an attempt to limit progression of PTLD, CyA was stopped in this animal at 48 days when clinical signs of PTLD first appeared. Clinical symptoms including fever, lethargy, anorexia, high WBC count, and palpable lymph nodes on physical examination all completely resolved in animal no. 13 629 by day 57. Figure 2A shows cell surface staining and FACS analysis of lymph node biopsy samples taken from this animal at the time of PTLD before CyA cessation (day 48) and after resolution of PTLD (day 69). At day 48, approximately 40% of the lymph node suspension consisted of CD3 donor-type
(PAA+) cells, which were almost completely absent by day
69. Figure 2B shows the histology of the same lymph node biopsy
samples. Typical polymorphous PTLD cells with a mixture of
immunoblasts, plasmacytoid cells, and plasma cells were seen throughout
the lymph node sample taken on day 48, but were not present in the day
69 sample. Immunohistochemistry revealed only CD3+ cells
remaining in the lymph node on day 69 with no distinct B-cell follicles
(data not shown).
Identification of a pGHV associated with PTLD Degenerate PCR primers were designed based on sequences of known gammaherpesviruses to amplify the highly conserved gpB envelope gene. DNA extracted from autopsy lymph node tissue of miniature swine that died of PTLD was amplified in a PCR by using the designed oligonucleotides. The resulting 627-bp PCR product was cloned and sequenced. The fragment was identified as a previously unknown gpB DNA sequence related to gammaherpesviruses. Amino acid sequence comparison revealed a high similarity (71% homology) to the gpB sequence of the wildebeest virus Alcelaphine herpesvirus (AHV) and also significant similarity to EBV and other gammaherpesviruses (Figure 3A and Table 3).
Using pGHV gpB sequence data, specific PCR primers were designed to detect pGHV in lymph node and peripheral blood samples. Figure 3B shows the results of PCR analysis of lymph node DNA from miniature swine that developed PTLD. All samples showed a significant increase in the level of pGHV (Figure 3B). Semiquantitative PCR indicated that the level of pGHV went from less than 1 copy in 103 cells prior to PTLD to approximately 10 to 100 copies per cell during PTLD (data not shown). Samples were not available for animal no. 13 100 or no. 12 757. A high copy number of pGHV could also be detected in DNA derived from PBMCs at the time of PTLD progression, as seen for animal no. 13 801. Figure 3C shows PCR results for a lymph node sample from animal no. 13 629 before and after PTLD resolution. After complete resolution of PTLD in animal no. 13 629, no pGHV sequences could be amplified in the DNA derived from lymph node samples.
We have identified a large-animal model of PTLD in miniature swine conditioned for hematopoietic stem cell transplantation. Here we describe the PTLD occurring in our partially inbred, MHC-defined herd of miniature swine and its close association with pGHV. Other animal models in which herpesvirus infection causes similar disease include human severe combined immunodeficiency models of EBV-induced PTLD,18 murine gammaherpesvirus 68 (MHV-68)-induced lymphoproliferation in mice,19 and simian herpesvirus-induced lymphomas in immunosuppressed primates.20 The availability of this reproducible miniature swine model of PTLD provides a unique opportunity to test approaches for diagnosis, treatment, and prevention of this disease. The PTLD observed in miniature swine closely resembles that observed in
humans undergoing transplantation (Table
4). The incidence of PTLD observed in our
transplant model, however, is much higher than that reported in human
allograft recipients.8,9 In human transplant recipients,
PTLD is generally associated with the intensity of immune suppression,
particularly the use of antilymphocyte preparations, with primary EBV
infection, and with coinfection by cytomegalovirus.6 The
high incidence in the miniature swine model may be related to the
absence of WBI or other myelosuppressive treatments in this protocol.
We have not observed PTLD or lymphoma in animals treated under any
other transplantation protocol used for miniature swine, including a
similar preparative regimen for PBSC transplantation that differs only
in the addition of 300 cGy WBI.13 Myelosuppressive host
conditioning including WBI results in a reduction of the host B-cell
pool, which may contribute to a reduction in the prevalence of
host-type PTLD. We observed only one case of donor-type PTLD among 21 animals treated with this regimen, and this prevalence is within the
range of what has been reported for patients having bone marrow
transplants.9 Of note, host-type PTLD is more often seen
in those receiving organ transplants (Table 4).8 This is
primarily due to the fact that the lymphoid system of organ transplant
recipients remains of host type, whereas that of bone marrow
transplants is generally converted to donor type. Because it is among
these cells that PTLD arises, it is understandable that organ
transplant recipients generally have PTLD of host type and bone marrow
transplant recipients have PTLD of donor type. In our model, where we
strive to achieve mixed chimerism rather than complete bone marrow
chimerism, we have seen PTLD of both host and donor types. Host PTLD
predominates, presumably because recipient B-cell precursors are more
prevalent at the time of origin of the disease than are the
corresponding donor elements.
Human neoplasms in immunosuppressed patients have been associated with gammaherpesviruses such as EBV (Burkitt lymphoma) and human herpesvirus 8 (Kaposi sarcoma, primary effusion lymphoma21). In this report, we identify a pGHV associated with lymphoproliferative disease in swine. The pGHV was found to be most similar to the wildebeest virus AHV with 71% homology at the amino acid level. AHV is known to cause wildebeest-associated malignant catarrhal fever,22 a fatal lymphoproliferative disease found in cattle. The pGHVs have only recently been identified23,24 and we are currently addressing whether the herein described pGHV is identical to either of the 2 other pGHVs described. The similarity of pGHV to a virus (AHV) known to infect ungulates other than its natural host may have important implications for xenotransplantation25 given that swine are currently favored as potential organ donors for xenotransplantation.26 However, pGHV transmission does not appear to occur in utero and can be prevented at birth by using clean-catch procedures (C. Patience, manuscript in preparation). Therefore, and because swine to be used for xenotransplantation will be screened extensively for all human pathogens, pGHV should pose a manageable risk. The observation of transient lymphoproliferation in 2 of the initial
animals (no. 12 757 and no. 13 100) receiving PBSC transplantation with a 30-day course of CyA was thought to be a hematopoietic graft-versus-host (GVH) or host-versus-graft (HVG) reaction that became
evident only after CyA was stopped at day 30.1 Despite a
marked increase in peripheral lymphocyte count, the absolute number of
donor-type lymphocytes in the peripheral blood in both animals remained constant suggesting that an expansion of
host-type lymphocytes had occurred. If this phenomenon were
due to an HVG reaction, an expansion of host T cells at this time would
be predicted. However, phenotypic analysis of the peripheral blood by
surface staining and FACS analysis on the day of maximal WBC in these animals revealed predominantly very large lymphocytes, the majority of
which were host-type non-T cells (PAA The elimination of WBI has greatly reduced the acute toxicity associated with preparative regimens for hematopoietic cell transplantation in miniature swine, which could, in turn, increase the potential clinical applicability of this approach.1,13 The frequent occurrence of PTLD among animals treated with this regimen, however, could have opposite implications. We are therefore currently working to identify the risk factors associated with PTLD. A similar lymphoproliferative disorder has recently been described in NIH miniature swine receiving liver allografts with a 12-day course of FK506.29 The degree of immunosuppression, viral exposure status of donor and recipient, and allogeneic transplantation could all play a role in development of lymphoproliferative disease in these animals. The availability of this genetically defined large-animal model of PTLD will allow us to study the role of the many potential contributing factors in the pathogenesis of PTLD. In this regard, we are planning to investigate the role of CyA, T-cell depletion, allogeneic stem cell transplantation, and the pretransplant pGHV and porcine cytomegalovirus exposure status of donor and recipient animals. The availability of a large-animal model of PTLD should facilitate the development of diagnostic and therapeutic approaches for prevention or treatment of PTLD in the clinical setting.
The authors would like to thank Drs Kai Sonntag and Yong-guang Yang
for critical review of the manuscript; Drs David M. Neville, Jr and
Joshua Scharff (NIH-NIMH, Bethesda, MD) for collaborating to provide
the pCD3-CRM9 used in these studies; Jason Bailey for excellent
technical assistance; and Lisa Bernardo for assistance in preparation
of the manuscript. We also wish to acknowledge the following companies
for generous gifts of their products
Submitted August 1, 2000; accepted October 31, 2000.
Supported by National Institutes of Health grant 1R01 HL63430-01 and by a grant from The Cure for Lymphoma Foundation. C.A.H. is a Cure for Lymphoma Foundation Fellow and a recipient of the MGH Claflin Distinguished Scholar Award.
C.A.H. and Y.F. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: David H. Sachs, Transplantation Biology Research Center, Massachusetts General Hospital, MGH-East, Bldg 149-9019, 13th St, Boston, MA 02129; e-mail: sachs{at}helix.mgh.harvard.edu.
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© 2001 by The American Society of Hematology.
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  N. C. Issa, R. A. Wilkinson, A. Griesemer, D. K. C. Cooper, K. Yamada, D. H. Sachs, and J. A. Fishman Absence of Replication of Porcine Endogenous Retrovirus and Porcine Lymphotropic Herpesvirus Type 1 with Prolonged Pig Cell Microchimerism after Pig-to-Baboon Xenotransplantation J. Virol., December 15, 2008; 82(24): 12441 - 12448. [Abstract] [Full Text] [PDF]
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 P. S. Cho, D. P. Lo, K. J. Wikiel, H. C. Rowland, R. C. Coburn, I. M. McMorrow, J. G. Goodrich, J. S. Arn, R. A. Billiter, S. L. Houser, et al. Establishment of transplantable porcine tumor cell lines derived from MHC- inbred miniature swine Blood, December 1, 2007; 110(12): 3996 - 4004. [Abstract] [Full Text] [PDF]
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H. Wang, J. VerHalen, M. L. Madariaga, S. Xiang, S. Wang, P. Lan, P.-A. Oldenborg, M. Sykes, and Y.-G. Yang Attenuation of phagocytosis of xenogeneic cells by manipulating CD47 Blood, January 15, 2007; 109(2): 836 - 842. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
Novartis Pharmaceuticals (Neoral)
and Abbot Laboratories (Ancef).