Alain Saliez

Although pig allogeneic mesenchymal stem cells are not immunogenic in vitro, intracardiac injection elicits an immune response in vivo.

Background. In vitro, mesenchymal stem cells (MSCs) have demonstrated a low immunogenic profile. In this study, we tested the immune response to allogeneic MSCs in immunocompetent swines both in vitro and in vivo. Methods. Major histocompatibility complex–controlled swine leukocyte antigen (SLA)cd and SLAdd were used as donor and recipient, respectively. In vitro, proliferative responses were tested by mixed lymphocyte reaction (MLR) or cocultures and cytokine profiling by enzyme-linked immunosorbent assay. In vivo, allogeneic MSCs were injected in cardiac infarcted area (n=3) and compared with subcutaneous injections of either MSCs (n=2) or peripheral blood mononuclear cells (PBMCs; n=2). Two additional animals received a skin graft as controls. No immunosuppression was used. Specific antidonor humoral responses were tested by flow cytometry and complement-dependent cytotoxicity assay. Results. In vitro, either unstimulated MSCs or interferon (IFN)-&ggr; stimulated MSC failed to elicit a proliferative response (stimulation index: 1.23 vs. 1.12 vs. 36.9 for controls, P<.001). Concomitantly to the absence of proliferation to MSCs, low production of IFN-&ggr; and interleukin-2 was evidenced in supernatants while the production of Th2 cytokines was comparable to controls. In vivo, all animals receiving skin grafts, subcutaneous PBMCs and intracardiac MSCs injections developed donor-specific cellular and humoral responses (immunoglobulins M and G) with antibody-complement-mediated cytotoxicity. Subcutaneous MSCs injection needed a rechallenge to similarly develop a cytotoxic humoral response. Conclusions. Intracardiac allogeneic porcine mesenchymal stem cells elicit an immune response despite their low immunogenic profile in vitro. This result suggests that in vivo characteristics of allogeneic MSCs might differ and emphasizes the importance of pursuing research both in vitro and in vivo.

Six-month survival of microencapsulated pig islets and alginate biocompatibility in primates: proof of concept.

Background. Pig islets xenotransplantation remains associated with a strong humoral and cellular xenogeneic immune responses. The aim of this study was to assess the long-term biocompatibility of alginate encapsulated pig islets after transplantation in primates Methods. Adult pig islets encapsulated in alginate under optimal conditions (n=7) or not (n=5) were transplanted under the kidney capsule of nondiabetic Cynomolgus maccacus. Additional primates received empty capsules (n=1) and nonencapsulated pig islets (n=2) as controls. Capsule integrity, cellular overgrowth, pig islet survival, porcine C-peptide and anti-pig IgM/IgG antibodies were examined up to 6 months after implantation Results. Nonencapsulated islets and islets encapsulated in nonoptimal capsules were rapidly destroyed. In seven primates receiving perfectly encapsulated pig islets, part of the islets survived up to 6 months after implantation without immunosuppression. Porcine C-peptide was detected after 1 month in 71% of the animals. The majority of grafts (86%) were intact and completely free of cellular overgrowth or capsule fibrosis. Explanted capsules, after 135 (n=2/2) and 180 (n=2/3) days, demonstrated residual insulin content and responses to glucose challenge (stimulation index of 2.2). Partial islet survival was obtained despite an elicited anti-pig IgG humoral response Conclusions. Optimal alginate encapsulation significantly prolonged adult pig islet survival into primates for up to 6 months, even in the presence of antibody response.

Streptozotocin-induced diabetes in large animals (pigs/primates): role of GLUT2 transporter and beta-cell plasticity.

Background. To induce irreversible diabetes in large animals, the efficiency of streptozotocin (STZ) was evaluated in pigs, primates and compared to the gold standard model in rats. Methods. Low (50 mg/kg) and high (150 mg/kg) doses of STZ were tested. Hepatic/renal function, glucose metabolism (intravenous glucose tolerance tests, fasting blood glucose) and histomorphometry were evaluated prior to, 1, and 4 weeks after STZ treatment. Results. In rats and primates, expressing a high level of GLUT2 expression on &bgr; cells, a dose of 50 mg/kg STZ induced irreversible diabetes (due to the 97% destruction of beta cell mass) without provoking liver or renal failure. In pigs, despite the use of high STZ dose, partial correction of hyperglycaemia was observed four weeks after STZ injection (decreased fasting blood glucose and intravenous glucose tolerance tests; increased insulin production). The correction of hyperglycaemia was associated with significant hypertrophy of immature pig &bgr;-cell clusters (+30%, P<0.05), whereas no hypertrophy was observed in rats/primates. Conclusion. These results demonstrated that STZ might be used to induce irreversible diabetes in rats and primates. In contrast, the low STZ sensitivity in pigs related to a low expression of GLUT2, higher number of immature &bgr; cells and compensatory &bgr;-cell hypertrophy, renders STZ-induced diabetes inappropriate for studying islet allografts in swine.

Steatosis is not sufficient to cause an impaired regenerative response after partial hepatectomy in rats.

BACKGROUND/AIMSnFatty liver is known to be associated with increased mortality and morbidity after liver resection. The ability of fatty liver to regenerate after two-thirds partial hepatectomy was studied in three different models of steatosis in rats: obese Zucker rats, orotic acid-fed Wistar rats and Wistar rats fed a methionine-low, choline-deficient diet.nnnMETHODSnLiver regeneration was assessed 24 h after partial hepatectomy by bromodeoxyuridine incorporation (immunohistochemistry), proliferating cell nuclear antigen, cyclin E and cyclin-dependent kinase 2 protein expression (Western blot analysis) and cyclin-dependent kinase 2 activity (kinase assays using histone H1 as a substrate).nnnRESULTSnNo significant difference of proliferative response was found between orotic acid or methionine-low, choline-deficient diet-fed and control Wistar rats 24 h after partial hepatectomy. In contrast, hepatocyte proliferation in obese Zucker rats after partial hepatectomy was significantly reduced when compared with their lean controls.nnnCONCLUSIONSnSteatosis per se does not impair liver regeneration. The reduced liver regeneration observed in obese Zucker rats may not be due to fatty infiltration itself but to other factors such as leptin receptor dysfunction.

Parameters favouring successful adult pig islet isolations for xenotransplantation in pig-to-primate models

Dufrane D, Dhoore W, Goebbels R‐M, Saliez A, Guiot Y, Gianello P. Parameters favouring successful adult pig islet isolations for xenotransplantation in pig‐to‐primate models.u2028Xenotransplantation 2006; 13: 204–214.

EUK-134, a synthetic superoxide dismutase and catalase mimetic, protects rat kidneys from ischemia-reperfusion-induced damage.

The effect of a new synthetic superoxide dismutase and catalase mimetic was investigated on renal ischemia-reperfusion syndrome in rats. Synthetic salen-manganese complexes have characteristics that might facilitate their potential usefulness as therapeutic agents: (1) unlike proteinaceous antioxidant enzymes, synthetic complexes, due to their low molecular weight, have a better stability and bioavailability; (2) they have a catalytic activity enhancing their efficiency over noncatalytic reactive oxygen metabolite scavengers; and finally, (3) exhibiting combined superoxide dismutase and catalase activity, they destroy both superoxide anions and hydrogen peroxides, thereby enhancing their protective effect on ischemically injured tissues. One such compound, EUK-134, was tested in uninephrectomized rats that underwent a left renal artery clamping. After a 75-min left renal artery clamping, a single intravenous injection of EUK-134 at 0.2 mg/kg, just before unclamping, provided significantly better renal function recovery during the week after the ischemic insult compared with recovery of untreated animals. Two hours after several periods of renal ischemia (30, 45, 60, and 75 min of left renal artery clamping), EUK-134 given at a similar dose significantly improved the glomerular filtration rate after an acute ischemia of 30 and 45 min, as assessed by EDTA 51Cr. Overall, these results show that synthetic superoxide dismutase-catalase mimetics such as EUK-134 can protect ischemically injured rat kidneys from ischemia-reperfusion syndrome when administered just before reperfusion.

The compensatory hyperplasia (liver regeneration) following ligation of a portal branch is initiated before the atrophy of the deprived lobes

BACKGROUND/AIMSnIn rats, partial ligation of portal branches produces atrophy of the deprived lobes and hypertrophy of the intact lobes. The hepatocyte proliferation observed in the nondeprived lobes is viewed as a compensatory hyperplasia, implying that the atrophy somewhat precedes the initiation of the proliferative response. As this has not been demonstrated, the time course and magnitude of those two sequences of events were investigated and compared with the well-defined response to a partial hepatectomy.nnnMETHODSnThe portal branch feeding the anterior liver lobes was ligated in male Wistar rats. One-third and two-thirds partial hepatectomies were also performed. Liver weight, the aminopyrine demethylation rate, an index of the liver mass, the DNA content and various indices of cell proliferation were measured.nnnRESULTSnResection of the anterior lobes (PH) or ligation of their portal blood supply (PBL) induced a marked DNA synthesis in the posterior lobes (3H-thymidine incorporation) reaching its maximum 24 h after both interventions. This response can even be accelerated by performing a sham operation 6 h before the PBL. The process leading to DNA synthesis thus seems to start as early after PBL as after a PH, although the weight of the liver or the aminopyrine demethylation rate was nearly unchanged 2 h following PBL. The initiation of the proliferative response clearly precedes and is thus independent of the reduction of the liver mass. On the other hand, the progressive reduction of the liver mass seems to determine the magnitude of the proliferative response, which is, for instance, greatly increased following the excision of the deprived lobes, as late as 10 h after ligation of their portal branches. In comparison with the results obtained after a 113 PH, the peak of DNA synthesis at the 24th hour is greater than predicted by the liver weight loss, but this parameter could underestimate the reduction of the functional liver mass.nnnCONCLUSIONnThe proliferative response following a PBL can be divided into an early phase occurring independently of the reduction of the liver mass and a late phase controlled by this reduction. The paradox of the proliferative response which seems to start before the atrophy to be compensated is resolved by this hypothesis.

Control of rate and extent of the proliferative response after partial hepatectomy.

To examine the role of the early changes occurring in the liver within the first hours after a partial hepatectomy and in an attempt to demonstrate the involvement of subsequent regulatory mechanisms, the size of the remnant liver was modified at various times and by different surgical techniques. Male Wistar rats were submitted to a two-thirds temporary partial hepatectomy produced by a 3-h occlusion of the pedicle of the anterior lobes protected by local hypothermia. Various indexes of cell proliferation ([3H]thymidine uptake and 5-bromo-2-deoxyuridine and proliferating cell nuclear antigen labeling) were not increased despite a c- myc expression as high as that observed after a two-thirds partial hepatectomy. The temporary partial hepatectomy and a sham operation induced modifications of the hepatocytes, allowing rapid DNA synthesis after a subsequent two-thirds partial hepatectomy. After this initial nonspecific response, the extent of the regenerative response is determined according to the size of the liver mass present approximately from the 10th to the 18th hour after the initial stimulus. For instance, when a one-third partial hepatectomy was converted into a two-thirds partial hepatectomy at the 10th hour, the DNA synthesis at the 24th hour reached the value observed after a straightforward two-thirds partial hepatectomy. Inversely, the regenerative response was significantly reduced when additional liver lobes were connected to neck vessels between the 14th and the 18th hour after a two-thirds partial hepatectomy. In conclusion, the actual liver mass present during the period corresponding to mid- to late G1 appears to control the magnitude of the proliferative response, which is not the simple consequence of the early changes following a partial hepatectomy.

After portal branch ligation in the rat, cellular proliferation is associated with selective induction of c-Ha-ras, p53, cyclin E, and Cdk2.

BACKGROUND In liver regeneration after portal branch ligation we previously showed that early cellular changes are observed in both the proliferating and atrophying liver lobes. They are therefore not indicative of future proliferative response. In this study we attempted to define precisely, in the same model, the time at which the cellular processes diverge between the lobes by measuring various parameters associated with cellular proliferation. We also investigated the possible role of inhibitors of cell proliferation in the absence of progression towards the S phase in the atrophying lobes. AIMS Expression of p53, c-Ha-ras, cyclin E, cyclin dependent kinase (Cdk2), transforming growth factor (TGF)-β, and interleukin (IL)-1α and IL-1β were assessed in relation to their potential role in proliferating and atrophying cellular phenomenons. METHODS Immunohistochemistry, northern blotting, western blotting, and reverse transcription-polymerase chain reaction were performed, mainly at time points corresponding to mid-G1/S phase progression (8–24 hours after surgery). RESULTS The common and thus most likely non-specific response was still evident 5–8 hours after surgery and included an increase in IL-1 mRNA as well as p53 and cyclin E proteins. From 12 hours onwards, p53, c-Ha-ras, cyclin E, and Cdk2 were selectively induced in proliferating lobes whereas IL-1β was predominantly activated in atrophying lobes. No changes in TGF-β or IL-1α expression were observed at the same time points in any of the liver lobes. CONCLUSIONS The initial response to portal branch ligation and thus probably to partial hepatectomy seems to be non-specific for at least eight hours. Thereafter, p53, c-Ha-ras, cyclin E, and Cdk2 seem to drive cellular proliferation while IL-1β is associated with cellular atrophy. In contrast, TGF-β and IL-1α do not seem to play a role in determining the commitment of cells towards atrophy or proliferation.

Inhibition of Humoral Response to Allogeneic Porcine Mesenchymal Stem Cell With 12 Days of Tacrolimus

Background. In vivo studies have highlighted allogeneic mesenchymal stem-cell (MSC) immunogenicity. We investigated in vitro MSC-immunosuppressive drugs interaction and further tested in vivo the humoral response to intracardiac allogeneic MSC transplantation in a mini-swine model receiving a short course of immunosuppression. Methods. For in vitro experiments, long-term culture MSCs were used. Immunosuppressive drugs tested were mycophenolate mofetil, cyclosporin, tacrolimus (TAC), sirolimus (SIR), and everolimus. Cell proliferation/viability was assessed on day 7. For each drug, the C50 was determined, and the agonistic effect between immunosuppressive drugs and MSCs on alloreactivity was measured in proliferation assay of MSC-peripheral blood mononuclear cell cultures. For in vivo experiments, one-haplotype swine leukocyte antigen class I and II mismatch (n=11) were used. Allogeneic MSCs were transplanted into ischemic myocardium. TAC was administered 12 days. Donor-specific antibody response was assessed by flow cytometry and complement-mediated cytotoxicity assay. Results. All drugs except TAC significantly decreased cell proliferation (from 17% to 62%). In MSC-peripheral blood mononuclear cell co-culture assay, MSCs’ immunomodulatory properties were maintained when TAC or SIR were used. In vivo experiments showed that only 2 of 11 animals under TAC developed donor-specific antibodies. Importantly, sera from those two animals did not elicit a complement-mediated cytotoxic response. Conclusions. Immunosuppressive drugs significantly affect proliferation and viability of MSCs, but neither TAC nor SIR had a detrimental impact on MSCs’ immunomodulatory properties. In this large-animal model, addition of short course of immunosuppression seems to overcome the immune response to intracardiac allogeneic MSCs, which was recently demonstrated to occur in the absence of immunosuppression.