Biomedical subjects
P Gianello
Publications and source records attributed to P Gianello.
Removal of anti-porcine natural antibodies from human and nonhuman primate plasma in vitro and in vivo by a Galalpha1-3Galbeta1-4betaGlc-X immunoaffinity column.
BACKGROUND: Natural antibodies (NAbs) against a terminal alpha1-3 galactosyl (alphaGal) epitope have been identified as the major human anti-pig NAbs. METHODS AND RESULTS: We used two synthetic alphaGal trisaccharides--type 6 (alphaGal6) and type 2(alphaGal2)--linked to an inert matrix to remove NAbs from human plasma in vitro. Flow cytometry indicated that an average of 85% of the NAb binding activity was depleted by adsorption with alphaGal6. By measuring the binding of NAbs to pig peripheral blood mononuclear cells and bone marrow cells, we demonstrated that alphaGal6 was more effective than alphaGal2 in removing NAbs, and the combination of alphaGal6 + alphaGal2 did not further increase removal of NAbs. The specificity of the removal of NAbs (IgM and IgG) reactive with the alphaGal epitope by alphaGal6 matrix was shown by enzyme-linked immunosorbent assay. In vivo studies in nonhuman primates compared plasma perfusion through a alphaGal6 immunoaffinity column with hemoperfusion through a pig liver for changes in blood pressure, hematocrit, platelets, and NAb adsorption. CONCLUSIONS: Both methods reduced the level of anti-pig IgM and IgG xenoreactive antibodies to nearly background, but column perfusion caused less hypotension and reduction in platelets than liver perfusion. Four pig kidneys transplanted into monkeys after column perfusion did not undergo hyperacute rejection, remaining functional for 2-10 days, with a mean functional period of 7 days, demonstrating that a pig kidney can support renal function in a primate.
Technical details for safer venous and biliary anastomoses for liver transplantation in the rat.
Although orthotopic liver transplantation in the rat is a well-established experimental model, no detailed illustrated description of the procedure is available in the literature. As a result, achieving success in the technique without prolonged learning time requires training in specialized centers where the step-by-step details essential to success in the technique can be learnt. The aim of this paper is to provide beginners with the culminative experience of 15 years of improvements made to Kamada's original model, clearly illustrating the necessary steps and fine detail of each anastomosis to help beginners avoid unnecessary complications. We hope that this complete description of orthotopic liver transplantation in the rat, which remains the most difficult rodent transplant model to master, will greatly help microsurgeons acquire this demanding procedure.
New technique of complete thymectomy in adult rats without tracheal intubation.
Adult rat thymectomy is frequently performed as part of the protocol for experiments in transplant immunology. Current methods used are either imprecise (suction methods) or require tracheal intubation (surgical technique) and are thus difficult to master. We have developed a safe technique of surgical removal of the thymus with resolution of any pneumothorax by suction through the xyphoid insertions of the diaphragm. This method is easy to learn and gives a 95% success rate.
Inadequate detection of accessory spleens and splenosis with laparoscopic splenectomy. A shortcoming of the laparoscopic approach in hematologic diseases.
BACKGROUND: The ultimate goal of surgery for hematological disorders is the complete removal of both the spleen and accessory spleens in order to avoid recurrence of the disease. Whereas splenectomy by open surgery provides excellent results, the validity of laparoscopic splenectomy in this regard remains unknown. OBJECTIVE: The purpose of this study was to evaluate the detection of accessory spleens during laparoscopic splenectomy for hematologic diseases. METHODS: We therefore evaluated the pre-, intra-, and postoperative detection of accessory spleens in a consecutive series of 18 patients treated by elective laparoscopic splenectomy for hematological diseases by using computed tomography (CT) and denatured red blood cell scintigraphy (DRBCS). RESULTS: Preoperative CT, DRBCS, and laparoscopic exploration detected 25%, 25%, and 75% of accessory spleens, respectively. At time of laparoscopy, 16 accessory spleens were detected in seven of the 18 patients (41%). In two patients (11%), laparoscopic exploration failed to detect accessory spleens, whereas preoperative CT (one case) and DRBCS (one case) did reveal them. Postoperatively, during a mean follow-up of 28 months (median, 24; range, 12-44 months), nine patients (50%) showed persistence of splenic tissue by DRBCS, and three of them had signs of disease recurrence. CONCLUSIONS: This prospective clinical study suggests that elective laparoscopic surgery for hematological diseases does not allow complete detection of accessory spleens. Moreover, after such a laparoscopic approach, residual splenic tissue is detectable in half of the patients during the follow-up.
Present status of laparoscopic splenectomy for hematologic diseases: certitudes and unresolved issues.
Laparoscopic splenectomy is considered to be the "gold-standard" treatment of benign hematologic diseases, with normal or slightly enlarged spleens. Laparoscopic treatment of malignant diseases and splenomegalies remains more controversial. The procedure requires a great surgeon's laparoscopic expertise, appropriate positioning of the patient and trocar insertion, and gentle and meticulous dissection of the spleen. The technique is feasible in 91% of the patients with a 0.9% operative mortality and a postoperative complications rate of 12%. When compared with open splenectomy in retrospective case-controlled studies, the laparoscopic approach includes a longer operative time and higher operative room costs. However, advantages include reduced postoperative hospital stay and faster return to normal activities. Despite scarce reported data, long-term hematologic cure rate seems to be equivalent in patients with idiopathic thrombocytopenic purpura. The accuracy of laparoscopic exploration to detect all accessory spleens is however questioned, and residual postoperative accessory splenic tissues have been observed. Prospective randomized controlled trials comparing short- and long-term results of open and laparoscopic splenectomies are required to confirm definitely the role of laparoscopy in the management of hematologic disorders.
Expression of an allogeneic MHC DRB transgene, through retroviral transduction of bone marrow, induces specific reduction of alloreactivity.
BACKGROUND: Transfer of MHC class II genes, through allogeneic bone marrow (BM) transplantation, induced long-lasting acceptance of renal allografts in miniature swine. To adapt this approach to the clinic, we have now examined whether somatic transfer of allogeneic class II DR genes, into otherwise autologous bone marrow cells (BMC), can provide the matching required for inducing immune tolerance. METHODS: Autologous BMC were transduced ex vivo with recombinant retroviruses for allogeneic DRB followed by BM transplantation. The recipients were then challenged with kidney allografts solely matched to the DRB transgene. RESULTS: Five miniature swine received autologous BMC conditioned with growth factors and transduced with recombinant retrovirus vectors containing allogeneic (n=4) or syngeneic (n=1) class II DRB genes and a drug-resistance marker. Expression of retrovirus-derived products in BM-derived cells was demonstrated by the detection of drug-resistant colony-forming progenitors and the presence of DRB retrovirus transcripts in peripheral cells. Analysis of selective mixed lymphocyte reaction responses to DR or DQ antigens indicated decreased reactivity toward the transduced DR gene product. Among all of the animals receiving fully mismatched kidney allografts, but with DRB matched to the transduced DRB, the one with the highest gene transduction rate showed stable allograft function and essentially normal renal histology for 2.5 years. A control animal, which received a syngeneic DRB gene, rejected its kidney allograft in 120 days after an earlier rejection crisis. CONCLUSIONS: These studies demonstrate that allogeneic MHC gene transfer into BM provides a new strategy for inducing tolerance across MHC barriers.
Effect of graft preservation and IgM depletion on guinea pig to rat cardiac xenograft survival.
BACKGROUND: Heterotopic guinea pig (GP) cardiac xenografts (XG) are hyperacutely rejected within minutes when transplanted into rats. METHODS: In this GP to rat cardiac XG model, we studied the effect on graft survival of a short cold preservation time (1 hr at 4 degrees C) in the presence or absence of rat anti-GP IgM preformed antibodies. The complete depletion of circulating IgM was obtained by two intraperitoneal injections of anti-rat IgM monoclonal antibody (MARM-4) on preoperative days -3 and -1. RESULTS: When the GP cardiac XG was cold preserved for 1 hr before transplantation, the mean graft survival time (MST) was 13.5+/-2.8 min, whereas without previous cold preservation, the MST was significantly prolonged to 51.5+/-12.3 min (P<0.001). Interestingly, the complete depletion of preformed circulating IgM before grafting significantly prolonged the MST of a cold-preserved XG to 37.1+/-11.3 min in comparison with a nondepleted recipient of a cold-preserved XG (P<0.02), but did not prolong the graft survival of a XG that was not cold preserved (42.5+/-14.1 min). To assess the effect of cold preservation and/or ischemia reperfusion, we intravenously injected a superoxide-dismutase mimetic (EUK-134) just before transplantation of a cold-preserved XG. This antioxidant regimen improved the MST from 13.5+/-2.8 min to 35.3+/-7.3 min (P<0.001). These results clearly suggested that either preservation lesions or preformed IgM are capable of accelerating the loss of the cardiac graft function, but also that the presence of preformed IgM seems to be especially deleterious when the cardiac XG has previously been ischemically injured. Analyzing the histological data, we also observed that the prompt cessation of cardiac function seen in cold-preserved grafts was uniformly associated with massive interstitial hemorrhage, thereby suggesting a particular susceptibility of the GP cardiac XG to cold preservation. To assess the effect of preservation on the GP cardiac function in a nonimmunological model, we performed syngeneic GP cardiac grafts and found that 1 hr of cold preservation provoked massive interstitial hemorrhage capable of promptly inducing the cessation of the heartbeat. CONCLUSIONS: Overall, this study demonstrated that both ischemic lesions and immunological processes might induce the cessation of cardiac graft function in the GP to rat model and this cessation of graft function is probably often misinterpreted as a XG rejection only.
Administration of exogenous interleukin-2 abrogates spontaneous rat liver allograft acceptance but does not affect long-term established graft survival.
BACKGROUND: Spontaneous tolerance to the orthotopic liver allograft uniformly occurs in the DA (RT1a) to PVG (RT1c) rat combination despite a fully allogeneic barrier. METHODS: To assess whether spontaneous acceptance might be the consequence of a T cell help deficit at the time of the first exposure of alloantigens to the host, we studied the effect of exogenous interleukin (IL)-2 injections at the time of liver transplantation and during long-term follow-up. RESULTS: Although spontaneous acceptance of the liver allograft constantly ensued in the DA to PVG combination, a daily injection of recombinant IL-2 (3 x 10(5) U) uniformly provoked acute cellular rejection of the liver allograft and consequently the death of animals by postoperative day 5-6. Simultaneous to the graft loss, hepatic enzymes (alanine aminotransferase) increased more than 50-fold in IL-2-treated recipients, whereas similar IL-2 treatment did not produce any hepatic dysfunction in syngeneic animals. By immunohistology, the expression of the alpha chain of the IL-2 receptor, usually undetectable in untreated animals, was evident on CD4 and CD8 lymphocytes infiltrating the liver graft. In contrast, a similar IL-2 regimen and even higher IL-2 doses (x 10(6) U) did not abrogate the liver allograft survival during long-term follow-up. CONCLUSIONS: Our results demonstrate that spontaneous rat liver allograft acceptance may be abolished by exogenous IL-2 injections, which suggests that an "inherent T cell help deficit" might be implicated in the spontaneous acceptance mechanisms of DA to PVG liver allografts.
[Is there a place for gene therapy in organ transplantation?].
The major research objectives in organ transplantation are to palliate the lack of organs, to decrease the adverse effects of chronic immunosuppression and to improve medium-term and long-term graft survival. Xenotransplantation and induction of a permanent and specific tolerance to an allograft therefore represent two main lines of research which could partly resolve the problems of organ transplantation. The objective of this article is to evaluate the possible role of gene therapy in the development of xenotransplantation and induction of allograft tolerance. They review the various gene vectors currently available as well as the routes of administration of these vectors specific to transplantation. The place of gene therapy is then evaluated in the context of allo- and xenotransplantation. In allotransplantation, transfection of certain genes of interest into the transplant organ before implantation or into the recipient's immune system is considered. Transfection into the transplant organ of genes coding for immunomodulating cytokines (TGF-beta, IL-4, IL-10, etc.), molecules which block the second signal (CTLA4-Ig) or molecules responsible for apoptosis (Fas/FasL) is discussed. The value of gene therapy in the recipient's immune system consists of transfection onto the recipient's bone marrow cells of genes coding for major histocompatibility system molecules (HLA-DR, DQ, etc.). In xenotransplantation, gene therapy will certainly play a major role in the development of transgenic pigs expressing, on the surface endothelium of their organs, certain human molecules which regulate the activity of complement (CD55, CD59, etc.) or which modify the expression of glycosylated xenoantigens (alpha-galactosyl) recognized by performed antibodies.
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.
Detailed modified technique for safer harvesting and preparation of liver graft in the rat.
Liver procurement and preparation for orthotopic liver transplantation (OLT) in the rat is the first stage for beginners aiming to acquire mastery of this model. It requires extensive and precise dissection in which each detail must provide security for the procurement and perfection for the future graft. Many technical details, "tricks and secrets," remain known only by expert teams, so that the learning phase can be long or unsuccessful if a period in such a team is not possible. We describe this procedure in great detail, including many improvements to the original technique. Complete illustration is provided to enable an easier appreciation of what remains the critical first part of the most difficult transplant model to perform in the rat.
Processing of IgM/Anti-IgM immune complexes by the liver of rats treated with anti-IgM monoclonal antibodies.
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[Surgical treatment of chronic pancreatitis with predominant cephalic involvement by double Wirsung duct diversion and restoration of permeability of the cephalic duct].
The aim of this study is to assess the long-term results of an original surgical technique for the treatment of patients suffering from painful chronic pancreatitis. From 1981 to 1994, 54 patients with chronic painful pancreatitis were operated, by means of an original duct drainage procedure, named by the authors "double drainage" because it consists of a large transduodenal sphincterotomy and a long pancreatic duct, accompanied by repermeabilization of the cephalic pancreatic duct. This procedure was used exclusively for type I pancreatitis with major lesions in the head of the gland (calcified stones, narrowing of the ducts, inflammatory process). There were 40 men and 14 females in this series. No perioperative mortality and a low morbidity (22%) were observed. Mean follow-up in 52 patients was 56 months (median: 59.5 months). The 5- year actuarial survival was 85.2% and 81% were free of pain (91% when the pancreatic duct was dilated to > 6 mm) versus 63% when the pancreatic duct was (6 mm) (p < 0.01). These excellent results should serve as a baseline for any alternative treatment of this category of painful chronic pancreatitis patients.
Molecular identification of porcine interleukin 10: regulation of expression in a kidney allograft model.
Clones encoding porcine interleukin 10 (IL-10) were isolated from a cDNA library produced from phytohemagglutinin-activated pig peripheral blood mononuclear cells. The porcine IL-10 nucleotide sequence was found to be highly homologous to the rat, mouse, and human IL-10 counterparts and to one of the open reading frames from the Epstein-Barr virus. In addition, pig IL-10 caused inhibition of gamma-interferon gene transcription as determined by a bioassay. To investigate the possible immunomodulatory role of IL-10, its expression during the induction of tolerance to kidney allografts by cyclosporin A in miniature swine was also investigated. Delayed expression and higher levels of IL-10 were observed in tolerant animals compared with animals rejecting their allografts. Since tolerance is achieved by a short course of cyclosporin A, we have also studied the in vitro effect of this drug on IL-10 gene transcription in blood mononuclear cells and have found that cyclosporin A inhibits IL-10 gene activation in T cells but does not interfere with IL-10 transcription in lipopolysaccharide-activated cells. These results suggest that the overexpression of IL-10, observed in cell populations infiltrating grafts from tolerant animals, may be a function of monocytes and/or B cells.
Humoral response of miniature swine tolerant to class I mismatched renal allografts.
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Effect of 1-28 alpha-h atrial natriuretic peptide on acute renal failure in cadaveric renal transplantation.
The efficacy and safety of (1-28) alpha-human ANP in preventing acute tubular necrosis (ATN) in cadaveric renal transplantation was tested by comparing ANP infusion with a maximal hydration (MH) regimen which we previously reported as effective in lowering the incidence of ATN (1, 2). Since the production of endogenous ANP increases with volume overloading (3), we hypothesized that increased endogenous ANP production may contribute to the beneficial effects of MH in renal transplant recipients. We thus conducted an open randomized study comparing the effect on early renal allograft function of MH (control group) versus moderate hydration plus ANP infusion (ANP group). Forty patients were blindly paired in two groups of 20 according to the duration of cold ischemia time (mean +/- 2 h). The demographic characteristics of donors and recipients were similar. Using a Swan-Ganz catheter, hemodynamic parameters were monitored for 4 h after transplantation. The group receiving ANP and moderate hydration was perfused to a mean pulmonary arterial pressure (PAP) of < or = 20 mmHg. The PAP in patients receiving MH was driven to > or = 25 mmHg. In the ANP group, a bolus of 100 micrograms of ANP was infused into the graft's renal artery at the time of unclamping, followed by 24 h of continuous intravenous infusion at 0.03 microgram/kg/min. Thereafter, the patients received ANP at a rate of 0.01 microgram/kg/min until the serum creatinine reached < 2 mg/dl. As a consequence of the hydration regimen, the PAP at unclamping was lower in the ANP group than in the control group; 20 +/- 3 and 26 +/- 4 mmHg, respectively (p < 0.05). The ANP plasma levels were significantly higher during the first 3 d in the ANP group (p < 0.001). The median recovery rate of renal function was similar in both groups. No patients in the ANP group experienced ATN while 4 patients (20%) in the control group did (p = 0.125). The need for hemodialysis was markedly reduced in the ANP group compared to the control group (1 ANP-treated patient required dialysis once whereas 5 patients from the control group underwent dialysis a total of 26 times; p = 0.068). ANP administration was well-tolerated and no hypotensive episodes were reported. This preliminary study suggests that ANP infusion is at least as effective as maximal hydration in preventing ATN and represents an efficient alternative for transplantation centers which do not use maximal hydration as a standard regimen in managing kidney allograft recipients.
Development of tolerance to class II-mismatched renal transplants after a short course of cyclosporine therapy in miniature swine.
Our laboratory has reported previously spontaneous acceptance of class II-matched, single haplotype (but not 2 haplotype), class I-mismatched renal allografts in miniature swine. All class II-mismatched animals rejected acutely regardless of class I matching. We have also demonstrated recently that a short course of high dose (10 mg/kg/day for 12 days) CsA uniformly induces donor-specific tolerance to 2-haplotype, class I-mismatched renal allografts. The survival of 2-haplotype, fully MHC mismatched renal allografts was prolonged by the same treatment, but tolerance was not induced, as all animals rejected eventually. We have now tested this short course of immunosuppressive therapy for its effect on renal allografts mismatched selectively for 2 haplotypes at class II. We have observed long-term graft survival in 5 of 7 animals under these conditions. Each of the 5 acceptor animals was demonstrated to be specifically tolerant by its response either to donor-matched skin grafts or to a second donor-matched kidney transplant without further immunosuppression. These data suggest the existence of a common pathway for induction of specific transplantation tolerance to MHC antigens when these antigens are recognized on vascular endothelium under conditions of altered cytokine production. They also suggest that tolerance induction under these conditions requires matching for either class I or class II antigens, which may have implications for the mechanism by which peripheral tolerance is induced, as well as practical implications for the extension of these results to potential clinical practice.