Lucian Jiga

Studying the immunosuppressive role of indoleamine 2,3‐dioxygenase: tryptophan metabolites suppress rat allogeneic T‐cell responses in vitro and in vivo

Pregnancy is a natural model of successful tolerance induction against allogeneic tissues. Recent studies pointed to a role of indoleamine 2,3‐dioxygenase (IDO), a tryptophan‐degrading enzyme expressed in the placenta, in mediation of T‐cell suppression. We want to apply to organ transplantation what nature has developed for suppression of fetal rejection during pregnancy. Here we analyze whether IDO‐induced tryptophan metabolites are able to suppress the allogeneic T‐cell response and allograft rejection in rats. Rat lymphocytes were stimulated with allogeneic dendritic cells in vitro in the presence of increasing amounts of tryptophan metabolites (kynurenine, 3‐hydroxykynurenine, anthranilic acid, 3‐hydroxyanthranilic acid and quinolinic acid) and T‐cell proliferation was determined. The findings showed that kynurenine, 3‐hydroxykynurenine and 3‐hydroxyanthranilic acid strongly suppress the T‐cell response, whereas anthranilic and quinolinic acid are noneffective. Vital staining of cells with subsequent fluorescence‐activated cell sorter analyses demonstrated that suppression is mediated by T‐cell death. Thereafter, the action of metabolites was analyzed in a skin allograft model (BN→LEW). Lewis recipients received daily s.c. injections of tryptophan metabolite mixture (kynurenine + 3‐hydroxyanthranilic acid), cyclosporin A (positive control), or no treatment (negative control). The metabolites induced a significant prolongation (P = 0.0018) of graft survival. We conclude that IDO‐induced tryptophan metabolites suppress the T‐cell response and prolong allograft survival in rats.

Generation of tolerogenic dendritic cells by treatment with mitomycin C: inhibition of allogeneic T-cell response is mediated by downregulation of ICAM-1, CD80, and CD86.

Background. Deliberately generated tolerogenic dendritic cells (DC) might be a useful tool for induction of donor-specific tolerance in transplantation. In this article, the authors study the effect of mitomycin C (MMC)-treated DC on rat T cells and delineate the mechanism of their conversion into tolerogenic cells. Methods. The influence of MMC treatment on the capacity of DC to activate allogeneic T cells was tested in vitro, and the expression of cell surface molecules was studied by flow cytometry. Results. MMC-treated DC lose their allostimulatory capacity, and this cannot be attributed to cell death or release of MMC. Interestingly, suppressed T cells cannot be restimulated, indicating that MMC-treated DC induce tolerance. MMC treatment selectively decreases adhesion (intercellular adhesion molecule [ICAM]-1) and co-stimulatory (CD80, CD86) molecules. Functional blocking of these molecules with specific antibodies confers to DC the same T-cell–suppressive properties as treatment with MMC. Conclusions. MMC treatment converts rat DC into tolerogenic cells. This mechanism is mediated by decrease of ICAM-1, CD80, and CD86.

Generation of suppressive blood cells for control of allograft rejection.

Our previous studies in rats showed that incubation of monocytic dendritic cells (DCs) with the chemotherapeutic drug mitomycin C (MMC) renders the cells immunosuppressive. Donor-derived MMC-DCs injected into the recipient prior to transplantation prolonged heart allograft survival. Although the generation of DCs is labour-intensive and time-consuming, peripheral blood mononuclear cells (PBMCs) can be easily harvested. In the present study, we analyse under which conditions DCs can be replaced by PBMCs and examine their mode of action. When injected into rats, MMC-incubated donor PBMCs (MICs) strongly prolonged heart allograft survival. Removal of monocytes from PBMCs completely abrogated their suppressive effect, indicating that monocytes are the active cell population. Suppression of rejection was donor-specific. The injected MICs migrated into peripheral lymphoid organs and led to an increased number of regulatory T-cells (Tregs) expressing cluster of differentiation (CD) markers CD4 and CD25 and forkhead box protein 3 (FoxP3). Tolerance could be transferred to syngeneic recipients with blood or spleen cells. Depletion of Tregs from tolerogenic cells abrogated their suppressive effect, arguing for mediation of immunosuppression by CD4⁺CD25⁺FoxP3⁺ Tregs. Donor-derived MICs also prolonged kidney allograft survival in pigs. MICs generated from donor monocytes were applied for the first time in humans in a patient suffering from therapy-resistant rejection of a haploidentical stem cell transplant. We describe, in the present paper, a simple method for in vitro generation of suppressor blood cells for potential use in clinical organ transplantation. Although the case report does not allow us to draw any conclusion about their therapeutic effectiveness, it shows that MICs can be easily generated and applied in humans.

Mitomycin C-treated antigen-presenting cells as a tool for control of allograft rejection and autoimmunity: From bench to bedside

Cells have been previously used in experimental models for tolerance induction in organ transplantation and autoimmune diseases. One problem with the therapeutic use of cells is standardization of their preparation. We discuss an immunosuppressive strategy relying on cells irreversibly transformed by a chemotherapeutic drug. Dendritic cells (DCs) of transplant donors pretreated with mitomycin C (MMC) strongly prolonged rat heart allograft survival when injected into recipients before transplantation. Likewise, MMC-DCs loaded with myelin basic protein suppressed autoreactive T cells of MS patients in vitro and prevented experimental autoimmune encephalitis in mice. Comprehensive gene microarray analysis identified genes that possibly make up the suppressive phenotype, comprising glucocorticoid leucine zipper, immunoglobulin-like transcript 3, CD80, CD83, CD86, and apoptotic genes. Based on these findings, a hypothetical model of tolerance induction by MMC-treated DCs is delineated. Finally, we describe the first clinical application of MMC-treated monocyte-enriched donor cells in an attempt to control the rejection of a haploidentical stem cell transplant in a sensitized recipient and discuss the pros and cons of using MMC-treated antigen-presenting cells for tolerance induction. Although many questions remain, MMC-treated cells are a promising clinical tool for controlling allograft rejection and deleterious immune responses in autoimmune diseases.

Immunosuppressive effect of tryptophan metabolites in composite tissue allotransplantation

Background: Hand transplantations have intensified immunological research into composite tissue allotransplantation to induce tolerance. Pregnancy is a successful, natural model of immunological tolerance. The enzyme indoleamine 2,3-deoxygenase plays an important role by catabolizing the amino acid tryptophan. The resulting metabolites have been shown to be immunosuppressive. The effect of tryptophan metabolites has not been investigated in vascularized organ transplantation before. In this study, the authors applied to composite tissue allotransplantation what nature has developed for pregnancy, and examined the immunosuppressive effect of tryptophan metabolites in a model of hind limb transplantation. Methods: Thirty-three allogeneic hind limb transplantations in the rat (Lewis → Brown-Norway) were performed in three groups. Group A (n = 12) received no immunosuppression, group B (n = 13) received tryptophan metabolites (kynurenine and 3-hydroxyanthranilic acid) locally and systemically, and group C (n = 8) served as a control group receiving FK506. The timing of rejection was assessed by clinical observation. Results: Rejection of the allogeneic hind limb occurred on average 6.58 days after transplantation in group A (no immunosuppression) and after 8.15 days in group B (tryptophan metabolites). Rejection was significantly delayed (log-rank test, p < 0.01). No rejection was seen with application of FK506 during the follow-up period of 21 days. Conclusions: For the first time, tryptophan metabolites have been applied in vascularized composite tissue allotransplantation and showed a significant immunosuppressive effect. These promising first results need further dose-effect and toxicological studies to increase the still limited immunosuppressive effect and define the clinical role these metabolites may play in the future.

Mastering the approach of internal mammary vessels: a new training model in pigs.

859e introduced under the skin. In 2001, Trepsat3 had already described blunt-tipped dissectors, which were in fact adapted scissors to whose extremities small heart-like metal pieces were coupled. Such an instrument is introduced under the skin and, as the scissors are opened, tissue disjunction occurs. We found such a wand type to be very interesting, but we observed that it really tires the surgeon’s hands because of repetitive opening movements. We developed new dissectors in which the opening movement has been inverted. The surgeon makes compression on this instrument’s handles, and its extremity determines skin avulsion. Instruments with four sizes (Fig. 1) enable lifting the whole face, including the retroauricular and the submental regions. This instrument is placed under the skin, specifically, under the areola layer, after the incision, and it is then introduced progressively as disruption occurs, leading to blunt dissection. Although it is large, this dissection is nontraumatic for the dermis vascular plexus, because it is below the subdermal vascular plane, superficial, but not too superficial to disrupt the vascular dermis plexus, and never deep, to prevent lesions in the nerves. The thickness of the flap is always the same, thus preventing cutaneous necrosis and nerve damage. Usually, all of the procedure is performed Fig. 1. Viterbos’s dissectors, 15, 17, 21, 21, and 36 cm in length.

Induction of therapeutic neoangiogenesis using In vitro–generated endothelial colony-forming cells: an autologous transplantation model in rat

BACKGROUND Accumulating evidence shows the potential of bone marrow-derived endothelial colony-forming cells (bmECFCs) as promising tools for vascular repair. However, knowledge about their in vitro expansion, characterization, and functional behavior is still controversial. We demonstrate the in vitro generation of rat bmECFCs and analyze their ability to promote tissue reperfusion in a chronic hind-limb ischemia model. METHODS Either in vitro-generated and characterized autologous bmECFCs or placebo was injected into ischemic hind limbs of Sprague-Dawley rats. Tissue perfusion was quantified by laser Doppler, in perfusion units (PU), at days 0, 15, and 30. RESULTS Rat bmECFCs acquire a typical phenotype (CD34(+)VEGFR2(+)CD133(+)CXCR4(+)CD45(-)), culture, and functional behavior (Dil-ac-LDL+) in vitro. Injection of autologous bmECFCs improves tissue perfusion in ischemic hind limbs (183.5 ± 3.29 PU(bmECFCs/day 30)versus 131 ± 3.9 PU(controls/day 30), P < 0.001). CONCLUSIONS We conclude that rat bmECFCs promote ischemic tissue reperfusion and their proangiogenic properties are a potential mechanism for this effect.

Mastering Lymphatic Microsurgery: A New Training Model in Living Tissue

Background Advanced microsurgical techniques have emerged as a promising approach for the treatment of lymphedema, but achieving international standards is limited by a scarcity of adequate training models. The purpose of this report is to describe our in vivo porcine training model for microsurgery. Study Design Five female common-breed pigs (Sus scrofa domesticus) weighing 20 to 28 kg were placed under general anesthesia, and blue patent violet dye was injected to highlight lymphatic structures and prepare the pigs for anatomical exploration and microsurgery. The number and type of patent anastomoses achieved and lymph node flaps created and any anatomical differences between porcine and human vessels were noted, in light of evaluating the use of pigs as a training model for microsurgery in living tissue. Results Multiple lymphatic-venous anastomoses were created at the site of a single incision made at the subinguinal region, running medial and parallel to the saphenous vessels. Ten multiple lymphatic-venous anastomoses were created in total, and all were demonstrated to be patent. Four lymph node flaps were prepared for lymph node transfer. The superficial lymphatic collector system in the caudal limb of the pig was identified and described with particular reference to the superficial, medial (dominant), and lateral branches along the saphenous vein and its accessory. Conclusions The authors present a safe and adaptable in vivo experimental microsurgical porcine model that provides the opportunity to practice several advanced lymphatic microsurgical techniques in the same animal. The ideal lymph node transfer training model can be developed from this anatomical detail, giving the opportunity to use it for artery-to-artery anastomoses, vein-to-vein anastomoses, and lymphatic-to-lymphatic anastomoses.

LOP01: Induction of therapeutic neoangiogenesis, using in-vitro generated endothelial colony-forming cells

498 LOP01: Induction of therapeutic neoangiogenesis, using in-vitro generated endothelial colony-forming cells: An autologous transplantation model in rat *L. Jiga, J. Jiga, B. Hoinoiu, S. Barac, A. Nistor, M. Ionac Victor Babes University of Medicine and Pharmacy, Division of Reconstructive Microsurgery, Timisoara, Romania Victor Babes University of Medicine and Pharmacy, Department for Transplantation Immunology and Cell Therapies, Timisoara, Romania