An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats.

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background. After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures. Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

In Vivo and In Vitro Evaluation of a Novel Hyaluronic Acid-Laminin Hydrogel as Luminal Filler and Carrier System for Genetically Engineered Schwann Cells in Critical Gap Length Tubular Peripheral Nerve Graft in Rats.

In the current study we investigated the suitability of a novel hyaluronic acid-laminin hydrogel (HAL) as luminal filler and carrier system for co-transplanted cells within a composite chitosan-based nerve graft (CNG) in a rat critical nerve defect model. The HAL was meant to improve the performance of our artificial nerve guides by giving additional structural and molecular support to regrowing axons. We filled hollow CNGs or two-chambered nerve guides with an inserted longitudinal chitosan film (CNG[F]s), with cell-free HAL or cell-free HA or additionally suspended either naïve Schwann cells (SCs) or fibroblast growth factor 2-overexpressing Schwann cells (FGF2-SCs) within the gels. We subjected female Lewis rats to immediate 15 mm sciatic nerve gap reconstruction and comprehensively compared axonal and functional regeneration parameters with the gold standard autologous nerve graft (ANG) repair. Motor recovery was surveyed by means of electrodiagnostic measurements at 60, 90, and 120 days post-reconstruction. Upon explantation after 120 days, lower limb target muscles were harvested for calculation of muscle-weight ratios. Semi-thin cross-sections of nerve segments distal to the grafts were evaluated histomorphometrically. After 120 days of recovery, only ANG treatment led to full motor recovery. Surprisingly, regeneration outcomes revealed no regeneration-supportive effect of HAL alone and even an impairment of peripheral nerve regeneration when combined with SCs and FGF2-SCs. Furthermore, complementary in vitro studies, conducted to elucidate the reason for this unexpected negative result, revealed that SCs and FGF2-SCs suspended within the hydrogel relatively downregulated gene expression of regeneration-supporting neurotrophic factors. In conclusion, cell-free HAL in its current formulation did not qualify for optimizing regeneration outcome through CNG[F]s. In addition, we demonstrate that our HAL, when used as a carrier system for co-transplanted SCs, changed their gene expression profile and deteriorated the pro-regenerative milieu within the nerve guides.

Recipient natural killer cells alter the course of rejection of allogeneic heart grafts in rats.

Rejection of solid organ grafts is regarded to be dependent on T cell responses. Nonetheless, numerous studies have focused on the contribution of NK cells in this process, resulting in contradictory theories. While some conclude that there is no participation of NK cells, others found an inflammatory or regulative role of NK cells. However, the experimental settings are rarely comparable with regard to challenged species, strain combinations or the nature of the graft. Thus, clear definition of NK cell contribution might be impeded by these circumstances. In this study we performed heterotopic heart transplantation (HTx) in rats, choosing one donor-recipient-combination leading to a fast and a second leading to a prolonged course of graft rejection. We intervened in the rejection process, by depletion of recipient NK cells on the one hand and by injection of activated NK cells syngeneic to the recipients on the other. The fast course of rejection could not be influenced by any of the NK cell manipulative treatments. However, the more prolonged course of rejection was highly susceptible to depletion of NK cells, resulting in significant acceleration of rejection, while injection of NK cells induced acceptance of the grafts. We suggest that, depending on the specific setting, NK cells can attenuate the first trigger of immune response, which allows establishing the regulatory activity leading to tolerance of the graft.

Pig to rat cell transplantation: reduced cellular and antibody responses to xenografts overexpressing PD-L1.

BACKGROUND: Programmed death-1 (PD-1) costimulation acts as a negative regulator of T-cell responses to allografts. However, the role of the PD-1 pathway in xenotransplantation is not well defined yet. We have shown previously that human in vitro T-cell responses to porcine transfectants overexpressing PD-Ligand1 (L23-PD-L1 cells) are remarkably weak. In this report, we asked whether the PD-1/PD-L1 pathway has the potential to diminish xenogeneic immune responses also in vivo. METHODS: L23-PD-L1 or mock transfected control cells (L23-GFP) were transplanted under the kidney capsule of rats. The occurrence of kidney-infiltrating rat leukocytes and the induction of anti-pig antibodies were monitored in grafted animals. RESULTS: Assessment of cellular infiltrates revealed similar numbers of macrophages in kidneys grafted with L23-PD-L1 or L23-GFP control cells. However, the level of MHC class-II molecules was reduced on macrophages responding to L23-PD-L1 grafts, suggesting a lower state of activation. Furthermore, less T cells were found in kidneys receiving L23-PD-L1 cells. In addition, the titers of induced anti-pig antibodies were significantly lower in rats grafted with L23-PD-L1 cells. CONCLUSIONS: These data suggest that signals triggered by PD-1-PD-L1 interaction interfere with activation pathways involved in the induction of cellular and antibody-mediated immune responses to xenografts in vivo. Targeting of PD-1 and/or PD-L1 may be a promising approach for immune modulation after xenotransplantation.

Exchange of cytosolic content between T cells and tumor cells activates CD4 T cells and impedes cancer growth.

BACKGROUND: T cells are known to participate in the response to tumor cells and react with cytotoxicity and cytokine release. At the same time tumors established versatile mechanisms for silencing the immune responses. The interplay is far from being completely understood. In this study we show contacts between tumor cells and lymphocytes revealing novel characteristics in the interaction of T cells and cancer cells in a way not previously described. METHODS/ FINDINGS: Experiments are based on the usage of a hydrophilic fluorescent dye that occurs free in the cytosol and thus transfer of fluorescent cytosol from one cell to the other can be observed using flow cytometry. Tumor cells from cell lines of different origin or primary hepatocellular carcinoma (HCC) cells were incubated with lymphocytes from human and mice. This exposure provoked a contact dependent uptake of tumor derived cytosol by lymphocytes--even in CD4⁺ T cells and murine B cells--which could not be detected after incubation of lymphocytes with healthy cells. The interaction was a direct one, not requiring the presence of accessory cells, but independent of cytotoxicity and TCR engagement. Electron microscopy disclosed 100-200 nm large gaps in the cell membranes of connected cells which separated viable and revealed astonishing outcome. While the lymphocytes were induced to proliferate in a long term fashion, the tumor cells underwent a temporary break in cell division. The in vitro results were confirmed in vivo using a murine acute lymphoblastic leukemia (ALL) model. The arrest of tumor proliferation resulted in a significant prolonged survival of challenged mice. CONCLUSIONS: The reported cell-cell contacts reveal new characteristics i.e. the enabling of cytosol flow between the cells including biological active proteins that influence the cell cycle and biological behaviour of the recipient cells. This adds a completely new aspect in tumor induced immunology.

Naïve rat NK cells control the onset of T cell response.

NK cell function in the rat is only defined in a rudimentary way due to missing tools for clear NK cell identification. The present study introduces the congenic LEW.BH-NKC rat strain which allows distinct detection of rat NK cells using commercial antibodies. LEW.BH-NKC rats were exposed in vivo to the porcine B cell line L23 by subcutaneous transfer of L23 cell suspension. We used Luciferase transgeneic L23 cells to follow the course of rejection by living imaging. L23 cells were rejected within five days after placement under the skin thus the rejection is mediated by innate immune responses in the first place. Indeed we found increased percentages of NK cells in the blood, spleen and in draining lymph nodes using flow cytometry methods. Surprisingly, we found as a consequence a decrease in proliferative T cell response in the draining lymph nodes. We identified NK cells as mediators of this regulation by in vitro performed mixed lymphocyte reactions. The remarkable feature was the naive state of NK cells exhibiting the regulative capacity. Furthermore, the regulation was not exclusively mediated by IL-10 as it has been reported before for influence of T cell response by activated NK cells but predominantly by TGF-ß. Interestingly, after initiation of the adaptive immune response, NK cells failed to take influence on the proliferation of T cells. We conclude that naive NK cells build up a threshold of activation impulse that T cells have to overcome.

Impact of Salinomycin on human cholangiocarcinoma: induction of apoptosis and impairment of tumor cell proliferation in vitro.

BACKGROUND: Cholangiocarcinoma (CC) is a primary liver cancer with increasing incidence worldwide. Despite all efforts made in past years, prognosis remains to be poor. At least in part, this might be explained by a pronounced resistance of CC cells to undergo apoptosis. Thus, new therapeutic strategies are imperatively required. In this study we investigated the effect of Salinomycin, a polyether ionophore antibiotic, on CC cells as an appropriate agent to treat CC. Salinomycin was quite recently identified to induce apoptosis in cancer stem cells and to overcome apoptosis-resistance in several leukemia-cells and other cancer cell lines of different origin. METHODS: To delineate the effects of Salinomycin on CC, we established an in vitro cell culture model using three different human CC cell lines. After treatment apoptosis as well as migration and proliferation behavior was assessed and additional cell cycle analyses were performed by flowcytometry. RESULTS: By demonstrating Annexin V and TUNEL positivity of human CC cells, we provide evidence that Salinomycin reveals the capacity to break apoptosis-resistance in CC cells. Furthermore, we are able to demonstrate that the non-apoptotic cell fraction is characterized by sustainable impaired migration and proliferation. Cell cycle analyses revealed G2-phase accumulation of human CC cells after treatment with Salinomycin. Even though apoptosis is induced in two of three cell lines of CC cells, one cell line remained unaffected in regard of apoptosis but revealed as the other CC cells decreased proliferation and migration. CONCLUSION: In this study, we are able to demonstrate that Salinomycin is an effective agent against previously resistant CC cells and might be a potential candidate for the treatment of CC in the future.

Human TNF-related apoptosis-inducing ligand-expressing dendritic cells from transgenic pigs attenuate human xenogeneic T cell responses.

BACKGROUND: Efficient and precise techniques for the genetic modification of pigs facilitate the generation of tailored donor animals for xenotransplantation. Numerous transgenic pig lines exist with the focus on inhibition of the complement system and of humoral immune responses. In addition, immune cell-based responses need to be controlled to prevent pig-to-primate xenograft rejection. Expression of human (hu) TNF-related apoptosis-inducing ligand (TRAIL) on porcine cells has the potential to ameliorate human T cell responses. METHODS: We generated transgenic pigs expressing human tumor necrosis factor (TNF)-related apoptosis-inducing ligand (huTRAIL) under the control of either the mouse H2K(b) promoter or a CMV enhancer/chicken ß-actin (CAG) promoter, the latter one (CAG-huTRAIL) on a GGTA1 knockout/huCD46 transgenic background. The biological activity of huTRAIL was demonstrated by its apoptosis-inducing effect on Jurkat lymphoma cells. To clarify whether huTRAIL affects also primary immune cells and whether its effects depend on the presence of co-stimulatory molecules, we exposed human peripheral blood mononuclear cells (PBMC) or isolated T cells to huTRAIL-expressing porcine fibroblasts or dendritic cells in vitro. RESULTS: H2Kb-huTRAIL transgenic pigs express huTRAIL mainly in the spleen and secondary lymphoid tissues. The CAG-huTRAIL construct facilitated huTRAIL expression in multiple organs, the level being at least one order of magnitude higher than in H2Kb-huTRAIL transgenic pigs. Incubation with huTRAIL-expressing H2Kb-huTRAIL transgenic porcine dendritic cells decreased human T cell proliferation significantly without any signs of apoptosis. In spite of the high transgene expression level, CAG-huTRAIL transgenic fibroblasts did not affect proliferation of human PBMC, independent of their activation state. CONCLUSIONS: These results suggest huTRAIL expression on porcine dendritic cells as a possible strategy to attenuate T cell responses against pig-to-primate xenografts.

The interplay between Leishmania promastigotes and human Natural Killer cells in vitro leads to direct lysis of Leishmania by NK cells and modulation of NK cell activity by Leishmania promastigotes.

NK cells represent one of the first lines of defence in the immune reaction after invasion of Leishmania parasites. Depletion of mouse natural killer (NK) cells dramatically enhances susceptibility of normally resistant mice. In this study we evaluated the fate of NK cells and parasites after contact formation. The hydrophilic fluorescent dye CMFDA (chloro-methylfluorescin diacetate) that allows analysis of cytotoxicity in flow cytometry and microscopy was used. Furthermore, these findings were confirmed with scanning and transmission electron microscopy. Direct contact points were found between Leishmania promastigotes and naïve human NK cells. These contacts were associated with transfer of cytosol by membrane bridges and cytotoxicity of NK cells against Leishmania. However, in contrast to other target cells which allow repeated exocytosis of lytic granules, contact with Leishmania causes immediate destruction of NK cells in a non-apoptotic way. Our results give a reasonable explanation for ex vivo observations of reduced NK cell numbers and impaired NK response in patients with acute cutaneous leishmaniasis. Animal models have clearly shown that NK cells play a key role in the induction and direction of the immune response. Thus inhibition of NK cells at the onset of infection would be advantageous for the survival of the parasite.

Invasion of Trypanosoma cruzi into host cells is impaired by N-propionylmannosamine and other N-acylmannosamines.

The etiologic agent of Chagas' disease, Trypanosoma cruzi, is widely distributed in South America, affecting millions of people with thousands of deaths every year. Adherence of the infectious trypomastigote to host cells is mediated by sialic acid. T. cruzi cannot synthesize sialic acids on their own but cleave them from the host cells and link them to glycans on the surface of the parasites using the trans-sialidase, a GPI-anchored enzyme. The infectivity of the protozoan parasites strongly depends on the activity of this enzyme. In this report, we investigated whether the transfer of sialic acids from the host to the parasites can be attenuated using novel sialic acid precursors. The cell line 86-HG-39 was infected with T. cruzi and treated with defined N-acylmannosamine analogues bearing an elongated N-acyl side-chain. By treatment of these cells the number of T. cruzi infected cell was reduced up to 60%. We also showed that the activity of the bacterial sialidase C was reduced with N-glycan substrates with elongated N-acyl side chains of the terminal sialic acids. The affinity of this sialidase decreased with the length of the N-acyl side-chain. The data presented suggest that N-acyl modified sialic acid precursors can change the transfer of sialic acids leading to modification of infection. Since the chemotherapy of this disease is inefficient and afflicted by side effects, the need of effective drugs is lasting. These findings propose a new path to prevent the dissemination of T. cruzi in the human hosts. These compounds or further modified analogues might be a basis for the search of new agents against Chagas' disease.

Systemic FasL and TRAIL neutralisation reduce leishmaniasis induced skin ulceration.

Cutaneous leishmaniasis (CL) is caused by Leishmania infection of dermal macrophages and is associated with chronic inflammation of the skin. L. aethiopica infection displays two clinical manifestations, firstly ulcerative disease, correlated to a relatively low parasite load in the skin, and secondly non-ulcerative disease in which massive parasite infiltration of the dermis occurs in the absence of ulceration of epidermis. Skin ulceration is linked to a vigorous local inflammatory response within the skin towards infected macrophages. Fas ligand (FasL) and Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expressing cells are present in dermis in ulcerative CL and both death ligands cause apoptosis of keratinocytes in the context of Leishmania infection. In the present report we show a differential expression of FasL and TRAIL in ulcerative and non-ulcerative disease caused by L. aethiopica. In vitro experiments confirmed direct FasL- and TRAIL-induced killing of human keratinocytes in the context of Leishmania-induced inflammatory microenvironment. Systemic neutralisation of FasL and TRAIL reduced ulceration in a model of murine Leishmania infection with no effect on parasitic loads or dissemination. Interestingly, FasL neutralisation reduced neutrophil infiltration into the skin during established infection, suggesting an additional proinflammatory role of FasL in addition to direct keratinocyte killing in the context of parasite-induced skin inflammation. FasL signalling resulting in recruitment of activated neutrophils into dermis may lead to destruction of the basal membrane and thus allow direct FasL mediated killing of exposed keratinocytes in vivo. Based on our results we suggest that therapeutic inhibition of FasL and TRAIL could limit skin pathology during CL.

NK cells contribute to the control of Trypanosoma cruzi infection by killing free parasites by perforin-independent mechanisms.

The protozoan parasite Trypanosoma cruzi circulates in the blood as trypomastigotes and invades a variety of cells to multiply intracellularly as amastigotes. The acute phase leads to an immune response that restricts the proliferation of the parasite. However, parasites are able to persist in different tissues, which causes the pathology of Chagas' disease. Natural killer (NK) cells play an important role in innate resistance to a variety of pathogens. In the present study we analyzed whether NK cells participated in the control of experimental T. cruzi infection. NK cells were depleted from C57BL/6 mice by antiasialo antibodies. This treatment caused an increased parasitemia during the acute phase, but tissue parasite burdens were not significantly altered according to quantitative real-time PCR. Our results demonstrated that NK cells were activated during the initial phase of a T. cruzi infection and exhibited a contact-dependent antiparasitic activity against extracellular parasites that was independent from perforin. Thus, NK cells limit the propagation of the parasite by acting on circulating T. cruzi trypomastigotes.