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author:("like, torsten")
1.  Exchange of Cytosolic Content between T Cells and Tumor Cells Activates CD4 T Cells and Impedes Cancer Growth  
PLoS ONE  2013;8(10):e78558.
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 100-200nm 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.
doi:10.1371/journal.pone.0078558
PMCID: PMC3813479  PMID: 24205259
2.  Naïve Rat NK Cells Control the Onset of T Cell Response 
PLoS ONE  2012;7(10):e47074.
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-β. 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.
doi:10.1371/journal.pone.0047074
PMCID: PMC3471963  PMID: 23077546
3.  Impact of Salinomycin on human cholangiocarcinoma: induction of apoptosis and impairment of tumor cell proliferation in vitro 
BMC Cancer  2012;12:466.
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.
doi:10.1186/1471-2407-12-466
PMCID: PMC3487825  PMID: 23057720
Salinomycin; Cholangiocarcinoma; Apoptosis; Tumor cell migration; Cell cycle
4.  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.
Author Summary
Cutaneous leishmaniases are associated with parasite-induced inflammatory lesions of the skin. The degree of clinical pathology is not associated with parasitic burden; on the contrary, ulcerative lesions are associated with low infectious load, and non-ulcerative lesions are associated with an abundant parasite infiltration. Leishmania are intracellular parasites in mammalian hosts and reside in macrophages in the deep layers of the skin, the dermis. The exact mechanism of ulceration in CL is not known and Leishmania parasites do not directly induce destruction of keratinocytes in the most superficial layer of the skin, the epidermis. In this study we investigated if ulcerated lesions were associated with higher expression of FasL- and TRAIL-induced cell-death of keratinocytes. We found a higher expression of FasL and TRAIL in human skin samples from ulcerative as compared to non-ulcerative leishmaniasis. In a mouse model of ulcerative leishmaniasis neutralisation of FasL and TRAIL reduced ulceration. We suggest that FasL and TRAIL participate in the ulcer formation during leishmaniasis both as a chemoattractant of activated neutrophils leading to tissue destruction and through direct killing of keratinocytes. Possible approaches to use this concept in therapeutical interventions with the aim to reduce immunopathology associated with leishmaniasis are discussed.
doi:10.1371/journal.pntd.0000844
PMCID: PMC2953481  PMID: 20967287
5.  NK Cells Contribute to the Control of Trypanosoma cruzi Infection by Killing Free Parasites by Perforin-Independent Mechanisms  
Infection and Immunity  2004;72(12):6817-6825.
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.
doi:10.1128/IAI.72.12.6817-6825.2004
PMCID: PMC529106  PMID: 15557602

Results 1-5 (5)