ABSTRACT Lycopene most likely contributes to the positive health effects of tomatoes on the cardiovascular system. However, elucidation of underlying cellular mechanisms is hampered by the intricate solubility of lycopene in aqueous solutions. Cells relevant to the cardiovascular system, including bovine aortic endothelial cells (BAECs), the monocytic cell line THP‐1, and RAT‐1 fibroblasts, were treated for various time periods (0–72 h) with different concentrations of lycopene (1, 5, and 10 µM), solubilized either in tetrahydrofuran (THF) or micelles as solvents. Incubation of all three cell types led to a concentration‐ and time‐dependent increase in cellular lycopene content. Both vehicles tested, THF and micelles, proved equally effective in the delivery of lycopene to cells. A marked difference in the amount of lycopene incorporated was observed among the various cell types. Compared with THP‐1 cells, the uptake of lycopene using both solvents was higher in BAECs and RAT‐1 fibroblasts for all concentrations and time points tested. PRACTICAL APPLICATIONS Epidemiological data indicate a beneficial effect for consumption of tomato products in the prevention of cardiovascular diseases. Only limited data are available on the cellular uptake of lycopene in vascular cells. Lycopene was successfully delivered to different cells relevant for the cardiovascular system. These results represent an important prerequisite for the study of molecular and cellular mechanisms by which lycopene may exert its beneficial effects on the cardiovascular system.
Purpose: In the context of kidney transplantation (KTx), little is known about the connection between donor-specific antibodies (DSA), Natural Killer (NK) cells and chemokines. The influence of immunosuppressive drugs (IS) on NK or B cell repertoires and their recruiting chemokines remains elusive. Therefore, we determined DSA in serum of patients after kidney transplantation (KTx) and correlated their presence with peripheral NK and B cell repertoires, cytokines and chemokines in order to define a novel immunological interface. Methods: PBMC of patients (3 months to 12 years after KTx) were analyzed for absolute cell numbers (n=122), NK and B cell subsets (n=21) by FACS. In serum, anti-HLA-Abs (DSA, n=122) were determined by multiplex assays. Cytokin, chemokines were determined by BioPlex technology (n=122). Results: According to the presence of HLA-specific mAb in serum, patients were separated into three groups: DSA+ (n=13) specific for allo-HLA of the grafted kidney, HLA+ (n=31) specific for HLA molecules not present in the graft and none (n=69). While the number of CD19+B cells/μl blood did not differ between the groups, significantly reduced CD56+ NK cells (p=0.009) were observed in DSA+ patients. Regarding NK cell recruiting chemokines, no significant differences were seen between the groups for plasma levels of CXCL1,9-12 or CCL5. Higher levels of the B cell chemokine CXCL13 (p=0.012) were detected in HLA+ patients versus DSA+ or negative patients. DSA+ patients had higher serum creatinine levels and, thus were classified as antibody-mediated rejection. Conclusions: The presence of serum DSA specific for the graft seems to be associated with alterations in the NK cell repertoire which raises the question whether FcgRIII+ NK cells may be recruited into the kidney. Even the presence of not donor-specific HLA antibodies correlated with higher levels of the B cell chemokine CXCL13 indicating that mAb production may be supported by the microenvironment. The association between DSA, chemokines and NK cells in KTx patients suggests an important pathophysiological mechanism that may be useful for future immune monitoring of kidney Tx patients.
Introduction: Following kidney transplantation, the involvement of NK cells in rejection episodes or immunological tolerance is discussed controversially. The peripheral NK cell repertoire of KTx patients at 3 or 6 months post Tx was investigated in parallel to the protocol biopsy for histopathological evaluation of the graft. The alterations were correlated to the biopsy status (“unsuspicious”, T cell-mediated rejection TCMR or borderline changes) and immunosuppression, respectively. Methods: Peripheral blood lymphocytes of KTx patients (N=100) at the time of the protocol biopsy at 3 or 6 month after Tx were isolated after informed consent of the patients. In addition to the “truecount” quantification of T, B, NK cells (cell/μl), the composition of the NK cell repertoire was determined by flow cytometry for the expression of several NK receptors including KIR and CD94/NKG2. In addition to NK cells, receptor expression was also addressed in T cell subsets. In some cases, protocol biopsies and peripheral blood was available at multiple time points after Tx and, thus, changes over time and immunosuppression (CsA vs. Tac vs. mTOR inhibitors) could be defined. Results: The numbers of NK cells/μl blood differed between patients after KTx with “unsuspicious” grafts and TCMR or borderline histopathology although not at a significant level. However, the distribution of CD56bright vs. CD56dim NK cells in these patient cohorts displayed significant differences compared to healthy donors. In addition, the NK subset analyses revealed substantial differences in the numbers of KIR+ NK cells between the patient cohorts. Conclusions: In our first analyses with patients between 3 and 12 months after KTx, we could demonstrate that the numbers of NK cells in peripheral blood differs between patients with kidney grafts unsuspicious of rejection and biopsy-proven TCMR or borderline changes. In addition, a more detailed NK cell subset analysis revealed significant differences in NK subset distribution between the patient cohorts and also, for some subsets, the immunosuppressive treatment groups mTOR inhibitors, in particular. Therefore, we assume that the composition of peripheral NK cells represents an important hallmark of the immune status, rejection vs. tolerance, in kidney transplantation. (This work was funded by the IFB-Tx, DFG TRR77, A3 snd SFB738 B8 projects and the HGF Alliance Immmunotherapy).
The role of endothelial cells in the pathophysiology of antibody-mediated rejection after renal transplantation has been widely investigated. We expand this scenario to the impact of epithelial cells on the microenvironment during rejection. Primary proximal tubular epithelial cells were stimulated via HLA class I, CD155 and CD166 based on their potential signal-transducing capacity to mediate back signaling after encounter with either T/NK cells or donor-specific antibodies. Upon crosslinking of these ligands with mAbs, PTEC secreted IL-6, CXCL1,8,10, CCL2, and sICAM-1. These proteins were also released by PTEC as consequence of a direct interaction with T/NK cells. Downmodulation of the receptor CD226 on effector cells confirmed the involvement of this receptor/ligand pair in back signaling. In vivo, CD155 and CD166 expression was detectable in proximal and distal tubuli of renal transplant biopsies, respectively. The composition of the protein microenvironment in these biopsies showed a substantial overlap with the PTEC response. Cluster and principal component analyses of the microenvironment separated unsuspicious from rejection biopsies and, furthermore, ABMR, TCMR, and borderline rejection. In conclusion, our results provide evidence that epithelial cells may contribute to the rejection process and pave the way to a better understanding of the pathomechanisms of kidney allograft rejection. The role of endothelial cells in the pathophysiology of antibody-mediated rejection after renal transplantation has been widely investigated. We expand this scenario to the impact of epithelial cells on the microenvironment during rejection. Primary proximal tubular epithelial cells were stimulated via HLA class I, CD155 and CD166 based on their potential signal-transducing capacity to mediate back signaling after encounter with either T/NK cells or donor-specific antibodies. Upon crosslinking of these ligands with mAbs, PTEC secreted IL-6, CXCL1,8,10, CCL2, and sICAM-1. These proteins were also released by PTEC as consequence of a direct interaction with T/NK cells. Downmodulation of the receptor CD226 on effector cells confirmed the involvement of this receptor/ligand pair in back signaling. In vivo, CD155 and CD166 expression was detectable in proximal and distal tubuli of renal transplant biopsies, respectively. The composition of the protein microenvironment in these biopsies showed a substantial overlap with the PTEC response. Cluster and principal component analyses of the microenvironment separated unsuspicious from rejection biopsies and, furthermore, ABMR, TCMR, and borderline rejection. In conclusion, our results provide evidence that epithelial cells may contribute to the rejection process and pave the way to a better understanding of the pathomechanisms of kidney allograft rejection.
Although trough levels of immunosuppressive drugs are largely used to monitor immunosuppressive therapy after solid organ transplantation, there is still no established tool that allows for a validated assessment of functional degree of immunosuppression or the identification of clinically relevant over- or under-immunosuppression, depending on graft homeostasis. Reliable non-invasive markers to predict biopsy proven acute rejection (BPAR) do not exist. Literature data suggest that longitudinal measurements of immune markers might be predictive of BPAR, but data in children are scarce. We therefore propose an observational prospective cohort study focusing on immune monitoring in children after liver transplantation. We aim to describe immune function in a cohort of children before and during the first year after liver transplantation and plan to investigate how the immune function profile is associated with clinical and laboratory findings. In an international multicenter prospective approach, children with end-stage liver disease who undergo liver transplantation are enrolled to the study and receive extensive immune monitoring before and at 1, 2, 3, 4 weeks and 3, 6, 12 months after transplantation, and whenever a clinically indicated liver biopsy is scheduled. Blood samples are analyzed for immune cell numbers and circulating levels of cytokines, chemokines and factors of angiogenesis reflecting immune cell activation. Statistical analysis will focus on the identification of trajectorial patterns of immune reactivity predictive for systemic non-inflammatory states, infectious complications or BPAR using joint modelling approaches. The ChilSFree study will help to understand the immune response after pLTx in different states of infection or rejection. It may provide insight into response mechanisms eventually facilitating immune tolerance towards the graft. Our analysis may yield an applicable immune panel for non-invasive early detection of acute cellular rejection, with the prospect of individually tailoring immunosuppressive therapy. The international collaborative set-up of this study allows for an appropriate sample size which is otherwise difficult to achieve in the field of pediatric liver transplantation.
In the context of kidney transplantation, little is known about the involvement of natural killer (NK) cells in the immune reaction leading to either rejection or immunological tolerance under immunosuppression. Therefore, the peripheral NK cell repertoire of patients after kidney transplantation was investigated in order to identify NK cell subsets that may be associated with the individual immune status at the time of their protocol biopsies for histopathological evaluation of the graft. Alterations in the peripheral NK cell repertoire could be correlated to the type of immunosuppression, i.e., calcineurin-inhibitors like Cyclosporin A vs. Tacrolimus with or without addition of mTOR inhibitors. Here, we could demonstrate that the NK cell repertoire in peripheral blood of kidney transplant patients differs significantly from healthy individuals. The presence of donor-specific antibodies was associated with reduced numbers of CD56(dim) NK cells. Moreover, in patients, down-modulation of CD16 and CD6 on CD56(dim) NK cells was observed with significant differences between Cyclosporin A- and Tac-treated patients. Tac-treatment was associated with decreased CD69, HLA-DR, and increased CD94/NKG2A expression in CD56(dim) NK cells indicating that the quality of the immunosuppressive treatment impinges on the peripheral NK cell repertoire. In vitro studies with peripheral blood mononuclear cells of healthy donors showed that this modulation of CD16, CD6, CD69, and HLA-DR could also be induced experimentally. The presence of calcineurin or mTOR inhibitors had also functional consequences regarding degranulation and interferon-γ-production against K562 target cells, respectively. In summary, we postulate that the NK cell composition in peripheral blood of kidney transplanted patients represents an important hallmark of the efficacy of immunosuppression and may be even informative for the immune status after transplantation in terms of rejection vs. drug-induced allograft tolerance. Thus, NK cells can serve as sensors for immunosuppression and may be utilized for future strategies of an individualized adjustment of immunosuppression.
The lymphocyte surface glycoprotein CD26 anchors adenosine deaminase to the lymphocyte surface and possesses dipeptidyl peptidase IV activity. A distinct subset of CD26++ lymphocytes in autologous hematopoietic progenitor cell transplants (HPCTs) was investigated with regard to clinical outcome after autologous HPCT. The phenotype of these cells was characterized in more detail.Forty-two eligible patients (multiple myeloma, n = 31; Hodgkin's disease, n = 3; non-Hodgkin's lymphoma, n = 6; peripheral neuroectodermal tumor, n = 1; acute myeloid leukemia, n = 1) were included in a retrospective analysis. Distinct cellular subsets, including CD26+/- and CD26++ subpopulations, were analyzed for correlations with kinetics of engraftment, progression-free survival, and overall survival.The numbers of CD26++ T lymphocytes in the autograft correlated inversely with progression-free survival (p = 0.013). CD26++ T lymphocytes transfused per kg of body weight were predictive for the occurrence of disease progression or relapse (p = 0.006). Importantly, the numbers of CD26++ cells showed a highly variable degree of enrichment in the autograft, but no significant variations in the peripheral blood before apheresis. The characterization of CD26++ cells revealed that CD26++/CD8+ cells form a homogeneous population with a distinct T memory cell phenotype (CD45RO+, CD161++, interleukin-18Rα++, CCR7-).CD26++ lymphocytes define a discrete phenotype of T memory cells with known chemoresistance and T-cell-repopulating capacity. Their enrichment during apheresis and corresponding depletion from the circulation are associated with an adverse outcome in autologous HPCT.
The inducible costimulator receptor (ICOS) is a third member of the CD28 receptor family that regulates T cell activation and function. ICOS binds to a newly identified ligand on antigen presenting cells different from the CD152 ligands CD80 and CD86. We used soluble ICOSIg and a newly developed murine anti-human ICOS ligand (ICOSL) monoclonal antibody to further characterize the ICOSL during ontogeny of antigen presenting cells. In a previous study, we found that ICOSL is expressed on monocytes, dendritic cells, and B cells. To define when ICOSL is first expressed on myeloid antigen presenting cells, we examined ICOSL expression on CD34+ cells in bone marrow. We found that CD34bright cells regardless of their myeloid commitment were ICOSL−, whereas ICOSL was first expressed when CD34 expression diminished and the myeloid marker CD33 appeared. However, acute myeloid leukemia cells were ICOSL-negative, whereas among B-cell malignancies only some cases of the most mature tumors such as prolymphocytic leukemia and hairy cell leukemia were positive. Next, we investigated purified CD34+ hematopoietic progenitor cells that did not constitutively express ICOSL but were induced to express ICOSL within 12 h after granulocyte/macrophage colony-stimulating factor/tumor necrosis factor α (TNF-α) stimulation. Interestingly, ICOSL was induced prior to CD80/CD86 induction on CD34+cells so that ICOSL was expressed in the absence of CD80/CD86. This suggests that ICOSL is an early differentiation marker along the monocytic/dendritic maturation pathway. Induction of ICOSL was dependent on TNF-α and was regulated via NF-κB as revealed by use of inhibitors specific for IκBα phosphorylation such as BAY 11-7082 and BAY 11-7085. The antigen presenting capacity of TNF-α stimulated CD34+ cells was strongly inhibited by ICOSIg fusion proteins or by NF-κB inhibition. Thus, TNF-α-induced ICOSL expression seemed to be functionally important for the costimulatory capacity of CD34+ hematopoietic progenitor cells.
