Generation and quality control of mature monocyte-derived dendritic cells for immunotherapy.

Dendritic cell vaccination is a form of active immunotherapy that aims to exploit the crucial role of DC in the initiation of T-cell responses. Numerous vaccination trials have been conducted targeting various tumor entities, including glioblastoma, the most frequent and aggressive malignant brain tumor in adults. They have demonstrated feasibility and safety and suggest improved survival, associated with induction of anti-tumoral immunity. Here, we describe in detail a large-scale 2-step protocol for successive GMP-compliant generation of immature and mature dendritic cells, yielding a highly homogenous population of CD83+ mature DC expressing CD40, CD80, CD86 and HLA-DR at high density, lacking activity of the immunosuppressive enzyme indoleamine-2,3-dioxygenase, migrating towards the chemokine CCL19 and showing highly potent T-cell stimulatory activity. Loaded with autologous tumor lysate, these cells are currently being evaluated in a phase II controlled randomized clinical trial (GlioVax) in glioblastoma patients.

Lymph node and tumor-associated PD-L1+ macrophages antagonize dendritic cell vaccines by suppressing CD8+ T cells.

Current immunotherapies provide limited benefits against T cell-depleted tumors, calling for therapeutic innovation. Using multi-omics integration of cancer patient data, we predict a type I interferon (IFN) responseHIGH state of dendritic cell (DC) vaccines, with efficacious clinical impact. However, preclinical DC vaccines recapitulating this state by combining immunogenic cancer cell death with induction of type I IFN responses fail to regress mouse tumors lacking T cell infiltrates. Here, in lymph nodes (LNs), instead of activating CD4+/CD8+ T cells, DCs stimulate immunosuppressive programmed death-ligand 1-positive (PD-L1+) LN-associated macrophages (LAMs). Moreover, DC vaccines also stimulate PD-L1+ tumor-associated macrophages (TAMs). This creates two anatomically distinct niches of PD-L1+ macrophages that suppress CD8+ T cells. Accordingly, a combination of PD-L1 blockade with DC vaccines achieves significant tumor regression by depleting PD-L1+ macrophages, suppressing myeloid inflammation, and de-inhibiting effector/stem-like memory T cells. Importantly, clinical DC vaccines also potentiate T cell-suppressive PD-L1+ TAMs in glioblastoma patients. We propose that a multimodal immunotherapy and vaccination regimen is mandatory to overcome T cell-depleted tumors.

Multiomics and spatial mapping characterizes human CD8+ T cell states in cancer.

Clinically relevant immunological biomarkers that discriminate between diverse hypofunctional states of tumor-associated CD8+ T cells remain disputed. Using multiomics analysis of CD8+ T cell features across multiple patient cohorts and tumor types, we identified tumor niche-dependent exhausted and other types of hypofunctional CD8+ T cell states. CD8+ T cells in "supportive" niches, like melanoma or lung cancer, exhibited features of tumor reactivity-driven exhaustion (CD8+ TEX). These included a proficient effector memory phenotype, an expanded T cell receptor (TCR) repertoire linked to effector exhaustion signaling, and a cancer-relevant T cell-activating immunopeptidome composed of largely shared cancer antigens or neoantigens. In contrast, "nonsupportive" niches, like glioblastoma, were enriched for features of hypofunctionality distinct from canonical exhaustion. This included immature or insufficiently activated T cell states, high wound healing signatures, nonexpanded TCR repertoires linked to anti-inflammatory signaling, high T cell-recognizable self-epitopes, and an antiproliferative state linked to stress or prodeath responses. In situ spatial mapping of glioblastoma highlighted the prevalence of dysfunctional CD4+:CD8+ T cell interactions, whereas ex vivo single-cell secretome mapping of glioblastoma CD8+ T cells confirmed negligible effector functionality and a promyeloid, wound healing-like chemokine profile. Within immuno-oncology clinical trials, anti-programmed cell death protein 1 (PD-1) immunotherapy facilitated glioblastoma's tolerogenic disparities, whereas dendritic cell (DC) vaccines partly corrected them. Accordingly, recipients of a DC vaccine for glioblastoma had high effector memory CD8+ T cells and evidence of antigen-specific immunity. Collectively, we provide an atlas for assessing different CD8+ T cell hypofunctional states in immunogenic versus nonimmunogenic cancers.

Trial watch: Dendritic cell (DC)-based immunotherapy for cancer.

Dendritic cell (DC)-based vaccination for cancer treatment has seen considerable development over recent decades. However, this field is currently in a state of flux toward niche-applications, owing to recent paradigm-shifts in immuno-oncology mobilized by T cell-targeting immunotherapies. DC vaccines are typically generated using autologous (patient-derived) DCs exposed to tumor-associated or -specific antigens (TAAs or TSAs), in the presence of immunostimulatory molecules to induce DC maturation, followed by reinfusion into patients. Accordingly, DC vaccines can induce TAA/TSA-specific CD8+/CD4+ T cell responses. Yet, DC vaccination still shows suboptimal anti-tumor efficacy in the clinic. Extensive efforts are ongoing to improve the immunogenicity and efficacy of DC vaccines, often by employing combinatorial chemo-immunotherapy regimens. In this Trial Watch, we summarize the recent preclinical and clinical developments in this field and discuss the ongoing trends and future perspectives of DC-based immunotherapy for oncological indications.

Crosstalk between ß-Catenin and CCL2 Drives Migration of Monocytes towards Glioblastoma Cells.

Isocitrate dehydrogenase (IDH)-wildtype glioblastoma (GBM) is a fast growing and highly heterogeneous tumor, often characterized by the presence of glioblastoma stem cells (GSCs). The plasticity of GSCs results in therapy resistance and impairs anti-tumor immune response by influencing immune cells in the tumor microenvironment (TME). Previously, ß-catenin was associated with stemness in GBM as well as with immune escape mechanisms. Here, we investigated the effect of ß-catenin on attracting monocytes towards GBM cells. In addition, we evaluated whether CCL2 is involved in ß-catenin crosstalk between monocytes and tumor cells. Our analysis revealed that shRNA targeting ß-catenin in GBMs reduces monocytes attraction and impacts CCL2 secretion. The addition of recombinant CCL2 restores peripheral blood mononuclear cells (PBMC) migration towards medium (TCM) conditioned by shß-catenin GBM cells. CCL2 knockdown in GBM cells shows similar effects and reduces monocyte migration to a similar extent as ß-catenin knockdown. When investigating the effect of CCL2 on ß-catenin activity, we found that CCL2 modulates components of the Wnt/ß-catenin pathway and alters the clonogenicity of GBM cells. In addition, the pharmacological ß-catenin inhibitor MSAB reduces active ß-catenin, downregulates the expression of associated genes and alters CCL2 secretion. Taken together, we showed that ß-catenin plays an important role in attracting monocytes towards GBM cells in vitro. We hypothesize that the interactions between ß-catenin and CCL2 contribute to maintenance of GSCs via modulating immune cell interaction and promoting GBM growth and recurrence.

Dendritic Cell Vaccination of Glioblastoma: Road to Success or Dead End.

Glioblastomas (GBM) are the most frequent and aggressive malignant primary brain tumor and remains a therapeutic challenge: even after multimodal therapy, median survival of patients is only 15 months. Dendritic cell vaccination (DCV) is an active immunotherapy that aims at inducing an antitumoral immune response. Numerous DCV trials have been performed, vaccinating hundreds of GBM patients and confirming feasibility and safety. Many of these studies reported induction of an antitumoral immune response and indicated improved survival after DCV. However, two controlled randomized trials failed to detect a survival benefit. This raises the question of whether the promising concept of DCV may not hold true or whether we are not yet realizing the full potential of this therapeutic approach. Here, we discuss the results of recent vaccination trials, relevant parameters of the vaccines themselves and of their application, and possible synergies between DCV and other therapeutic approaches targeting the immunosuppressive microenvironment of GBM.

Ciprofloxacin enhances phototoxicity of 5-aminolevulinic acid mediated photodynamic treatment for chordoma cell lines.

BACKGROUND: Chordoma are uncommon aggressive tumors of the skeleton. Surgical resection is often subtotal and adjuvant treatment possibilities are limited as chordomas are highly chemo- and radioresistant. In the present study we examined the impact of 5-ALA PDT on different human chordoma cell lines. Furthermore, we investigated the variation of two parameters: (1.) 5-ALA incubation time and (2.) supplemental use of ciprofloxacin as iron chelator. METHODS: Experiments were realized in vitro with three different human chordoma cell lines: U-CH2, U-CH2B and U-CH14. After pre-incubation for 24 h with various concentrations of ciprofloxacin (1.5 - 5.0 µg/ml), different amounts of 5-ALA (15 - 50 µg/ml) were applied to the cells either for a brief (4 h) or a long (6 h) incubation time. Subsequently cells were exposed to photodynamic radiation. Cell viability was exploited by WST-1 assay. Thus, for each of the three cell lines, two drug combinations (ciprofloxacin plus 5-ALA and 5-ALA only) and two incubation times (short, 4 h and long, 6 h) were tested. Negative control groups were also examined. RESULTS: Supplemental use of ciprofloxacin led to increased cell death in each of the cell lines. Different 5-ALA incubation times (4 h vs. 6 h) showed no significant differences in cell viability except for U-CH2. CONCLUSION: Ciprofloxacin as an ordinary applied antibiotic, enhanced the effect of 5-ALA PDT on different human chordoma cell lines in vitro. The impact was dependent on the dose of ciprofloxacin-5-ALA. There were no notable differences for the tested 5-ALA incubation times. In human chordoma cell lines 5-ALA PDT may effectively be amended by ciprofloxacin.

Diagnostic impact of additional O-(2-[18F]fluoroethyl)-L-tyrosine (18F-FET) PET following immunotherapy with dendritic cell vaccination in glioblastoma patients.

OBJECTIVE: Vaccination therapy using tumour antigen-loaded, autologous dendritic cells (DC) is a promising therapeutic approach alongside standard treatment for glioblastoma (GBM). However, reliable diagnostic criteria regarding therapy monitoring are not established. Here, we analysed the impact of additional 18F-fluoroethyl-tyrosine positron emission tomography (18F-FET PET) imaging following DC vaccination therapy. METHODS: We analysed data of GBM patients who received DC vaccination therapy. Following MRI diagnosis of tumour recurrence, additional static and dynamic 18F-FET PET imaging was performed. Vaccination was performed five times by intradermal injections, either weekly between concomitant radio/-chemotherapy and intermittent chemotherapy or after tumour recurrence, before re-radiation therapy. MRI and 18F-FET PET results were compared and correlated with clinical data. RESULTS: Between 2003 and 2016, 5 patients were identified who received DC vaccination and 18F-FET PET imaging (1 female/4 males; mean age: 44 ± 14 y). 3/5 patients showed congruent results of tumour progression. In three patients 18F-FET PET indicated treatment related changes, which was in contrast to MRI findings that indicated tumour progression. In these patients 18F-FET PET results could be confirmed by either neuropathological diagnosis or according to the RANO criteria. CONCLUSIONS: Despite the small patients number our results indicate an additional impact of 18F-FET PET for monitoring outcome following vaccination therapy.

Human iPSC-derived MSCs (iMSCs) from aged individuals acquire a rejuvenation signature.

BACKGROUND: Primary mesenchymal stem cells (MSCs) are fraught with aging-related shortfalls. Human-induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) have been shown to be a useful clinically relevant source of MSCs that circumvent these aging-associated drawbacks. To date, the extent of the retention of aging-hallmarks in iMSCs differentiated from iPSCs derived from elderly donors remains unclear. METHODS: Fetal femur-derived MSCs (fMSCs) and adult bone marrow MSCs (aMSCs) were isolated, corresponding iPSCs were generated, and iMSCs were differentiated from fMSC-iPSCs, from aMSC-iPSCs, and from human embryonic stem cells (ESCs) H1. In addition, typical MSC characterization such as cell surface marker expression, differentiation capacity, secretome profile, and trancriptome analysis were conducted for the three distinct iMSC preparations-fMSC-iMSCs, aMSC-iMSCs, and ESC-iMSCs. To verify these results, previously published data sets were used, and also, additional aMSCs and iMSCs were analyzed. RESULTS: fMSCs and aMSCs both express the typical MSC cell surface markers and can be differentiated into osteogenic, adipogenic, and chondrogenic lineages in vitro. However, the transcriptome analysis revealed overlapping and distinct gene expression patterns and showed that fMSCs express more genes in common with ESCs than with aMSCs. fMSC-iMSCs, aMSC-iMSCs, and ESC-iMSCs met the criteria set out for MSCs. Dendrogram analyses confirmed that the transcriptomes of all iMSCs clustered together with the parental MSCs and separated from the MSC-iPSCs and ESCs. iMSCs irrespective of donor age and cell type acquired a rejuvenation-associated gene signature, specifically, the expression of INHBE, DNMT3B, POU5F1P1, CDKN1C, and GCNT2 which are also expressed in pluripotent stem cells (iPSCs and ESC) but not in the parental aMSCs. iMSCs expressed more genes in common with fMSCs than with aMSCs. Independent real-time PCR comparing aMSCs, fMSCs, and iMSCs confirmed the differential expression of the rejuvenation (COX7A, EZA2, and TMEM119) and aging (CXADR and IGSF3) signatures. Importantly, in terms of regenerative medicine, iMSCs acquired a secretome (e.g., angiogenin, DKK-1, IL-8, PDGF-AA, osteopontin, SERPINE1, and VEGF) similar to that of fMSCs and aMSCs, thus highlighting their ability to act via paracrine signaling. CONCLUSIONS: iMSCs irrespective of donor age and cell source acquire a rejuvenation gene signature. The iMSC concept could allow circumventing the drawbacks associated with the use of adult MSCs und thus provide a promising tool for use in various clinical settings in the future.

Accumulation of protoporphyrin IX in medulloblastoma cell lines and sensitivity to subsequent photodynamic treatment.

BACKGROUND: Medulloblastoma (MB) is the most common malignant primary brain tumor of childhood. High risk patients still have a poor outcome, and especially young patients suffer from standard therapy induced sequelae. Therefore, other therapeutic options need to be explored. In glioblastoma (GBM), application of 5-aminolaevulinic acid (5-ALA) results in selective accumulation of protoporphyrin IX (PPIX) in the tumor cells, which can be exploited during fluorescence-guided surgery to increase the extent of resection or for photodynamic therapy (PDT) induced phototoxicity. It is not entirely clear, whether MB cells accumulate PPIX and are sensitive to PDT. METHODS: Human MYC-amplified (Med8A and D283) and non-amplified (UW228-2 and ONS76) MB cell lines were incubated for 2, 4 or 6 h with increasing doses (0-100 µg/ml) of 5-ALA, and PPIX accumulation was determined by flow cytometry. To assess sensitivity to 5-ALA/PDT, cells were incubated with 5-ALA and subsequently exposed to laser light of 635 nm wavelength (18.75 J/cm2). After an additional 24 h culture period, viability of cells was quantified using the WST-1 assay. Expression of ferrochelatase was detected by reverse transcription and quantitative polymerase chain reaction. Ferrochelatase activity was quantified by measuring the enzymatic conversion of PPIX to zinc-protoporphyrin. Expression of the ABCG2 transporter protein CD338 was detected by flow cytometry. RESULTS: All MB cell lines showed a time- and dose-dependent accumulation of PPIX after exposure to exogenous 5-ALA and became sensitive to 5-ALA/PDT-induced phototoxicity. PPIX accumulation was reduced compared to U373 GBM cells at shorter incubation periods and limiting 5-ALA doses. Moreover, not all MB cells became PPIX positive and overall phototoxicity was lower in the MB cell lines. Notably, the MYC-amplified MB cells demonstrated a more pronounced photosensitivity compared to their non-amplified counterparts. There was no difference in expression of ferrochelatase, but enzymatic activity appeared to be reduced in the MB cells compared to U373 GBM cells, whereas CD338 was expressed on the MB cells only. CONCLUSION: Medulloblastoma cell lines accumulate PPIX after application of 5-ALA and become sensitive to PDT, associated with low ferrochelatase expression and activity. Photosensitivity is more pronounced in MYC-amplified cell lines. In contrast to GBM cells, however, PPIX accumulation appears to be reduced, restricted to a subset of cells and associated with lower photosensitivity of the MB cell lines, possibly due to expression of the ABCG2 transporter protein CD338 on MB cells.

A randomized controlled phase II trial of vaccination with lysate-loaded, mature dendritic cells integrated into standard radiochemotherapy of newly diagnosed glioblastoma (GlioVax): study protocol for a randomized controlled trial.

BACKGROUND: Despite the combination of surgical resection, radio- and chemotherapy, median survival of glioblastoma multiforme (GBM) patients only slightly increased in the last years. Disease recurrence is definite with no effective therapy existing after tumor removal. Dendritic cell (DC) vaccination is a promising active immunotherapeutic approach. There is clear evidence that it is feasible, results in immunological anti-tumoral responses, and appears to be beneficial for survival and quality of life of GBM patients. Moreover, combining it with the standard therapy of GBM may allow exploiting synergies between the treatment modalities. In this randomized controlled trial, we seek to confirm these promising initial results. METHODS: One hundred and thirty-six newly diagnosed, isocitrate dehydrogenase wildtype GBM patients will be randomly allocated (1:1 ratio, stratified by O6-methylguanine-DNA-methyltransferase promotor methylation status) after near-complete resection in a multicenter, prospective phase II trial into two groups: (1) patients receiving the current therapeutic "gold standard" of radio/temozolomide chemotherapy and (2) patients receiving DC vaccination as an add-on to the standard therapy. A recruitment period of 30 months is anticipated; follow-up will be 2 years. The primary objective of the study is to compare overall survival (OS) between the two groups. Secondary objectives are comparing progression-free survival (PFS) and 6-, 12- and 24-month OS and PFS rates, the safety profile, overall and neurological performance and quality of life. DISCUSSION: Until now, close to 500 GBM patients have been treated with DC vaccination in clinical trials or on a compassionate-use basis. Results have been encouraging, but cannot provide robust evidence of clinical efficacy because studies have been non-controlled or patient numbers have been low. Therefore, a prospective, randomized phase II trial with a sufficiently large number of patients is now mandatory for clear evidence regarding the impact of DC vaccination on PFS and OS in GBM. TRIAL REGISTRATION: Protocol code: GlioVax, date of registration: 17. February 2017. Trial identifier: EudraCT-Number 2017-000304-14. German Registry for Clinical Studies, ID: DRKS00013248 (approved primary register in the WHO network) and at ClinicalTrials.gov , ID: NCT03395587 . Registered on 11 March 2017.

5-Aminolevulinic acid-based photodynamic therapy of chordoma: In vitro experiments on a human tumor cell line.

BACKGROUND: Chordomas are very rare tumors of the skull base and the sacrum. They show infiltrating and destructive growth and are known to be chemo- and radio-resistant. After surgical resection, the recurrence rate is high and overall survival limited. As current adjuvant treatments are ineffective, new treatment concepts are urgently needed. 5-aminolevulinic acid-based photodynamic therapy (5-ALA based PDT) showed promising results for malignant gliomas. However, it is unknown so far, whether chordomas accumulate protoporphyrin IX (PPIX) after application of 5-ALA and whether they are sensitive to subsequent 5-ALA based PDT. METHODS: The immortalized human chordoma cells U-CH2 were used as in vitro model. After incubation for 4h or 6h with different 5-ALA concentrations, PPIX accumulation was determined by flow cytometry. To assess sensitivity to PDT, chordoma cells were incubated at 30.000cells/well (high cell density) or 15.000cells/well (low cell density) with graded doses of 5-ALA (0-50µg/ml) in 96-well plates and subsequently exposed to laser light of 635nm wavelength (18.75J/cm2). Cell survival was measured 24h after exposure to laser light using the WST-1 assay. RESULTS: U-CH2 cells dose-dependently accumulated PPIX (ANOVA; p<0.0001). PPIX fluorescence was significantly higher, when cells were incubated with 5-ALA for 6h compared to 4h at higher 5-ALA concentrations (ANOVA/Bonferroni; p≤0.05 for≥30µg/ml 5-ALA). For both cell densities, a 5-ALA dose-dependent decline in viability was observed (ANOVA; p<0.0001). Viability was significantly lower at higher 5-ALA concentrations, when 30.000 cells/wells were treated compared to 15.000cells/well (ANOVA/Bonferroni; p≤0.001 for≥30µg/ml 5-ALA). LD50 was 30.25µg/ml 5-ALA. CONCLUSION: The human UCH-2 cell line was a very useful in vitro model to study different effects of 5-ALA based PDT. For the first time, it could be shown that human chordoma cells may be destroyed by 5-ALA/PDT.

Human glioblastoma stem-like cells accumulate protoporphyrin IX when subjected to exogenous 5-aminolaevulinic acid, rendering them sensitive to photodynamic treatment.

Glioblastoma (GBM) is the most frequent and lethal primary brain tumor in adults. Despite multimodal therapy combining resection, radio- and alkylating chemotherapy, disease recurrence is universal and prognosis of patients is poor. Glioblastoma stem-like cells (GSC), which can be grown as neurospheres from primary tumors in vitro, appear to be resistant to the established therapies and are suspected to be the driving force for disease recurrence. Thus, efficacy of emerging therapies may depend on targeting GSC. 5-aminolaevulinic acid-mediated photodynamic therapy (5-ALA/PDT) is a promising therapeutic approach in GBM. It utilizes the selective accumulation of the photosensitizer protoporphyrin IX (PPIX) in GBM cells after application of 5-ALA. When exposed to laser light of 635nm wavelength, PPIX initiates a photochemical reaction resulting in the generation of reactive oxygen species, which kill the tumor cells. Whether GSC accumulate PPIX and are sensitive to 5-ALA/PDT is currently unknown. Therefore, human GSC were derived from primary tumors and grown as neurospheres under serum free conditions. When subjected to exogenous 5-ALA, a dose- and time-dependent accumulation of PPIX in GSC was observed by flow cytometry, which varied between individual GSC preparations. Subsequent exposure to laser light of 635nm wavelength substantially killed GSC, whereas treatment with 5-ALA or exposure to laser light only had no effect. LD50 values differed between GSC preparations, but were negatively correlated with PPIX accumulation in GSC. In summary, we report for the first time that glioblastoma stem-like cells accumulate PPIX when subjected to 5-aminolaevulinic acid and are sensitive to 5-aminolaevulinc acid based photodynamic therapy.

Unreserved application of epigenetic methods to define differences of DNA methylation between urinary cellular and cell-free DNA.

Urinary DNA is increasingly gaining importance in diagnosis of urological malignancies. Especially cell-free DNA originating from apoptotic and necrotic cells of the early tumor could become a key target for early stage tumor diagnosis. Aberrant DNA methylation forms tumor cell characteristic epigenetic profiles which are covalently established before any tumor related aberration at transcriptional or protein level has occurred. In addition, these epigenetic signatures are alterably adapted to and accompanying the individual stages of multistep, progressive tumorigenesis. Hence, they seem very promising for diagnosis as well as for monitoring the patient's follow-up care and even for decisions regarding personalized therapeutic options. The essential prerequisite at this approach will be a reliable methodological handling of the biological material of interest. In this study we present detailed analyses of LINE-1 DNA methylation profiles and demonstrate the sensitive detection of LINE-1 DNA methylation differences as well as between cancer patients and healthy individuals, between urinary cellular and cell-free DNA. In addition, we show methylome differences between both DNA fractions from a healthy individual and bladder cancer patients. In conclusion, we demonstrate here the unrestricted amenability of urinary cell-free DNA for both, a detailed characterization of a distinct DNA methylation alteration and its sensitive detection and a comprehensive global, array-based screening for DNA methylation differences.

Cytomegalovirus infection impairs immunosuppressive and antimicrobial effector functions of human multipotent mesenchymal stromal cells.

Human mesenchymal stromal cells (MSC) possess immunosuppressive and antimicrobial effects that are partly mediated by the tryptophan-catabolizing enzyme indoleamine-2,3-dioxygenase (IDO). Therefore MSC represent a promising novel cellular immunosuppressant which has the potential to control steroid-refractory acute graft versus host disease (GvHD). In addition, MSC are capable of reducing the risk of infection in patients after haematopoietic stem cell transplantation (HST). Recent data indicate that signals from the microenvironment including those from microbes may modulate MSC effector functions. As Cytomegalovirus (CMV) represents a prominent pathogen in immunocompromised hosts, especially in patients following HST, we investigated the impact of CMV infection on MSC-mediated effects on the immune system. We demonstrate that CMV-infected MSC lose their cytokine-induced immunosuppressive capacity and are no longer able to restrict microbial growth. IDO expression is substantially impaired following CMV infection of MSC and this interaction critically depends on intact virus and the number of MSC as well as the viral load. Since overt CMV infection may undermine the clinical efficacy of MSC in the treatment of GvHD in transplant patients, we recommend that patients scheduled for MSC therapy should undergo thorough evaluation for an active CMV infection and receive CMV-directed antiviral therapy prior to the administration of MSC.

Activated platelets interfere with recruitment of mesenchymal stem cells to apoptotic cardiac cells via high mobility group box 1/Toll-like receptor 4-mediated down-regulation of hepatocyte growth factor receptor MET.

Recruitment of mesenchymal stem cells (MSC) following cardiac injury, such as myocardial infarction, plays a critical role in tissue repair and may contribute to myocardial recovery. However, the mechanisms that regulate migration of MSC to the site of tissue damage remain elusive. Here, we demonstrate in vitro that activated platelets substantially inhibit recruitment of MSC toward apoptotic cardiac myocytes and fibroblasts. The alarmin high mobility group box 1 (HMGB1) was released by platelets upon activation and mediated inhibition of the cell death-dependent migratory response through Toll-like receptor (TLR)-4 expressed on the MSC. Migration of MSC to apoptotic cardiac myocytes and fibroblasts was driven by hepatocyte growth factor (HGF), and platelet activation was followed by HMGB1/TLR-4-dependent down-regulation of HGF receptor MET on MSC, thereby impairing HGF-driven MSC recruitment. We identify a novel mechanism by which platelets, upon activation, interfere with MSC recruitment to apoptotic cardiac cells, a process that may be of particular relevance for myocardial repair and regeneration.

Cytomegalovirus impairs the induction of indoleamine 2,3-dioxygenase mediated antimicrobial and immunoregulatory effects in human fibroblasts.

Human fibroblasts provide immunosuppressive functions that are partly mediated by the tryptophan-catabolizing enzyme indoleamine-2,3-dioxygenase (IDO). Moreover, upon stimulation with inflammatory cytokines human fibroblasts exhibit broad-spectrum antimicrobial effector functions directed against various clinically relevant pathogens and these effects are also IDO-dependent. Therefore human fibroblasts are suggested to be involved in the control of immune reactions during infectious diseases. As human cytomegalovirus (HCMV) represents a pathogen frequently found in immunocompromised hosts and IDO is involved in the control of HCMV growth, we here investigated the impact of HCMV infection on IDO-mediated antimicrobial and immunoregulatory effects. We show that infection with HCMV substantially impairs IFN-γ-induced IDO-activity in human fibroblasts in a dose and time dependent fashion. Consequently, these cells are no longer able to restrict bacterial and parasitic growth and, furthermore, loose their IDO-mediated immunosuppressive capacity. Our results may have significant implications for the course of HCMV infection during solid organ transplantation: we suggest that loss of IDO-mediated antimicrobial and immunoregulatory functions during a HCMV infection might at least in part explain the enhanced risk of organ rejection and infections observed in patients with HCMV reactivation after solid organ transplantation.

Migration of mesenchymal stem cells towards glioblastoma cells depends on hepatocyte-growth factor and is enhanced by aminolaevulinic acid-mediated photodynamic treatment.

Hepatocyte-growth factor (HGF) is expressed by glioblastomas and contributes to their growth, migration and invasion. HGF also mediates migration of mesenchymal stem cells (MSC) to sites of apoptotic cell death. Moreover, MSC show tropism for glioblastomas, which is exploited in gene therapy to deliver the therapeutics to the tumor cells. Here, we have studied whether HGF contributes to the recruitment of MSC by glioblastoma cells and whether aminolaevulinic acid-mediated photodynamic therapy (ALA/PDT), a novel therapeutic approach that induces apoptosis in glioblastoma cells, affects HGF release and this migratory response. MSC expressed the HGF receptor MET and migrated towards U87 and U251 glioblastoma spheroids. Migration increased significantly when spheroids were subjected to ALA/PDT, which was associated with induction of apoptosis and up-regulation of HGF. Neutralizing HGF resulted in significant inhibition of MSC migration towards untreated as well as ALA/PDT-treated spheroids. Thus, glioblastoma cells express HGF, which contributes to the attraction of MSC. ALA/PDT induces apoptosis and augments HGF release causing enhanced MSC migration towards the tumor cells. ALA/PDT may therefore be exploited to improve targeting of MSC delivered gene therapy, but it may also constitute a risk in terms of beneficial effects for the tumor.

Platelet transfusion alters CD40L blood level and release capacity in patients suffering from thrombocytopenia.

BACKGROUND: Platelet (PLT)-derived cytokines, such as soluble CD40 ligand (sCD40L), play an important role in the development of adverse transfusion reactions associated with the administration of PLT products. In this study, we determined sCD40L concentration and release capacity in patients with thrombocytopenia before and after receiving a PLT transfusion. STUDY DESIGN AND METHODS: The study included 12 patients suffering from chemotherapy-induced thrombocytopenia. sCD40L levels and release capacity were measured in plasma samples of the patients before and after PLT administration as well as in the respective plateletpheresis concentrates by enzyme-linked immunosorbent assay. Sixteen healthy blood donors served as a control group. RESULTS: In PLT concentrates, elevated sCD40L levels (2567±134 pg/mL) were observed in comparison to plasma sCD40L levels in controls (238.4±35.3 pg/mL). sCD40L plasma concentration of patients with thrombocytopenia was significantly reduced (86.3±16.7 pg/mL) before transfusion and increased to nearly normal levels (204.4±24.8 pg/mL) after PLT administration. In parallel, the sCD40L release capacity per PLT showed no significant difference between controls and patients with thrombocytopenia before transfusion (33.3±2.6 and 29.3±4.6 ag/PLT, respectively) but was significantly reduced after PLT transfusion (22.4±2.7 compared to 29.3±4.6 ag/PLT). CONCLUSIONS: In patients with thrombocytopenia, sCD40L levels were clearly influenced by PLT transfusions: PLT administration led to a normalization of sCD40L plasma concentration. Nevertheless, adverse transfusion reactions did not occur in these patients. The sCD40L release capacity was enhanced by PLT administration dependent on the increase in the amount of PLT count.

Modulation of migratory activity and invasiveness of human glioma spheroids following 5-aminolevulinic acid-based photodynamic treatment. Laboratory investigation.

OBJECT: Five-aminolevulinic acid-mediated photodynamic therapy (ALA/PDT) can improve the clinical outcome in patients suffering from glioblastoma. Besides direct phototoxicity, additional mechanisms may contribute. Therefore, the authors studied the influence of ALA/PDT on glioblastoma's migratory and invasive behavior in a human glioma cell spheroid model. METHODS: Glioma spheroids were grown from human U373 and A172 cell lines. After ALA/PDT of spheroids, the authors assessed the migration of tumor cells and their capacity to invade a collagen matrix, as well as changes in their viability, morphology, and expression of matrix metalloproteinases (MMPs). RESULTS: The authors found that ALA/PDT caused long-lasting, nearly complete suppression of glioma cell migration and matrix invasion compared with nontherapeutic controls, including either irradiation or incubation with ALA only. Although ALA/PDT induced tumor cell apoptosis, suppression of migration/invasion was not simply due to phototoxicity because 50% of tumor cells remained vital throughout the observation period. Moreover, the morphology of ALA/PDT-treated cells changed significantly toward a polygonal, epithelial-like appearance, which was associated with alterations in the actin cytoskeleton. Furthermore, downregulation of MMP-7 and -8 was observed after treatment whereas other MMPs remained unchanged. CONCLUSIONS: In addition to directly eliminating glioma cells through apoptosis, ALA/PDT alters their invasiveness, possibly due to the effects on the cytoskeletal organization and MMP expression.