A New Human Somatic Stem Cell from Placental Cord Blood with Intrinsic Pluripotent Differentiation Potential

Here a new, intrinsically pluripotent, CD45-negative population from human cord blood, termed unrestricted somatic stem cells (USSCs) is described. This rare population grows adherently and can be expanded to 1015 cells without losing pluripotency. In vitro USSCs showed homogeneous differentiation into osteoblasts, chondroblasts, adipocytes, and hematopoietic and neural cells including astrocytes and neurons that express neurofilament, sodium channel protein, and various neurotransmitter phenotypes. Stereotactic implantation of USSCs into intact adult rat brain revealed that human Tau-positive cells persisted for up to 3 mo and showed migratory activity and a typical neuron-like morphology. In vivo differentiation of USSCs along mesodermal and endodermal pathways was demonstrated in animal models. Bony reconstitution was observed after transplantation of USSC-loaded calcium phosphate cylinders in nude rat femurs. Chondrogenesis occurred after transplanting cell-loaded gelfoam sponges into nude mice. Transplantation of USSCs in a noninjury model, the preimmune fetal sheep, resulted in up to 5% human hematopoietic engraftment. More than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion and substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected many months after USSC transplantation. No tumor formation was observed in any of these animals

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

Abstract 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

Diagnostic impact of additional O-(2-[18F]fluoroethyl)-L-tyrosine ( 18 F-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 criteriaConclusions: Despite the small patients number our results indicate an additional impact of 18F-FET PET for monitoring outcome following vaccination therapy

HCMV infection abrogates IFN-γ-induced IDO activity and subsequent antimicrobial effects.

<p>(A) HFF (3×10⁴/well) were stimulated with different amounts of IFN-γ in cell culture medium containing 0.6 mM tryptophan. The cultures were infected with HCMV (MOI 5) at the time point of IFN-γ stimulation, after 24 hours or 48 hour, respectively. After three days the kynurenine production by the cells was determined to directly measure IDO enzyme activity using Ehrlich’s reagent. Data are given as mean kynurenine production +/− SEM of five independent experiments, each done in triplicates. The OD measured in the negative control (unstimulated HFF) is subtracted in all groups. (B) HFF were stimulated with IFN-γ and infected with HCMV as described above. After three days cultures were infected with <i>S. aureus</i> (10–100 cfu/well) and bacterial growth was determined photometrically 24 hours later. Data are given as mean optical density +/− SEM of three independent experiments, each done in triplicates. (C) HFF were stimulated with IFN-γ and infected with HCMV as described above. Three days after activation the cells were infected with <i>T. gondii</i> (2×10⁴/well) and parasite growth was determined using [³H]-uracil three days later. Data are given as mean cpm +/− SEM of four independent experiments, each done in triplicates. HCMV added within the first 24 h of culture significantly reduced (p<0.05) IFN-γ-induced IDO activity and subsequent antibacterial and antiparasitic effects. (D) HFF cultures were co-infected with <i>T. gondii</i> and HCMV. After two days an immunofluorescence analysis was performed. While <i>T. gondii</i> parasites were detected with an anti-GRA9 antibody stained in green within the parasitophorous vacuole, HCMV was detected with an anti-HCMV-pp72 antibody stained in red [panel a)] within the nucleus of the host cell. As a control panel b) shows DAPI nuclear staining in blue with merged phase contrast.</p

HCMV blocks IDO activity and subsequent antibacterial effects observed in co-cultures of activated T cells and HFF.

<p>Peripheral blood mononuclear cells (PBMC; 1×10⁵/well), stimulated with a CD3-directed mAb (OKT3), were co-cultured with HFF (2×10⁴/well) in the absence or presence of HCMV (MOI 5). As control UV-inactivated HCMV (uvCMV) was used. (A) After three days IDO activity was determined and is presented as mean kynurenine production +/− SEM of 4 independent experiments, each done in triplicates. The OD measured in the negative control (unstimulated HFF) is subtracted in all groups. (B) HFF/PBMC co-cultures were set up as described above. After three days cultures were infected with <i>S. aureus</i> (10–100 cfu/ml) and bacterial growth was determined photometrically 24 hours later. As a control L-tryptophan (100 µg/ml) was added at the time point of bacterial infection. Data are given as mean OD_{(620 nm)} +/− SEM of three experiments, each done in triplicates. Significant differences (p<0.05) as compared to the positive control are marked by asterisks.</p

HCMV infection abrogates the immunosuppressive effect of human fibroblasts

<p>. (A) PBMC (2×10⁵/well) were activated with a CD3-directed mAb (OKT3) and cultured in the presence or absence of HFF which were infected with HCMV (MOI 5) or not. After three days T cell proliferation was assessed using [³H]-thymidin. Data are given as mean cpm +/− SD of a representative experiment performed in triplicates. As a control 1- MT was used. (B) Different amounts of HFF, either HCMV infected (MOI 5) or not, were co-cultured with OKT3-activated PBMC. Thereafter T cell proliferation was determined as described above. Data are given as % of positive control without HFF. Each dot represents a single experiment (n = 7), each performed in triplicates. A significant inhibition of T cell proliferation by HFF is marked with asterisks.</p