Ralf Schubert

Molecular and secretory profiles of human mesenchymal stromal cells and their abilities to maintain primitive hematopoietic progenitors.

Mesenchymal stromal cells (MSC) provide a supportive cellular microenvironment and are able to maintain the self‐renewal capacity of hematopoietic progenitor cells (HPC). Isolation procedures for MSC vary extensively, and this may influence their biologic properties. In this study, we have compared human MSC isolated from bone marrow (BM) using two culture conditions, from cord blood (CB), and from adipose tissue (AT). The ability to maintain long‐term culture‐initiating cell frequency and a primitive CD34+CD38− immunophenotype was significantly higher for MSC derived from BM and CB compared with those from AT. These results were in line with a significantly higher adhesion of HPC to MSC from BM and CB versus MSC from AT. We have compared the cytokine production of MSC by cytokine antibody arrays, enzyme‐linked immunosorbent assay, and a cytometric bead array. There were reproducible differences in the chemokine secretion profiles of various MSC preparations, but there was no clear concordance with differences in their potential to maintain primitive function of HPC. Global gene expression profiles of MSC preparations were analyzed and showed that adhesion proteins including cadherin‐11, N‐cadherin, vascular cell adhesion molecule 1, neural cell adhesion molecule 1, and integrins were highly expressed in MSC preparations derived from BM and CB. Thus, MSC from BM and CB are superior to MSC from AT for maintenance of primitive HPC. The latter property is associated with specific molecular profiles indicating the significance of cell‐cell junctions but not with secretory profiles.

Elevated Oxidative Stress in Patients with Ataxia Telangiectasia

Ataxia telangiectasia (AT) is a pleiotropic genetic disorder characterized by progressive neurodegeneration, especially of cerebellar Purkinje cells, immunodeficiency, increased incidence of cancer, and premature aging. The disease is caused by functional inactivation of the ATM (AT-mutated) gene product, which is thought to act as a sensor of reactive oxygen species and oxidative damage of cellular macromolecules and DNA. The compound phenotype of AT might thus be linked to a continuous state of oxidative stress leading to an increase of programmed cell death (apoptosis). To assess this hypothesis, we analyzed lipid peroxidation products and the oxidative stress associated DNA base damage 8-hydroxy-2-deoxyguanosine in patients with AT. Oxidative damage to lipids and DNA was found to be markedly increased in AT patients. These results indicate that ATM might play an important role in the maintenance of cell homeostasis in response to oxidative damage. In this context, a better control of levels of reactive oxygen species could be a rational foundation of therapeutic intervention to help alleviate some of the symptoms associated with AT.

Selective inhibition of tumor growth by clonal NK cells expressing an ErbB2/HER2-specific chimeric antigen receptor.

Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy.

Efficacy of probiotic Lactobacillus GG on allergic sensitization and asthma in infants at risk

Background Probiotics are perceived to exert beneficial effects in the prevention and treatment of allergic diseases.

Sputum biomarker profiles in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) and association between pulmonary function.

Lung diseases like cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are associated with chronic airway inflammation. The aim of our study was to compare a complex biomarker profile in order to characterize specific inflammatory patterns in sputum of patients with CF and COPD. Induced sputum samples of 19 CF-, 26 COPD patients and 21 healthy controls were analyzed for concentrations of IL-1beta, IL-2, IL-6, IL-8, IL-13, IP-10, MCP-1, IFN-gamma and TNF-alpha using the new cytometric bead array (CBA) technology. Significant differences in airway biomarker profiles of CF and COPD were detected. Patients with CF showed a significant increase in IL-1beta, IL-6, IL-8, IL-13, TNF-alpha, IFN-gamma and MCP-1. COPD patients showed an increase in IL-6, IL-8, IL-13 and MCP-1 compared to healthy controls. CF and COPD compared to each other exhibited differences in IL-1beta, IL-2, IL-8, TNF-alpha, IFN-gamma and MCP-1 levels. Significant correlations between the parameters of lung function and sputum biomarker levels were found. Analyzing induced sputum allows characterization of specific airway biomarker profiles in CF and COPD and can be related to the clinical status of the patient. CBA of induced sputum seems to be a pivotal tool to characterize pulmonary inflammation.

Human adipose-derived mesenchymal stem cells in vitro: evaluation of an optimal expansion medium preserving stemness

BACKGROUND AIMS The potential of cultured adipose-derived stem cells (ASC) in regenerative medicine and new cell therapeutic concepts has been shown recently by many investigations. However, while the method of isolation of ASC from liposuction aspirates depending on plastic adhesion is well established, a standard expansion medium optimally maintaining the undifferentiated state has not been described. METHODS We cultured ASC in five commonly used culture media (two laboratory-made media and three commercially available media) and compared them with a standard medium. We analyzed the effects on cell morphology, proliferation, hepatocyte growth factor (HGF) expression, stem cell marker profile and differentiation potential. Proliferation was measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a fluorescent assay. Release of HGF was assessed by an immunoassay. Expression of characteristic stem cell-related transcription factors and markers was evaluated by quantitative polymerase chain reaction (qPCR) (Nanog, Sox-2, Rex-1, nestin and Oct-4) and flow cytometry (CD44, CD73, CD90, CD105 and CD166), and differentiation was shown by adipogenic medium. RESULTS The morphology and expansion of ASC were significantly affected by the media used, whereas none of the media influenced the ASC potential to differentiate into adipocytes. Furthermore, two of the media induced an increase in expression of transcription factors, an increased secretion of HGF and a decrease in CD105 expression. CONCLUSIONS Culture of ASC in one of these two media before using the cells in cell therapeutic approaches may have a benefit on their regenerative potential.

Comparison of cytokine levels from undiluted vitreous of untreated patients with retinal vein occlusion

Purpose:  To compare cytokines in undiluted vitreous of treatment‐naïve patients with macular oedema without vitreomacular traction secondary to branch (BRVO), central (CRVO) and hemi‐central (H‐CRVO) retinal vein occlusion.

Comprehensive Phenotypic Characterization of Human Adipose-Derived Stromal/Stem Cells and Their Subsets by a High Throughput Technology

The characterization of adipose-derived stromal/stem cells (ASCs) remains difficult due to the lack of a definitive and unique cellular marker. Therefore, a combination of markers is necessary to identify the cells. No comprehensive analysis of the immunophenotype of expanded plastic adherent ASCs has been published. Therefore, the aim of this study was to characterize the general phenotype of cultured ASCs and to further analyze cellular subsets. ASCs were isolated from lipoaspirates from patients undergoing cosmetic liposuction and cultured in standard cell culture. A comprehensive phenotype characterization was done with the BD Lyoplate™ Human Cell Surface Marker Screening Panel containing 242 antibodies and isotype controls. Cultured ASCs not only showed the characteristic expression profile of mesenchymal stem cells (MSCs), but also revealed donor-specific variability in the expression of 49 other markers. We further detected markers with a scattering in the fluorescence intensity, indicating subpopulations with different expression profiles. Therefore, a multi-color flow cytometric analysis was done after staining the cells with direct-labeled antibodies against CD73, CD90, CD105, and either CD34, CD140b, CD200, CD201, or CD36 to verify the selected subpopulations of ASCs. We detected no CD34-CD36 double-positive population, but CD34(+)-CD36(-) and CD34(-)CD36(+) subpopulations, both of which are positive for the 3 main MSC markers, CD73, CD90, and CD105. All other detected subpopulations also co-expressed the 3 main MSC markers, and therefore fulfill the minimal phenotypic criteria for the definition of cultured MSCs. Our study demonstrates the first comprehensive phenotypic characterization of ASCs and clearly highlights donor-specific variability in ASC preparations.

Effect of n–3 Polyunsaturated Fatty Acids in Asthma after Low-Dose Allergen Challenge

Background: We investigated the anti-inflammatory potential of n–3 polyunsaturated fatty acids (PUFA) on specific bronchial inflammation. Allergic asthmatics were challenged using a low-dose allergen provocation model. Methods: Our parallel double-blinded study randomly assigned 23 house dust mite-allergic asthmatics (aged 22–29 years; 13 females, 10 males) to dietary supplementation with either an n–3 PUFA-enriched fat blend (0.69 g/day) or placebo for 5 weeks. After 3 weeks, the patients were challenged daily with low doses of mite allergen for 2 weeks. Primary outcome parameters were effects on lung function (forced expiratory volume in 1 s, FEV1) and exhaled nitric oxide (eNO) as a marker of bronchial inflammation. Results: Even before the bronchial challenge, eNO was significantly lower in the n–3 PUFA group (p = 0.014). Levels of eNO increased during allergen exposure in both groups, but differences in means were significantly lower in the n–3 PUFA group (p = 0.022). During the low-dose allergen challenge, there were no differences between the groups with regard to symptoms, FEV1 or the allergen dose required to induce deterioration of lung function (PD20). Numbers of sputum eosinophils did not differ significantly, while serum eosinophils (10.1 ± 0.1.84 vs. 5.79 ± 0.69%) as well as changes in eosinophilic cationic protein (20.5 ± 9.93 vs. –1.68 ± 4.36 ng/ml) and in vitro cysteinyl leukotriene release (2,889 ± 872 vs. 1,120 ± 173 ng/ml) were significantly lower in the n–3 PUFA group (p < 0.05 each). Conclusion: Our results provide evidence that dietary supplementation with n–3 PUFA is able to reduce bronchial inflammation even after low-dose allergen challenge.

Tolerance induction after specific immunotherapy with pollen allergoids adjuvanted by monophosphoryl lipid A in children.

Specific immunotherapy (SIT) is a well‐established and clinically effective treatment for allergic diseases. A pollen allergoid formulated with the T helper type 1 (Th1)‐inducing adjuvant monophosphoryl lipid A (MPL) facilitates short‐term SIT. Little is known about mechanisms of tolerance induction in this setting. In a prospective study, 34 patients allergic to grass pollen (25 male, nine female, median age 10·2 years) received a total of 44 SIT courses (20 in the first, 24 in the second) with MPL‐adjuvanted pollen allergoids. Immunogenicity was measured by levels of specific immunoglobulin G (IgGgrass) and IgG4grass by antibody blocking properties on basophil activation, and by induction of CD4+, CD25+ and forkhead box P3 (FoxP3+) regulatory T cells (Treg). Specific IgG and IgG4 levels increased only slightly in the first year of SIT. In the second year these changes reached significance (P < 0·0001). In keeping with these findings, we were able to show an increase of Treg cells and a decreased release of leukotrienes after the second year of treatment. In the first year of treatment we found little evidence for immunological changes. A significant antibody induction was seen only after the second course of SIT. Short‐course immunotherapy with pollen allergoids formulated with the Th1‐inducing adjuvant MPL needs at least two courses to establish tolerance.