Christine Brostjan

Dendritic Cell-Based Vaccination in Solid Cancer

PURPOSE Dendritic cell (DC)-based immunotherapy is rapidly emerging as a viable tool in cancer treatment. This approach has been used mostly in patients in the presence of defined tumor antigens such as melanoma. In this study, cancer patients with advanced disease that lacks defined tumor antigens were vaccinated with tumor lysate-pulsed DCs. PATIENTS AND METHODS Twenty patients (pancreatic, hepatocellular, cholangiocellular, and medullary thyroid carcinoma) with stage IV disease were enrolled in the study. In 3-week intervals, freshly isolated autologous CD14 magnetic bead-selected monocytes were cultured in granulocyte-macrophage colony-stimulating factor and interleukin-4 to obtain immature DCs. These cells were pulsed with autologous tumor lysate and matured with tumor necrosis factor alpha. Mature DCs were applied into a groin lymph node, under ultrasound guidance. Adjuvant interleukin-2 (20,000 U/kg) was given subcutaneously daily, for 12 days, after each vaccination. Toxicity, tumor marker profile, immune response, and clinical response were determined. RESULTS Vaccination was well tolerated. No physical signs of autoimmunity were detected. DC vaccination induced delayed-type hypersensitivity reactivity in 18 patients. Tumor marker responses were observed in eight patients. In addition, in three patients the generation of interferon gamma-positive T cells was induced during the vaccination. Objective changes in measurable lesions or tumor markers were evident in seven of 20 assessed patients. None of the patients was found to meet the criteria for partial or complete responses. CONCLUSION These data indicate that vaccination with autologous tumor-pulsed DCs generated from peripheral blood is safe and can induce tumor-specific cellular cytotoxicity. Clinical responses are achievable, even in patients with advanced disease.

Catecholamines up-regulate lipopolysaccharide-induced IL-6 production in human microvascular endothelial cells

The catecholamine‐mediated modulation of the cytokine network has primarily been demonstrated for leukocytes. Whereas catecholamines decrease the LPS‐induced production of IL‐6 by leukocytes, serum levels of IL‐6 are dramatically increased by the catecholamine epinephrine in animal endotoxemia models. We now demonstrate that epi‐nephrine as well as norepinephrine can induce IL‐6 in an endothelial cell line (HMEC‐1). Furthermore, these catecholamines could even potentiate the LPS‐induced IL‐6 protein production. The synergistic effect of cat‐echolamines and LPS could be reproduced in primary human skin microvascular endothelial cells. The cate‐cholamine‐induced IL‐6 stimulation is based on increased IL‐6 mRNA levels. RNA stability assays revealed that this regulation is not a result of enhanced RNA stability and therefore is most likely due to an increased transcription. Treatment with cycloheximide indicated that new protein synthesis is not necessary for this transcriptional up‐regulation of IL‐6 mRNA. Prein‐cubation with α and β receptor antagonists showed that the effect is mediated by β1‐ and β2‐adrenergic receptors. Thus, endothelial cells might be a possible source of increased IL‐6 production observed in situations such as stress or septic shock, in which catecholamines are elevated due to endogenous production or exogenous application.—Gornikiewicz, A., Sautner, T., Brostjan, C., Schmierer, B., Függer, R., Roth, E., Muhlbacher, F., Bergmann, M. Catecholamines up‐regu‐late lipopolysaccharide‐induced IL‐6 production in human microvascular endothelial cells. FASEB J. 14, 1093–1100 (2000)

Dendritic cell vaccination in medullary thyroid carcinoma.

Purpose: Prognosis and treatment effectiveness for medullary thyroid carcinoma (MTC) are strictly related to tumor stage. Palliative treatment options show no significant benefit. A promising treatment approach for human cancer is based on the vaccination of autologous dendritic cells (DCs). Experimental Design: The objective of this study was to evaluate the effectiveness of DC vaccines in MTC patients. Therefore, we generated autologous tumor lysate-pulsed DCs from 10 patients suffering from advanced MTC for repeated vaccination. Mature DCs were derived from peripheral blood monocytes by using CD14 magnetic bead selection and subsequent culture in the presence of granulocyte macrophage colony-stimulating factor, interleukin 4, and tumor necrosis factor α with or without addition of IFN-γ. DCs were loaded with tumor lysate and further injected into a groin lymph node. Toxicity, tumor marker profile, immune response, and clinical response were determined. Results: Vaccination was well tolerated and induced a positive immunological response in all of the tested patients as evaluated by in vivo delayed-type hypersensitivity reactivity or in vitro intracytoplasmic IFN-γ detection assay. Three patients had a partial response, 1 patient presented a minor response, and 2 patients showed stable disease. The remaining 4 patients had progressive disease. Conclusions: These data provide strong evidence that vaccination with tumor-lysate pulsed DCs results in the induction of a specific immune response in patients suffering from MTC. Objective clinical responses could be observed even for far-advanced disease. Therefore, we suggest that MTC is particularly suited for DC-based immunotherapy.

Evidence for serotonin as a relevant inducer of liver regeneration after liver resection in humans

Liver regeneration (LR) involves a complex interplay of growth factors and antagonists. In this context, platelet‐derived serotonin (5‐HT) has been identified as a critical inducer of LR in mice. Clinical evidence for a role of 5‐HT in LR in humans is lacking. Accordingly, serum and plasma 5‐HT was monitored perioperatively in 60 patients undergoing liver resection, of which 35 served as exploration and 25 as validation sets. Intraplatelet (IP) levels of 5‐HT were calculated by subtraction of plasma 5‐HT from serum values. Serum markers of liver function were used to evaluate LR and liver dysfunction (LD). In the exploration setting, IP 5‐HT levels significantly decreased after liver resection (P < 0.001) and gradually recovered during the first week. IP 5‐HT measured before surgery specifically predicted LD in the subsequent 7 days (area under the curve: 0.721; P = 0.029). Patients suffering from postoperative LD and morbidity were found to have reduced IP 5‐HT levels during the entire perioperative period. Furthermore, we validated that reduced preoperative IP 5‐HT (<73 ng/mL) was associated with an increased incidence of postoperative LD and morbidity (P =0.045 and P = 0.021) and were able to demonstrate that IP 5‐HT levels were an independent predictor of poor clinical outcome. Conclusions: These findings provide evidence that IP 5‐HT correlates with LR in humans: Patients with low IP 5‐HT before liver resection suffered from delayed hepatic regeneration. Therefore, IP 5‐HT levels may prove a helpful clinical marker to predict postoperative LD and clinical outcome before hepatic resection and initiate suitable interventions. (Hepatology 2014;60:257‐266)

Thrombospondin-1: a unique marker to identify in vitro platelet activation when monitoring in vivo processes.

Summary.  Background:  Measuring platelet activation in patients has become a potent method to investigate pathophysiological processes. However, the commonly applied markers are sensitive to detrimental influences by in vitro platelet activation during blood analysis.

Differential expression of inhibitory and activating CD94/NKG2 receptors on NK cell clones

Natural killer cells are known to express a variety of surface receptors involved in HLA class I monitoring. It is thus of interest to investigate the clonal distribution and relative expression levels of activating versus inhibitory NK receptors. We have developed a quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay designed to determine specific and absolute mRNA levels for NKG2-A/B, -C, -E, -F, -H and NKG2-D. When analyzing NK cell clones derived from a single donor we found differential expression of inhibitory (NKG2-A/B) versus triggering (NKG2-C and potentially -E, -F, -H) NK receptor chains. The generation of the splice variants NKG2-E and -H seemed to occur at a constant ratio. We further compared NKG2 transcript levels to surface receptor expression as monitored by flow cytometric analysis and to NK cell cytotoxicity as detected by reverse ADCC: a clear correlation was observed. Thus, the data obtained reveal a substantial variability in the NKG2 repertoire among NK cell subpopulations, which is likely to affect the sensitivity and reactivity towards the ligand HLA-E.

Linkage of the NKG2 and CD94 receptor genes to D12S77 in the human natural killer gene complex

Abstract The human natural killer (NK) gene complex is located on the short arm of chromosome 12 and contains a number of genes encoding C-type lectin receptors important for natural killer cell function. Among these are CD94 and the five NKG2 genes. The CD94 protein associates with different NKG2 isoforms to heterodimeric receptors which function to inhibit or trigger cytotoxicity of NK cells depending on the NKG2 isoform. We selected two yeast artificial chromosome clones comprising approximately 1.5 Mb of the NK gene complex and established a contig of underlying P1-derived artificial chromosome clones containing all NKG2 and the CD94 genes. A detailed analysis shows that all six genes are found within a region of 100 to 200 kilobases proximal of the marker D12S77. The gene order established is D12S77 – CD94 – NKG2D – NKG2F – NKG2E – NKG2C – NKG2A. The NKG2 genes are of identical transcriptional orientation, whereas the CD94 gene is placed in opposite orientation. The tight genomic linkage of these genes and the identical orientation of the NKG2 genes suggest coordinate regulation of expression during the differentiation of natural killer cells.

The centromeric part of the human natural killer (NK) receptor complex: lectin‐like receptor genes expressed in NK, dendritic and endothelial cells

Summary: The human natural killer (NK) receptor complex encompasses a region of about 2 Mb on the short arm of chromosome 12. It contains at least 18 lectin‐like receptor genes, of which some are expressed in NK and NK/T cells and function as NK receptors. Close to the CD94 and NKG2 NK receptor genes in the centromeric part, a novel family of genes, expressed in myeloid, dendritic and/or endothelial cells, recently became evident. These genes encode a receptor for oxidized low density lipoprotein in endothelial cells and three other receptors potentially serving regulatory functions in dendritic cells. Although the overall structure of the human NK receptor complex is similar to the syntenic rodent regions, the centromeric part lacks the cluster of Ly49 genes. This supports the notion that recognition of MHC class Ia molecules has evolved separately in rodents and humans in the lectin‐like Ly49 and the killer immunoglobulin‐like receptors, respectively. In the telomeric part, other lectin‐like genes expressed in different hematopoietic lineages are found. The receptors of the NK receptor complex apparently serve important functions in several leukocytes and in endothelial cells, and the exact role of these receptors, their ligands, and their distinct and co‐ordinate regulation in different cell lineages warrants further investigation.( )

The differential activity of interferon-α subtypes is consistent among distinct target genes and cell types

IFN-α proteins have been described to originate from 14 individual genes and allelic variants. However, the exceptional diversity of IFN-α and its functional impact are still poorly understood. To characterize the biological activity of IFN-α subtypes in relation to the cellular background, we investigated the effect of IFN-α treatment in primary fibroblasts and endothelial cells of vascular or lymphatic origin. The cellular response was evaluated for 13 distinct IFN-α proteins with respect to transcript regulation of the IFN-stimulated genes (ISGs) IFIT1, ISG15, CXCL10, CXCL11 and CCL8. The IFN-α proteins displayed a remarkably consistent potency in gene induction irrespective of target gene and cellular background which led to the classification of IFN-α subtypes with low (IFN-α1), intermediate (IFN-α2a, -4a, -4b, -5, -16, -21) and high (IFN-α2b, -6, -7, -8, -10, -14) activity. The differential potency of IFN-α classes was confirmed at the ISG protein level and the functional protection of cells against influenza virus infection. Differences in IFN activity were only observed at subsaturating levels of IFN-α proteins and did not affect the time course of ISG regulation. Cell-type specific responses were apparent for distinct target genes independent of IFN-α subtype and were based on different levels of basal versus inducible gene expression. While fibroblasts presented with a high constitutive level of IFIT1, the expression in endothelial cells was strongly induced by IFN-α. In contrast, CXCL10 and CXCL11 transcript levels were generally higher in endothelial cells despite a pronounced induction by IFN-α in fibroblasts. In summary, the divergent potency of IFN-α proteins and the cell-type specific regulation of individual IFN target genes may allow for the fine tuning of cellular responses to pathogen defense.

Neoadjuvant Treatment of Colorectal Cancer with Bevacizumab: The Perioperative Angiogenic Balance Is Sensitive to Systemic Thrombospondin-1 Levels

Purpose: Colorectal cancer patients receiving neoadjuvant treatment with bevacizumab, a monoclonal antibody neutralizing vascular endothelial growth factor (VEGF), may suffer from wound healing complications after surgery as the antibody persists in patient blood. We characterized the systemic angiogenic balance in the perioperative period to evaluate its effect on physiologic angiogenesis. Experimental Design: Nineteen patients receiving combination chemotherapy and bevacizumab for six neoadjuvant cycles were compared with 14 patients receiving chemotherapy without bevacizumab. Plasma from perioperative days −1, +1, +7, and +21 was analyzed for VEGF, thrombospondin-1 (TSP-1), and PD-ECGF concentrations. The angiogenic capacity was further tested in an in vitro assay of endothelial cell proliferation and migration. Results: On day +1, the onset of wound healing was reflected in a change of balance, i.e., an increase of proangiogenic factors VEGF and platelet-derived endothelial cell growth factor compared with low TSP-1 inhibitor levels in both treatment groups. Patients with bevacizumab therapy showed significantly higher blood levels of total VEGF throughout the evaluation period. However, most VEGF molecules were inactive, i.e., complexed with antibody. Nevertheless, the capacity to stimulate endothelial growth was higher for these plasma samples and was reflected in low TSP-1 levels and an altered TSP-1 sensitivity. When purified TSP-1 protein was added, plasma samples of the bevacizumab but not the chemotherapy group showed reduced endothelial growth. Conclusions: Feedback mechanisms of bevacizumab therapy are not restricted to VEGF expression but seem to involve additional factors, such as TSP-1, which influences the systemic angiogenic balance and permits endothelial growth.