Stefan Hacker

Bevacizumab protects against sinusoidal obstruction syndrome and does not increase response rate in neoadjuvant XELOX/FOLFOX therapy of colorectal cancer liver metastases

AIM In patients suffering from colorectal cancer liver metastases, 5-fluorouracil-based chemotherapy plus oxaliplatin ensures superior response rates at the cost of hepatic injury. Knowledge about the consequences of bevacizumab on chemotherapy-induced hepatic injury and tumor response is limited. METHODS Resected liver specimens from patients of two prospective, non-randomized trials (5-fluorouracil/oxaliplatin+/-bevacizumab) were analyzed retrospectively. Hepatotoxicity to the non-tumor bearing liver was evaluated for sinusoidal obstruction syndrome, hepatic steatosis and fibrosis. Tumor response under chemotherapy was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST). RESULTS Bevacizumab decreased the severity of the sinusoidal obstruction syndrome. Bevacizumab had no impact on hepatic steatosis and fibrosis. The addition of bevacizumab to chemotherapy had no effect on tumor response compared to combination chemotherapy alone. CONCLUSIONS This analysis shows that bevacizumab protects against the sinusoidal obstruction syndrome and thus provides the histological explanation of the safe use of bevacizumab prior to liver resection. Furthermore, we show that bevacizumab does not improve tumor response according to RECIST.

T cell senescence and contraction of T cell repertoire diversity in patients with chronic obstructive pulmonary disease

Pathogenetic mechanisms leading to chronic obstructive pulmonary disease (COPD) remain poorly understood. Because clonogenic T cells (CD4+CD28null) were shown to be increased in autoimmune diseases we hypothesized that CD4+CD28null T cells play a role in COPD. Here we describe that enhanced presence of CD4+CD28null cells is associated with impaired lung function. Sixty‐four patients and controls were included. T cell phenotype was analysed using flow cytometry. Enzyme‐linked immunosorbent assays were utilized to determine cytokines. Statistical evaluations were performed using non‐parametric group comparisons and correlations. A logistic regression model was used to determine predictive values of CD4+CD28null in the diagnosis of COPD. Populations of CD4+ T cells lacking surface co‐stimulatory CD28 were enlarged significantly in evaluated patients when compared with controls. Natural killer (NK)‐like T cell receptors (CD94, 158) and intracellular perforin, granzyme B were increased in CD4+CD28null cells. Cytokine production after triggering of peripheral blood mononuclear cells (PBMCs) was elevated in patients at early disease stages. Receiver operating characteristic curve plotting revealed that presence of CD4+CD28null T cells has a diagnostic value. These CD4+CD28null T cells show increased expression of NK‐like T cell receptors (CD94, 158) and intracellular perforin and granzyme B. Furthermore, triggering of PBMCs obtained from patients with mild COPD led to increased interferon‐γ and tumour necrosis factor‐α production in vitro compared with controls. Our finding of increased CD4+CD28null T cells in COPD indicates that chronic antigen exposure, e.g. through contents of smoke, leads to loss of CD28 and up‐regulation of NK cell receptors expression on T cells in susceptible patients.

Irradiated cultured apoptotic peripheral blood mononuclear cells regenerate infarcted myocardium

Background  Acute myocardial infarction (AMI) is followed by post AMI cardiac remodelling, often leading to congestive heart failure. Homing of c‐kit+ endothelial progenitor cells (EPC) has been thought to be the optimal source for regenerating infarcted myocardium.

Discrimination of clinical stages in non-small cell lung cancer patients by serum HSP27 and HSP70: a multi-institutional case-control study.

INTRODUCTION Lung cancer represents a major healthcare problem. Accordingly, there is an urgent need to identify serum biomarkers for early diagnosis of lung pathology. We have recently described that patients with manifest COPD evidence elevated levels of heat shock proteins (HSPs). Based on these data, we speculated whether HSPs are also increased in patients with diagnosed lung cancer. METHODS Serum levels of HSP27, phospho-HSP27 (pHSP27) and HSP70 in patients with non-small cell lung cancer (NSCLC) diagnosed at an early (stages I-II, n=37) or advanced (stages IIIA-IV, n=72) stage were determined by using ELISA. Healthy smokers (n=24), healthy never-smoker volunteers (n=33) and COPD patients (n=34) according to GOLD classification served as control population. RESULTS Serum levels of HSP27 were elevated in patients with NSCLC diagnosed at an early or advanced stage when compared with both healthy control groups (P<0.005 and P<0.0001 respectively). Statistically significant differences were furthermore found between the groups of patients with early vs. advanced stage NSCLC (P=0.0021). Serum levels of HSP70 were also significantly elevated in patients with NSCLC diagnosed at an early or at an advanced stage when compared with either healthy control groups (P=0.0028 and P<0.0001 respectively). In univariate logistic regression models including healthy subjects and patients with NSCLC, HSP70 had an area under the curve (AUC) of 0.779 (P<0.0001) and HSP27 showed an AUC of 0.870 (P<0.0001). CONCLUSION Our data suggest that serum HSP27 levels might serve as a possible tool to discriminate between early and advanced stages NSCLC.

Alpha-Gal specific IgG immune response after implantation of bioprostheses.

BACKGROUND We have previously shown that the alpha-Gal (Galalpha1.3-Galbeta1-4GlcNAc-R) epitope is a relevant xenoantigen present on bioprostheses utilized in cardiac surgery and elicits an alpha-Gal specific IgM immune response. We sought to investigate whether that immune response continues after valve implantation. MATERIALS AND METHODS We collected plasma samples from patients who underwent bioprosthesis implantation (n = 19) or mechanical valve replacement (n = 8), respectively, prior to, at 10 days and at 3 months after cardiac surgery. ELISA was utilized to quantify alpha-Gal specific IgG and IgG subclasses. 3 bioprosthetic tissue samples were obtained from patients who had to undergo re-operation within 1 week (n = 1) or at 12-15 months (n = 2) after the initial operation. We utilized confocal laser scanning microscopy (CLSM) to detect the presence of alpha-Gal epitopes (IB4) and cell nuclei (DAPI). RESULTS alpha-Gal specific IgG was significantly increased 3 months after implantation of bioprostheses compared to preoperative values (p < 0.001) and was significantly higher than alpha-Gal specific IgG levels of the control group (p < 0.05). IgG3 was the major subclass directed against alpha-Gal (p < 0.05, pre- vs. postoperative values). In CLSM analysis we demonstrated that bioprostheses explanted 1 week after implantation contained IB4/DAPI positive cells within the collagen matrix. In contrast, in patients who underwent reoperation after 12 months, porcine tissue showed a complete lack of IB4/DAPI. CONCLUSION Our results indicate that the implantation of bioprostheses elicits a specific humoral immune response against alpha-Gal bearing cells compared to controls within 3 months after cardiac surgery. The complete absence of IB4/DAPI positive structures 12 months after implantation indicates a specific degradation of alpha-Gal bearing cells through previous exposure to the human blood circuit.

Increased soluble serum markers caspase-cleaved cytokeratin-18, histones, and ST2 indicate apoptotic turnover and chronic immune response in COPD.

Introduction: Chronic obstructive pulmonary disease (COPD) is a worldwide burden and a major cause of death. The disease is accompanied by chronic inflammation and increased cellular turnover that is partly due to an overwhelming induction of apoptosis. In this study, we hypothesized that systemic markers of apoptosis are altered in patients with mild‐to‐severe COPD.

Secretome of peripheral blood mononuclear cells enhances wound healing.

Non-healing skin ulcers are often resistant to most common therapies. Treatment with growth factors has been demonstrated to improve closure of chronic wounds. Here we investigate whether lyophilized culture supernatant of freshly isolated peripheral blood mononuclear cells (PBMC) is able to enhance wound healing. PBMC from healthy human individuals were prepared and cultured for 24 hours. Supernatants were collected, dialyzed and lyophilized (SECPBMC). Six mm punch biopsy wounds were set on the backs of C57BL/6J-mice and SECPBMC containing emulsion or controls were applied daily for three days. Morphology and neo-angiogenesis were analyzed by H&E-staining and CD31 immuno-staining, respectively. In vitro effects on diverse skin cells were investigated by migration assays, cell cycle analysis, and tube formation assay. Signaling pathways were analyzed by Western blot analysis. Application of SECPBMC on 6 mm punch biopsy wounds significantly enhanced wound closure. H&E staining of the wounds after 6 days revealed that wound healing was more advanced after application of SECPBMC containing emulsion. Furthermore, there was a massive increase in CD31 positive cells, indicating enhanced neo-angiogenesis. In primary human fibroblasts (FB) and keratinocytes (KC) migration but not proliferation was induced. In endothelial cells (EC) SECPBMC induced proliferation and tube-formation in a matrigel-assay. In addition, SECPBMC treatment of skin cells led to the induction of multiple signaling pathways involved in cell migration, proliferation and survival. In summary, we could show that emulsions containing the secretome of PBMC derived from healthy individuals accelerates wound healing in a mouse model and induce wound healing associated mechanisms in human primary skin cells. The formulation and use of such emulsions might therefore represent a possible novel option for the treatment of non-healing skin ulcers.

HSP27 and HSP70 serum and urine levels in patients suffering from chronic kidney disease.

BACKGROUND Chronic kidney disease (CKD) is a condition associated with inflammation and high levels of uremic toxins and reactive oxygen species. As a counterregulation to systemic stress heat shock proteins (HSP) are increased expressed to minimize cell death and preserve cell integrity by inhibiting apoptotic pathways. The aim of this study was to determine HSP27 and HSP70 concentrations in sera and urine of patients suffering from CKD. METHODS Concentrations of HSP27 and HSP70 in urine and serum were determined in 119 patients with CKD stages 1 to 5 and 23 healthy volunteers by using ELISA technique. RESULTS HSP27 serum levels were significantly elevated in patients suffering from CKD stages 3 to 5 as well as fractional HSP27 excretion in stages 2-5 versus healthy controls. Absolute HSP70 urinary values were significantly elevated in stages 4 and 5 and fractional HSP70 excretion was increased in stage 5 compared to controls. Moreover, ROC curve analysis showed the potential of urine and especially serum HSP levels to identify various stages of CKD. CONCLUSION We provide evidence for elevated HSP27 concentrations in serum and urine and increased HSP70 excretion levels in patients suffering from CKD. Moreover, our results show that HSP levels might offer potential to examine the stages of CKD as well as the disease course which could further promote individually adjusted treatment planning.

Age-related changes in expression and function of Toll-like receptors in human skin

Toll-like receptors (TLRs) initiate innate immune responses and direct subsequent adaptive immunity. They play a major role in cutaneous host defense against micro-organisms and in the pathophysiology of several inflammatory skin diseases. To understand the role of TLRs in the acquisition of immunological competence, we conducted a comprehensive study to evaluate TLR expression and function in the developing human skin before and after birth and compared it with adults. We found that prenatal skin already expresses the same spectrum of TLRs as adult skin. Strikingly, many TLRs were significantly higher expressed in prenatal (TLRs 1-5) and infant and child (TLRs 1 and 3) skin than in adult skin. Surprisingly, neither dendritic cell precursors in prenatal skin nor epidermal Langerhans cells and dermal dendritic cells in adult skin expressed TLRs 3 and 6, whereas the staining pattern and intensity of both TLRs in fetal basal keratinocytes was almost comparable to those of adults. Stimulation of primary human keratinocytes from fetal, neonatal and adult donors with selected TLR agonists revealed that the synthetic TLR3 ligand poly (I:C) specifically, mimicking viral double-stranded RNA, induced a significantly enhanced secretion of CXCL8/IL8, CXCL10/IP-10 and TNFα in fetal and neonatal keratinocytes compared with adult keratinocytes. This study demonstrates quantitative age-specific modifications in TLR expression and innate skin immune reactivity in response to TLR activation. Thus, antiviral innate immunity already in prenatal skin may contribute to protect the developing human body from viral infections in utero in a scenario where the adaptive immune system is not yet fully functional.

Elevated levels of interleukin-1β-converting enzyme and caspase-cleaved cytokeratin-18 in acute myocardial infarction

Background  Systemic inflammation and apoptosis‐specific immune activation play a major role in acute coronary syndromes (ACS) including acute myocardial infarction (AMI). The role of systemic and coronary obtained inflammatory plasma protein interleukin‐1β precursor (IL‐1βp), IL‐1β‐converting enzyme (ICE) and the apoptosis‐specific caspase‐cleaved cytokeratin‐18 (ccCK‐18) are not known in ACS.