X-linked inhibitor of apoptosis protein represents a promising therapeutic target for relapsed/refractory ALL.

Acute lymphoblastic leukemia (ALL) represents the most frequent malignancy in children, and relapse/refractory (r/r) disease is difficult to treat, both in children and adults. In search for novel treatment options against r/r ALL, we studied inhibitor of apoptosis proteins (IAP) and Smac mimetics (SM). SM-sensitized r/r ALL cells towards conventional chemotherapy, even upon resistance against SM alone. The combination of SM and chemotherapy-induced cell death via caspases and PARP, but independent from cIAP-1/2, RIPK1, TNFα or NF-κB. Instead, XIAP was identified to mediate SM effects. Molecular manipulation of XIAP in vivo using microRNA-30 flanked shRNA expression in cell lines and patient-derived xenograft (PDX) models of r/r ALL mimicked SM effects and intermediate XIAP knockdown-sensitized r/r ALL cells towards chemotherapy-induced apoptosis. Interestingly, upon strong XIAP knockdown, PDX r/r ALL cells were outcompeted in vivo, even in the absence of chemotherapy. Our results indicate a yet unknown essential function of XIAP in r/r ALL and reveal XIAP as a promising therapeutic target for r/r ALL.

A Novel Method to Achieve Precision and Reproducibility in Exposure Parameters for Low-Frequency Pulsed Magnetic Fields in Human Cell Cultures.

The effects of extremely low-frequency electromagnetic field (ELF-MF) exposure on living systems have been widely studied at the fundamental level and also claimed as beneficial for the treatment of diseases for over 50 years. However, the underlying mechanisms and cellular targets of ELF-MF exposure remain poorly understood and the field has been plagued with controversy stemming from an endemic lack of reproducibility of published findings. To address this problem, we here demonstrate a technically simple and reproducible EMF exposure protocol to achieve a standardized experimental approach which can be readily adopted in any lab. As an assay system, we chose a commercially available inflammatory model human cell line; its response to magnetic fields involves changes in gene expression which can be monitored by a simple colorimetric reporter gene assay. The cells were seeded and cultured in microplates and inserted into a custom-built, semi-automated incubation and exposure system which accurately controls the incubation (temperature, humidity, CO2) and magnetic-field exposure conditions. A specific alternating magnetic field (<1.0% spatial variance) including far-field reduction provided defined exposure conditions at the position of each well of the microplate. To avoid artifacts, all environmental and magnetic-field exposure parameters were logged in real time throughout the duration of the experiment. Under these extensively controlled conditions, the effect of the magnetic field on the cell cultures as assayed by the standardized operating procedure was highly reproducible between experiments. As we could fully define the characteristics (frequency, intensity, duration) of the pulsed magnetic field signals at the position of the sample well, we were, for the first time, able to accurately determine the effect of changing single ELF-MF parameters such as signal shape, frequency, intensity and duty cycle on the biological response. One signal in particular (10 Hz, 50% duty cycle, rectangular, bipolar, 39.6µT) provided a significant reduction in cytokine reporter gene expression by 37% in our model cell culture line. In sum, the accuracy, environmental control and data-logging capacity of the semi-automated exposure system should greatly facilitate research into fundamental cellular response mechanisms and achieve the consistency necessary to bring ELF-MF/PEMF research results into the scientific mainstream.

Pulsed Electromagnetic Field Therapy Improves Osseous Consolidation after High Tibial Osteotomy in Elderly Patients-A Randomized, Placebo-Controlled, Double-Blind Trial.

Extremely low-frequency pulsed electromagnetic field (ELF-PEMF) therapy is proposed to support bone healing after injuries and surgical procedures, being of special interest for elderly patients. This study aimed at investigating the effect of a specific ELF-PEMF, recently identified to support osteoblast function in vitro, on bone healing after high tibial osteotomy (HTO). Patients who underwent HTO were randomized to ELF-PEMF or placebo treatment, both applied by optically identical external devices 7 min per day for 30 days following surgery. Osseous consolidation was evaluated by post-surgical X-rays (7 and 14 weeks). Serum markers were quantified by ELISA. Data were compared by a two-sided t-test (α = 0.05). Device readouts showed excellent therapy compliance. Baseline parameters, including age, sex, body mass index, wedge height and blood cell count, were comparable between both groups. X-rays revealed faster osseous consolidation for ELF-PEMF compared to placebo treatment, which was significant in patients ≥50 years (∆mean = 0.68%/week; p = 0.003). Findings are supported by post-surgically increased bone-specific alkaline phosphatase serum levels following ELF-PEMF, compared to placebo (∆mean = 2.2 µg/L; p = 0.029) treatment. Adverse device effects were not reported. ELF-PEMF treatment showed a tendency to accelerate osseous consolidation after HTO. This effect was stronger and more significant for patients ≥50 years. This ELF-PEMF treatment might represent a promising adjunct to conventional therapy supporting osseous consolidation in elderly patients.

NEC is likely a NETs dependent process and markers of NETosis are predictive of NEC in mice and humans.

Necrotizing enterocolitis (NEC) is one of the most devastating diseases affecting premature and mature infants. It is hypothesized that NEC is the result of neutrophils' active role in hyperinflammation after bacterial gut colonization, through their nuclear DNA release and formation of neutrophil extracellular traps (NETs) to combat pathogens. The aim of this study was to evaluate the importance of NETs in NEC pathogenesis, as well as to identify and validate markers of NETosis to predict NEC. NEC was induced in mice by gavage feeding of Neocate and lipopolysaccharide, followed by ten minutes of hypoxia (5% O2) q12h for five days, starting on day four postpartum (p.p.). The interrelation of NEC and neutrophils, including NETs, was assessed macroscopically (i.e. NEC score, SYTOX Orange), microscopically (i.e. Chiu score, citrullinated histone H3, neutrophil elastase), and in blood samples (i.e. cell-free DNA (cfDNA), DNase). In order to determine the exact role of NETs in NEC pathogenesis, a protein arginine deiminase (PAD) inhibition model was established (preventing NETs formation in mice) by injecting BB-Cl-amidine once daily, starting on day one p.p. Additionally, human intestinal samples of diagnostically verified NEC were analyzed. In total, 76 mice were analyzed in the experiment. Serum cfDNA correlated positively with NEC manifestation, as measured by macroscopic NEC score (r = 0.53, p = 0.001), and microscopic evaluation with Chiu score (r = 0.56, p < 0.001). Markers of neutrophil activation and NETosis were significantly increased in animals with NEC and in human samples as compared to controls. Further, prevention of NETosis by protein arginine deiminase (PAD) inhibition in mice significantly reduced mortality, tissue damage, and inflammation in mice induced with NEC. Our results suggest that the hyperinflammation observed in NEC is a NETs-dependent process, as NEC severity was significantly reduced in mice incapable of forming NETs (PAD inhibition) and markers for NEC and NETs correlated positively during the time course of NEC induction. Further, serum surrogate markers of NETosis (such as cfDNA and DNase) appear to predict NEC in neonatal mice. As findings of the mouse NEC model correlate positively with human NEC samples immunohistochemically, the hyperinflammation reaction observed in mice could potentially be applied to human NEC pathogenesis.

Vincristine resistance in relapsed neuroblastoma can be efficiently overcome by Smac mimetic LCL161 treatment.

PURPOSE: In spite of good initial therapy response neuroblastomas often spread to distant organs or relapse after periods of remission. Dysregulation of apoptosis, a hallmark of cancer, is often effected by elevated levels of antiapoptotic signals leading to resistance against chemotherapeutic drugs. Inhibitors of apoptosis proteins (IAPs) are crucial cellular apoptosis regulators. Targeting IAPs with Smac mimetics has been demonstrated as a promising strategy for treatment of neuroblastoma and other tumors. METHODS: In paired neuroblastoma cell lines, obtained from the same patient at time of diagnosis (CHLA-15) and postchemotherapy during progressive disease (CHLA-20), expression of crucial IAPs was determined. Furthermore, effects of vincristine on viability, cytotoxicity, apoptosis induction and caspase-3/7 activation were determined. RESULTS: Cellular IAP-1 (cIAP-1) and X-linked IAP (XIAP) expression was increased in cell line CHLA-20. Moreover, biological effects of vincristine were significantly lower in these cells. Treatment of cells with Smac mimetic LCL161 increased the effects of vincristine in CHLA-15 cells and more importantly was able to overcome vincristine resistance in CHLA-20 cells. CONCLUSIONS: These findings demonstrate the potential of Smac mimetics for the development of novel therapeutic approaches for the treatment of relapsed/resistant neuroblastoma.

Sensitization of neuroblastoma for vincristine-induced apoptosis by Smac mimetic LCL161 is attended by G2 cell cycle arrest but is independent of NFκB, RIP1 and TNF-α.

We demonstrated sensitization for chemotherapy by Smac mimetic (SM) LCL161, a potent antagonist of inhibitor of apoptosis proteins (IAP), in neuroblastoma (NB). Vinca alkaloids, particularly vincristine (VCR), displayed the strongest impact on inhibition of proliferation and apoptosis induction in combination with LCL161. The underlying signaling pathways remain elusive, though. LCL161 induces a quick degradation of cellular IAP 1 (cIAP-1). Combination of LCL161 with VCR had only marginal effects on X-linked IAP (XIAP) protein expression. Cell death is accompanied by activation of intrinsic (caspase-9 and MMP) and extrinsic (caspase-8) pathways of apoptosis, repression of migratory potential and cell cycle arrest in G2 phase. LCL161-induced cIAP degradation leads to activation of non-canonical and blockade of canonical NF-κB pathways but not induction of apoptosis. Surprisingly NF-κB and TNF-α signaling is negligible for VCR- and VCR/LCL161-induced apoptosis since chemical inhibition of NF-κB using BAY-7085 and PBS-1086, as well as application of TNF-α blocking antibody Humira (adalimumab) has no relevant effect on cell death. Recently formation of a TNF-α-independent complex (ripoptosome) consisting of RIP1, FADD and caspase-8 following IAP inhibition by SM has been described. However, targeting of RIP1 by Necrostatin was not sufficient to influence apoptosis induced by VCR/LCL161.

Therapeutic targeting of extracellular DNA improves the outcome of intestinal ischemic reperfusion injury in neonatal rats.

Thrombosis and inflammation cooperate in the development of intestinal infarction. Recent studies suggest that extracellular DNA released by damaged cells or neutrophils in form of extracellular traps (NETs) contributes to organ damage in experimental models of ischemia-reperfusion injury. Here we compared the therapeutic effects of targeting fibrin or extracellular DNA in intestinal infarction after midgut volvulus in rats. Following iatrogenic midgut volvulus induction for 3 hours, we treated animals with a combination of tissue plasminogen activator (tPA) and low molecular weight heparin (LMWH) to target fibrin or with DNase1 to degrade extracellular DNA. The therapeutic effects of tPA/LMWH and DNase1 were analyzed after 7 days. We observed that both therapeutic interventions ameliorated tissue injury, apoptosis, and oxidative stress in the intestine. DNase1, but not tPA/LMWH, reduced intestinal neutrophil infiltration and histone-myeloperoxidase-complexes, a surrogate marker of NETs, in circulation. Importantly, tPA/LMWH, but not DNase1, interfered with hemostasis as evidenced by a prolonged tail bleeding time. In conclusion, our data suggest that the therapeutic targeting of fibrin and extracellular DNA improves the outcome of midgut volvulus in rats. DNase1 therapy reduces the inflammatory response including NETs without increasing the risk of bleeding. Thus, targeting of extracellular DNA may provide a safe therapy for patients with intestinal infarction in future.

Degradation of Extracellular DNA by DNase1 Significantly Reduces Testicular Damage After Testicular Torsion in Rats.

OBJECTIVE: To examine the effects of DNase1 treatment on testicular damage after testicular torsion (TT). It has been demonstrated that TT induces thrombus formation and that anticoagulation significantly reduces testicular damage after TT. It was hypothesized that these thrombi are dependent on neutrophil extracellular traps (NETs) and thus NETs disintegration would reduce testicular cell damage. METHODS: A sham operation was performed in 10 rats. Thirty-four rats underwent induction of iatrogenic TT for 3 hours. After de-torsion and randomization, 24 rats received DNase1 or inactivated DNase1. The following parameters were assessed: testicular damage via Cosentino grading; spermatogenesis via Johnsen score; stem cell factor and c-Kit, apoptosis via Bax, Bcl2, Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling assay, and cleaved caspase3 staining; oxidative stress via superoxide dismutase, catalase, glutathione peroxidase, and malondialdehyde; neutrophil recruitment via myeloperoxidase and neutrophil elastase staining; and NET formation via cell-free DNA. RESULTS: Forty-three rats were included in the study. Subjects treated with DNase1 showed significantly less cellular damage, oxidative stress, and apoptosis. Further, DNase1-treated rats demonstrated a significant improvement of spermatogenesis, compared with the controls. CONCLUSION: The results of the study indicate that thrombus formation during TT is quite likely NET associated, and that dissolution of cell-free DNA (including NETs) significantly improves testicular damage in rats. As treatment with DNase1 reduced apoptosis, oxidative stress, and inflammation, without adversely affecting coagulation, it might be a suitable treatment for (neonatal) TT and ought to be evaluated in humans.

Smac mimetic LCL161 supports neuroblastoma chemotherapy in a drug class-dependent manner and synergistically interacts with ALK inhibitor TAE684 in cells with ALK mutation F1174L.

Neuroblastoma is the most common extracranial solid tumor during infancy and childhood.Outcome of high-risk and late-stage disease remains poor despite intensive treatment regimens.Suppressing inhibitor of apoptosis proteins (IAPs) using Smac mimetics (SM) significantly sensitizes neuroblastoma (NB) cells for chemotherapy, however strongly dependent on the cytotoxic drug combined with SM.Therefore, a systematic analysis of the impact of SM in combination with different classes of chemotherapeutics was of crucial importance. Treatment of NB cell lines with SM LCL161 and vinca alkaloids revealed a strong synergistic inhibition of proliferation and significant induction of apoptosis in virtually all established and de novo NB cell lines (n=8).In contrast, combination of anthracyclines or topoisomerase inhibitors with LCL161 showed a synergism for single drugs and/or cell lines only.Furthermore, we could show that insensibility to LCL161-mediated sensitization for chemotherapeutics is associated with aberrant activation of anaplastic lymphoma kinase (ALK) by common mutation F1174L. Inhibition of ALK using TAE684 is able to overcome this resistance in a synergistic fashion, a finding that could be highly relevant for improvement of neuroblastoma therapy.

Modulation of Thrombosis Significantly Reduces Testicular Damage after Testicular Torsion in Rats: Anti-Thrombotic Treatment and Testicular Torsion.

OBJECTIVE: To evaluate the effects of thrombolysis and/or anticoagulation on testicular viability after testicular tortion (TT) was the aim of this study. It has been suggested that alterations of circulation during TT result in thrombus formation that might prevent sufficient perfusion after detorsion. Due to the narrow safety margin of testicular perfusion, even moderate disturbances in blood supply can cause major testicular damage. METHODS: In 112 rats, the right testicle was torsed for 3 or 6 hours. After detorsion and randomization, they received either enoxaparin, alteplase, both, or placebo, according to their subgroup. Thrombus formation was accessed via D-dimers, pDNA, oxidative testicular damage was evaluated via glutathione peroxidase and malondialdehyde, and cellular damage via inhibin B, testosterone, histological analysis (Johnsen score, Cosetino grading), and TUNEL assay. RESULTS: One hundred and twelve rats were included in the study. The treatment with alteplase or enoxaparin showed significantly less testicular damage and significantly improved Sertoli cell function. Enoxaparin significantly reduced oxidative impairment. CONCLUSION: The results of the study indicate that TT induces thrombus formation and demonstrate that modulation of thrombosis significantly ameliorates testicular damage in rats. Hence, this treatment option after TT ought to be evaluated in humans.

Targeting of MYCN by means of DNA vaccination is effective against neuroblastoma in mice.

The MYCN oncogene is a strong genetic marker associated with poor prognosis in neuroblastoma (NB). Therefore, MYCN gene amplification and subsequent overexpression provide a possible target for new treatment approaches in NB. We first identified an inverse correlation of MYCN expression with CD45 mRNA in 101 NB tumor samples. KEGG mapping further revealed that MYCN expression was associated with immune-suppressive pathways characterized by a down-regulation of T cell activation and up-regulation of T cell inhibitory gene transcripts. We then aimed to investigate whether DNA vaccination against MYCN is effective to induce an antigen-specific and T cell-mediated immune response. For this purpose, we generated a MYCN-expressing syngeneic mouse model by MYCN gene transfer to NXS2 cells. MYCN-DNA vaccines were engineered based on the pCMV-F3Ub plasmid backbone to drive ubiquitinated full-length MYCN-cDNA and minigene expression. Vaccines were delivered orally with attenuated S. typhimurium strain SL7207 as a carrier. Immunization with both MYCN-DNA vaccines significantly reduced primary tumor growth of MYCN-expressing NB cells in contrast to negative controls. The immune response was mediated by tumor-infiltrating T cells in vivo, which revealed MYCN-specific and MHC class I-restricted lysis of inducible MYCN-expressing NB target cells in vitro. Finally, these antigen-specific T cells also killed MYCN-negative mammary carcinoma cells pulsed with MYCN peptides in contrast to controls. In summary, we demonstrate proof of concept that MYCN can be targeted by DNA vaccination, which may provide an approach to overcoming MYCN immune-suppressive activities in patients with MYCN-amplified disease.

A chicken embryo model for the study of umbilical and supraumbilical body wall malformations.

UNLABELLED: BACKGROUND/PURPOSE; The embryology of ventral body wall malformations is only partially understood, although their incidence is relatively common. As only few experimental data exist on the development of those defects, the aim of our study was to compare the teratogenic effect of trypan blue (TB) and suramin (SA) in their capability to induce umbilical and supraumbilical abdominal wall malformations in a chicken egg model. MATERIALS AND METHODS: A total of 255 fertilized chicken eggs were incubated at 38 °C and 75% relative humidity. Embryos were treated in ovo on incubation day 2.5 (Hamburger/Hamilton (HH) stage 13). The eggshell was windowed, and solutions of TB or SA were injected into the coelomic cavity at the region of the umbilicus. The window was closed and the embryos reincubated until examination on day 8 (HH 34). RESULTS: A total of 60 embryos survived in each group. The largest number of embryos presented with defects in the umbilical and supraumbilical region (25% in the SA group and 40% in the TB group). A combination of both defects (thoracoabdominoschisis) was seen in 20% of the TB and 8.3% of the SA groups, respectively. Associated anomalies found in both groups were head and eye defects, abnormal pelvic configurations, leg deformities, and mild forms of cloacal exstrophies. CONCLUSIONS: TB and SA have both a high potential to induce umbilical and supraumbilical ventral body wall malformations in chicken embryos. This novel animal model might help to establish a more profound understanding of the developmental steps in ventral body wall formation and the embryology for its malformations.

Taurolidine specifically inhibits growth of neuroblastoma cell lines in vitro.

BACKGROUND: Neuroblastoma is a common pediatric solid tumor with poor outcome for metastatic disease. Thus, novel therapeutic options are of main interest. The anti-neoplastic properties of taurolidine have been demonstrated on a variety of human cancer cells. However, data on neuroblastoma is lacking. Therefore, our aim was to evaluate the effect of taurolidine on growth of neuroblastoma cell lines. MATERIALS AND METHODS: Neuroblastoma SK-N-BE(2)-M17 and SK-N-SH cells and nonmalignant human umbilical vein endothelial cells as controls were incubated with increasing concentrations of taurolidine (100, 250, 500 µM). Cell growth was examined after 12, 24, and 48 hours of exposure. RESULTS: Inhibition of cell growth by taurolidine was seen in both malignant cell lines. When compared with human umbilical vein endothelial cells, the neuroblastoma cell lines were significantly more responsive to taurolidine. CONCLUSIONS: The observed negative impact on cell growth, highly distinctive in SK-N-BE(2)-M17 and SK-N-SH, implies a taurolidine-specific mode of action that appears dependent on differences on cellular and molecular level. Further investigations are warranted to evaluate its mechanism and probable clinical use.

Gut inflammation and expression of ICC in a fetal lamb model of fetoscopic intervention for gastroschisis.

BACKGROUND: The pathogenesis of intestinal dysmotility in gastroschisis is not completely understood. Peel formation and disorganization of interstitial Cajal cells (ICC) have been proposed in humans. The aim of this study was to evaluate the impact of prenatal coverage of gastroschisis on gut inflammation and expression of ICC in a fetal lamb model. METHODS: Twenty-one German blackhead sheep with an abdominal wall defect that was created fetoscopically on day 77 of 145 days gestation were used in this study. Intrauterine surgery with the aim to cover the defect was performed 3 weeks later; two fetuses were covered completely, 5 partially and 11 remained uncovered. Three fetuses without gastroschisis were used as controls. All fetuses were retrieved by cesarean section at day 135. Samples of the small intestine were stained with hematoxylin and eosin for histologic analysis of peel formation and serosal and muscular thickness. For ICC detection, immunohistochemistry using anti-CD117 (c-Kit) antibody was used. RESULTS: In all samples with exposure to amniotic fluid, peel formation and significantly decreased ICC were found. Complete coverage reduced peel formation and disorganization of ICC compared to uncovered animals almost to the level of controls. CONCLUSIONS: Peel formation and ICC derangement were significantly reduced by prenatal coverage of gastroschisis. Moreover, this animal model mimics the histopathological bowel changes as seen in human gastroschisis and may, therefore, be used for further research on the pathophysiology and fetal therapy of this malformation.

Prenatal latex sensitization in patients with spina bifida: a pilot study.

OBJECT: Patients with spina bifida are particularly vulnerable to developing immunoglobulin E (IgE)-mediated latex sensitization. Even though many risk factors leading to latex allergy in these patients have been described, it is still unclear whether the increased prevalence of latex sensitization is disease associated or due to the procedures used to treat spina bifida. The aim of this study was to assess prenatal latex sensitization in patients with spina bifida by examining IgE levels in umbilical cord blood. METHODS: Patients with spina bifida and matched healthy infants were recruited from the University Medical Center Hamburg-Eppendorf and Children's Hospital Altona. Latex-specific and total IgE were assessed in umbilical cord blood using ImmunoCAP testing to evaluate the degree of prenatal latex sensitization. RESULTS: Twenty-two subjects, 10 with spina bifida and 12 healthy individuals, were included. Subjects were selected after matching for sex, gestational age, weight, parental allergy profile, number of prenatal examinations, and utilization of latex tools during pregnancy (propensity score estimates, p = 0.36). In patients with spina bifida, latex-specific and total IgE levels were significantly higher than those in healthy individuals (p = 0.001). After normalization to total IgE, latex-specific IgE levels were higher, yet not significantly increased (p = 0.085). CONCLUSIONS: Perinatally, there is a significant augmentation of total and latex-specific IgE in patients with spina bifida. After correcting for total IgE, latex-specific IgE was increased, yet not significantly higher than in matched, healthy controls. This pilot study gives novel insights in the immunological reactions related to spina bifida. The increased latex-specific IgE levels could possibly be associated with the occurrence of a latex allergy in the future.

Taurolidine cooperates with antineoplastic drugs in neuroblastoma cells.

Neuroblastoma is the most common extracranial tumor in childhood. Outcome of stage 4 disease remains poor and the development of novel therapeutic approaches is thus urgently needed. Taurolidine (TRD), originally invented to avoid catheter infections, has shown to exhibit antineoplastic activity in various cancers. The growth of neuroblastoma cell lines is inhibited by TRD as recently demonstrated. Further analysis disclosed a significant negative growth effect of TRD on the four neuroblastoma cell lines SH-EP TET21N, SK-N-AS, SK-N-BE(2)-M17 and SK-N-SH. Detected IC50 (51-274 µM; 48 h) are promising and correspond to clinically-achievable plasma levels. Apoptosis was induced (76-86%; 48 h) in a time-dependent manner mediated by a simultaneous activation of the intrinsic and extrinsic pathways. This was confirmed by cleavage of caspases -3, -8 and -9 and abrogation of apoptosis by pan-caspase inhibition. Application of TRD resulted in a significant enhancement of cytotoxic drugs vincristine/doxorubicin (2/3 of 4 cell lines) making TRD a promising candidate to be included in neuroblastoma therapy regimens in the future.

Mucosal loss with increased expression of IL-6, IL-8, and COX-2 in a formula-feeding only neonatal rat model of necrotizing enterocolitis.

INTRODUCTION: The aim of our study is to establish a reliable neonatal rat model by formula feeding only for evaluation of early surgical intervention on the course of experimental necrotizing enterocolitis (NEC). MATERIAL AND METHODS: Newborn Sprague-Dawley rats were divided into 50 breast-fed (group 1) and 38 formula fed (Similac/Esbilac, group 2) animals. The pups were sacrificed on the 4th, 5th, and 6th day of life and the terminal intestine examined for macroscopic and histologic changes as well as cytokine expression. RESULTS: The histological mucosal damage was significantly higher of group 2 compared to group 1. The area of the vital mucosa of group 2 was significantly (58.57%, p<0.001) lower compared to group 1 (75.12%). The mRNA expression of the inflammatory cytokines IL-6, IL-8 and COX-2 was significantly 2-, 5- and 10-fold increased in group 2 compared to group 1. DISCUSSION: Formula fed newborn rats displayed an inflammatory enterocolitis similar to human NEC. Our study demonstrates a significant loss of mucosa in animals with NEC having increased expression levels of IL-6, IL-8 and COX-2. Mucosal loss appears to be a distinct feature of experimental NEC and has to be correlated with the human disease.

Aberrant ZNF423 impedes B cell differentiation and is linked to adverse outcome of ETV6-RUNX1 negative B precursor acute lymphoblastic leukemia.

Differentiation arrest is a hallmark of acute leukemia. Genomic alterations in B cell differentiation factors such as PAX5, IKZF1, and EBF-1 have been identified in more than half of all cases of childhood B precursor acute lymphoblastic leukemia (ALL). Here, we describe a perturbed epigenetic and transcriptional regulation of ZNF423 in ALL as a novel mechanism interfering with B cell differentiation. Hypomethylation of ZNF423 regulatory sequences and BMP2 signaling result in transactivation of ZNF423α and a novel ZNF423ß-isoform encoding a nucleosome remodeling and histone deacetylase complex-interacting domain. Aberrant ZNF423 inhibits the transactivation of EBF-1 target genes and leads to B cell maturation arrest in vivo. Importantly, ZNF423 expression is associated with poor outcome of ETV6-RUNX1-negative B precursor ALL patients. Our work demonstrates that ALL is more than a genetic disease and that epigenetics may uncover novel mechanisms of disease with prognostic implications.

Smac mimetic LBW242 sensitizes XIAP-overexpressing neuroblastoma cells for TNF-α-independent apoptosis.

Despite intensive treatment regimens, high-risk and late-stage neuroblastoma tends to have a poor survival outcome. Overexpression of the apoptotic regulator, X-linked inhibitor of apoptosis protein (XIAP), has been associated with chemotherapy resistance in several cancers including neuroblastoma. Here, we report preclinical evidence that XIAP offers an effective therapeutic target in neuroblastoma. Human and murine neuroblastoma cells were treated with the Smac mimetic LBW242 alone or in combination with cytotoxic drugs used clinically to treat neuroblastoma. Expression of XIAP protein, but not mRNA, was highly increased in neuroblastoma cells compared to healthy adrenal gland tissue, consistent with a posttranscriptional regulation of XIAP expression. Treatment with LBW242 sensitized human and murine neuroblastoma cells to chemotherapy-induced apoptosis, which was mediated by activation of both the intrinsic and extrinsic apoptosis pathways. Although Smac mimetics have been reported to stimulate TNF-α-induced apoptosis by degradation of cellular IAP (cIAP)-1/2, we found that LBW242-mediated sensitization in neuroblastoma cells occurred in a TNF-α-independent manner, despite induction of cIAP-1/2 degradation and TNF-α expression. Together, our findings show that XIAP targeting sensitizes neuroblastoma to chemotherapy-induced apoptosis, suggesting a novel therapeutic approach to treat this childhood malignancy.

DNA vaccination: using the patient's immune system to overcome cancer.

Cancer is one of the most challenging diseases of today. Optimization of standard treatment protocols consisting of the main columns of chemo- and radiotherapy followed or preceded by surgical intervention is often limited by toxic side effects and induction of concomitant malignancies and/or development of resistant mechanisms. This requires the development of therapeutic strategies which are as effective as standard therapies but permit the patients a life without severe negative side effects. Along this line, the development of immunotherapy in general and the innovative concept of DNA vaccination in particular may provide a venue to achieve this goal. Using the patient's own immune system by activation of humoral and cellular immune responses to target the cancer cells has shown first promising results in clinical trials and may allow reduced toxicity standard therapy regimen in the future. The main challenge of this concept is to transfer the plethora of convincing preclinical and early clinical results to an effective treatment of patients.