Leopold Ludwig

RAG Mutations in Human B Cell-Negative SCID

Patients with human severe combined immunodeficiency (SCID) can be divided into those with B lymphocytes (B+ SCID) and those without (B− SCID). Although several genetic causes are known for B+ SCID, the etiology of B− SCID has not been defined. Six of 14 B− SCID patients tested were found to carry a mutation of the recombinase activating gene 1 (RAG-1), RAG-2, or both. This mutation resulted in a functional inability to form antigen receptors through genetic recombination and links a defect in one of the site-specific recombination systems to a human disease.

SHARP is a novel component of the Notch/RBP-Jκ signalling pathway

Notch proteins are the receptors for an evolutionarily highly conserved signalling pathway that regulates numerous cell fate decisions during development. Signal transduction involves the presenilin‐dependent intracellular processing of Notch and nuclear translocation of the intracellular domain of Notch, Notch‐IC. Notch‐IC associates with the DNA‐binding protein RBP‐Jκ/CBF‐1 to activate transcription of Notch target genes. In the absence of Notch signalling, RBP‐Jκ/CBF‐1 acts as a transcriptional repressor through the recruitment of histone deacetylase (HDAC) corepressor complexes. We identified SHARP as an RBP‐Jκ/CBF‐1‐interacting corepressor in a yeast two‐hybrid screen. In cotransfection experiments, SHARP‐mediated repression was sensitive to the HDAC inhibitor TSA and facilitated by SKIP, a highly conserved SMRT and RBP‐Jκ‐interacting protein. SHARP repressed Hairy/Enhancer of split (HES)‐1 promoter activity, inhibited Notch‐1‐mediated transactivation and rescued Notch‐1‐induced inhibition of primary neurogenesis in Xenopus laevis embryos. Based on our data, we propose a model in which SHARP is a novel component of the HDAC corepressor complex, recruited by RBP‐Jκ to repress transcription of target genes in the absence of activated Notch.

Mitogenic and antiapoptotic role of constitutive NF‐κB/Rel activity in pancreatic cancer

The transcription factor NF‐κB/Rel was found to be constitutively activated in human pancreatic cancer. RelA is present in the nucleus in primary human pancreatic cancer samples as well as in pancreatic cancer cell lines. NF‐κB/Rel–binding activity consists of NF‐κB1(p50) and RelA(p65). Constitutive NF‐κB/Rel activity correlates with IκB kinase (IKK) activity and can be blocked by dominant negative mutants of IKKβ and to a lesser extent by IKKα. Constitutive NF‐κB/Rel activity and the transactivation potential of RelA(p65) can be inhibited by dominant negative mutant Ras, the PI3 kinase inhibitor LY294002, or dominant negative mutant Akt kinase. Transfection of a dominant negative mutant epidermal growth factor receptor (EGF‐R), EGF‐R kinase inhibitor Tyrphostin and LY 294002 blocked IKK activity and NF‐κB–dependent transcription. Inhibition of constitutive IKK or NF‐κB/Rel activity increased the number of apoptotic cells. Stably expressing a nondegradable form of IκBα inhibited anchorage‐dependent and ‐independent proliferation in MiaPaCa2 and Panc1 cells. Our data demonstrate that an EGF‐R/Ras/PI3 kinase/Akt/IKK‐dependent pathway contributes to constitutive NF‐κB/Rel activity in pancreatic cancer. Inhibition of NF‐κB/Rel activity reveals a mitogenic and antiapoptotic role for NF‐κB/Rel in pancreatic cancer.

Glial-derived neurotrophic factor regulates apoptosis in colonic epithelial cells

BACKGROUND & AIMS Ablation of the enteric glia leads to a fulminant hemorrhagic jejunoileitis. We hypothesized that glial-derived neurotrophic factor (GDNF) may be involved in mucosal protection of the gut. Therefore, we examined the regulation of GDNF and its receptor (GFR-alpha1) in colonic inflammation and its effects on colonic epithelial cell apoptosis. METHODS The expression of GDNF and GFR-alpha1 was investigated in experimental colitis of rats and in human inflammatory bowel disease (IBD). GDNF-induced activation of Akt (protein kinase B [PKB]) and mitogen-activated protein kinase (MAPK) in the colonic epithelial cell lines HT-29 and SW480 was studied. Furthermore, the antiapoptotic potency of GDNF in SW480 cells was evaluated. RESULTS GDNF was specifically up-regulated in experimental rat colitis and in IBD. In contrast, GFR-alpha1 was constitutively expressed in rat and human colonic epithelium. GDNF potently activated MAPK and Akt (PKB) in colonic epithelial cells. Moreover, GDNF strongly prevented apoptosis in SW480 cells. Our data show that GDNF-mediated protection against apoptosis depends on activation of the MAPK and phosphatidylinositol 3-kinase/Akt (PKB) pathways. CONCLUSIONS GDNF is up-regulated in IBD and has strong antiapoptotic properties in colonic epithelial cells. This points to a novel role of the neurotrophic factor GDNF for mucosal protection and regeneration in IBD.

Auditory evoked potentials compared with bispectral index for monitoring of midazolam and propofol sedation during colonoscopy.

OBJECTIVES:The purpose of this study was to evaluate and compare Bispectral index (BIS) and A-line auditory evoked potential index (AAI) for monitoring depth of low-dose midazolam and propofol sedation during colonoscopy.METHODS:A total of 115 consecutive patients (ASA I–IV), receiving low-dose midazolam and propofol sedation for colonoscopy, were evaluated. BIS and AAI levels, Observers Assessment of Alertness/Sedation (OAA/S) scores, blood pressure, heart rate, oxygen saturation, as well as the presence or absence of eyelash reflex, patient reaction to an external noxious stimulus and to procedure-related pain were recorded every 1–3 min by a single trained observer.RESULTS:There was a positive correlation between BIS and OAA/S scores (correlation coefficient=0.77) and to a lesser extent AAI and OAA/S scores (correlation coefficient=0.47). BIS and AAI showed significant differences between subsequent levels of sedation (P<0.001). The clustered receiver operating characteristic curve estimate of BIS for the detection of deep sedation was significantly better than that of AAI (P<0.001). Regarding the presence or absence of eyelash reflex and patient reaction to an external noxious stimulus and to procedure-related pain, significant different levels were found for BIS as well as AAI, respectively. Only small changes were observed in hemodynamic variables and oxygen saturation. Overall, our data suggest target BIS levels of slightly above 73 for moderate sedation (defined as OAA/S scores 2 and 3).CONCLUSIONS:BIS and AAI correlated with the level of sedation. Hemodynamic variables were poor indicators of the hypnotic–anesthetic status of the patient. BIS discriminated best between moderate and deep sedation and could complement clinical observation for guidance of moderate sedation.

Grap-2, a novel RET binding protein, is involved in RET mitogenic signaling

Signal transduction of the RET receptor tyrosine kinase is involved in developmental processes as well as in neoplastic transformation. Activation of RET initiates receptor autophosphorylation on specific tyrosines that act as docking sites for downstream signaling molecules. Using the cytoplasmatic part of RET as bait in a yeast two-hybrid screen, we identified a novel SH2 and SH3 domain containing adaptor protein previously termed Grap-2/Grf40/GrpL/GRID and its murine homologue as Gads/Mona, respectively. This protein, predominantly expressed in cells of hematopoietic origin, is involved in signaling downstream of the T-cell receptor and the receptor for monocyte colony-stimulating factor. Here, we show that Grap-2 is also expressed in neuroendocrine tumors and cell lines known to bear mutated forms of RET. Endogenously expressed RET and Grap-2 coimmunoprecipitate from lysates of a medullary thyroid carcinoma cell line. Grap-2 directly associates with RET in pull-down experiments using in vitro translated proteins. Overexpression of Grap-2 inhibits RET-induced NF-κB activation, and cotransfection of Grap-2 significantly reduces focus formation induced by oncogenic RET in NIH 3T3 cells. Taken together, these results suggest that besides being involved in tyrosine kinase signaling in hematopoietic cells, Grap-2 plays a tissue-specific role as an inhibitor of RET mitogenic signaling.

Menin mutations in the diagnosis and prediction of multiple endocrine neoplasia type 1

Introduction: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the development of multiple endocrine adenomas, typically in the pancreas, anterior pituitary, and parathyroid glands. The disease is associated with germ-line mutations of the menin gene, a putative tumor-suppressor gene located on human chromosome 11q13. Methods: To facilitate the diagnosis and prediction of MEN1 in patients and their relatives, we developed a molecular two-step strategy to screen for menin gene mutations. DNA fragments covering the entire menin coding sequence are generated from patient cDNA by polymerase reaction (PCR) and subsequently analyzed by single-strand conformational polymorphism electrophoresis (SSCP). Fragments with aberrant SSCP migration are DNA-sequenced to directly characterize menin mutations. In a second diagnostic step, genomic DNA of healthy relatives of the corresponding MEN1 index patient is analyzed by PCR, with only the specific exon amplified harboring the family-specific mutation. Mutation-specific restriction enzyme digestion of this PCR product finally allows the identification of mutation carriers through pathological restriction fragment patterns. Results: Using this approach, we identified an in-frame deletion mutation (Δ Tyr Met) located in menin exon 4 (codon 227 – 228) that co-segregates with the disease phenotype in a large MEN1 family from Southern Germany. Conclusion: It is likely that the direct molecular analysis of menin gene mutations will replace the genetic and biochemical screening tests currently used in the clinical management of MEN1 families. In addition, these studies may provide clues to the tumor biology of both sporadic and MEN1-associated endocrine adenomas.

Bi-allelic inactivation of the MEN1 tumor suppressor gene in human grade II astrocytoma.

In humans and gene-targeted mice, loss of multiple endocrine neoplasia type 1 (MEN1) tumor suppressor gene function causes neuroendocrine tumors, frequently of the parathyroid and pituitary glands and the pancreas. The MEN1 gene product interacts with glial fibrillary acidic protein (GFAP) in the brain. We here demonstrate bi-allelic MEN1 inactivation in a grade II astrocytoma in an individual carrying a heterozygous MEN1 germ line deletion mutation (788del6). This tumor represents a novel, non-endocrine MEN1-phenotype, compatible with a role of MEN1-GFAP in glial oncogenesis. Clinically, a genetic predisposition to variant neoplasias should be considered in the management of MEN1 patients.

Genetic Biopsy for Prediction of Surveillance Intervals after Endoscopic Resection of Colonic Polyps: Results of the GENESIS Study

Background and objective Current surveillance strategies for colorectal cancer following polypectomy are determined by endoscopic and histopathological factors. Such a distinction has been challenged. The present study was designed to identify molecular parameters in colonic polyps potentially defining new sub-groups at risk. Methods One hundred patients were enrolled in this multicentre study. Polyps biopsies underwent formalin-free processing (PAXgene, PreAnalytiX) and targeted next generation sequencing (38 genes (QIAGEN), NextSeq 500 platform (Illumina)). Genetic and histopathological analyses were done blinded to other data. Results In 100 patients, 224 polyps were removed. Significant associations of genetic alterations with endoscopic or histological polyp characteristics were observed for BRAF, KRAS, TCF7L2, FBXW7 and CTNNB1 mutations. Multivariate analysis revealed that polyps ≥ 10 mm have a significant higher relative risk for harbouring oncogene mutations (relative risk 3.467 (1.742–6.933)). Adenomas and right-sided polyps are independent risk factors for CTNNB1 mutations (relative risk 18.559 (2.371–145.245) and 12.987 (1.637–100.00)). Conclusions Assessment of the mutational landscape of polyps can be integrated in the workflow of current colonoscopy practice. There are distinct genetic patterns related to polyp size and location. These results suffice to optimise individual risk calculation and may help to better define surveillance intervals.