NAC functions as a modulator of SRP during the early steps of protein targeting to the endoplasmic reticulum.

Nascent polypeptide-associated complex (NAC) was initially found to bind to any segment of the nascent chain except signal sequences. In this way, NAC is believed to prevent mistargeting due to binding of signal recognition particle (SRP) to signalless ribosome nascent chain complexes (RNCs). Here we revisit the interplay between NAC and SRP. NAC does not affect SRP function with respect to signalless RNCs; however, NAC does affect SRP function with respect to RNCs targeted to the endoplasmic reticulum (ER). First, early recruitment of SRP to RNCs containing a signal sequence within the ribosomal tunnel is NAC dependent. Second, NAC is able to directly and tightly bind to nascent signal sequences. Third, SRP initially displaces NAC from RNCs; however, when the signal sequence emerges further, trimeric NAC·RNC·SRP complexes form. Fourth, upon docking to the ER membrane NAC remains bound to RNCs, allowing NAC to shield cytosolically exposed nascent chain domains not only before but also during cotranslational translocation. The combined data indicate a functional interplay between NAC and SRP on ER-targeted RNCs, which is based on the ability of the two complexes to bind simultaneously to distinct segments of a single nascent chain.

TNFα inhibitors restrict T cell activation and cycling via Notch-1 signalling in inflammatory bowel disease.

BACKGROUND: Tumour necrosis factor α (TNFα) inhibitors such as adalimumab and infliximab are frequently prescribed for inflammatory bowel disease (IBD). Despite the clinical success of TNFα inhibitors, their physiological mode of action is not fully understood. The aim of this study was to investigate the mode of action of anti-TNFα agents in IBD. METHODS: It was hypothesised that Notch mediates anti-TNFα action in T cells. A study was carried out to identify Notch-1 as a link by which anti-TNFα antibodies mediate their inhibitory functions. RESULTS: TNFα inhibitors induced T cell apoptosis, inhibited activation, reduced cytokine secretion and restricted cell cycling. TNFα blockade at several levels showed that TNFα is responsible for inducing apoptosis by anti-TNFα but not for cell cycle restriction. By linking Notch and TNFα it was shown that (1) Notch-1 mucosal expression differs in inflamed and non-inflamed mucosa and increases in response to anti-TNFα treatment; (2) Notch-1 function is regulated by TNFα inhibitors; (3) Notch-1 binds to TNFα; and (4) Notch-1 inhibition prevents anti-TNFα-induced T cell cycle arrest but not apoptosis. CONCLUSIONS: TNFα inhibitors potently inhibit T cell function. By demonstrating for the first time that Notch-1 mediates the inhibitory effects of adalimumab and infliximab on T cell cycling, this study reveals a new mode of action and also an underlying signalling pathway by which biological agents act in IBD.

Comparative Multi-Epitope-Ligand-Cartography reveals essential immunological alterations in Barrett's metaplasia and esophageal adenocarcinoma.

BACKGROUND: Barrett's esophagus (BE) is caused by gastroesophageal reflux with consecutive mucosal inflammation, predisposing patients to the development of esophageal adenocarcinoma (EAC). We investigated changes in T cell-related mucosal combinatorial molecular protein patterns in both diseases using the novel Multi-Epitope-Ligand-Cartography, a unique robotic whole-cell imaging technology that simultaneously visualizes dozens of proteins in structurally intact tissues and correlates cellular localization of proteins with function. RESULTS: Biopsies were taken during endoscopy from BE, EAC, and normal control tissue, and proteomic microscopy was performed on 32 different epitopes. When the significance level was set to p < 0.0005 and the search depth to five antibody combinations, controls and BE can be differentiated by 63, controls and EAC by 3222, and BE from EAC by 1521 distinct protein combinations.For example, the number of activated apoptotic naïve and memory T cells was significantly increased only in BE, whereas the number of activated apoptotic helper and regulatory T cells was significantly elevated in BE and EAC. In contrast, the number of activated apoptotic cytotoxic T cells was significantly elevated only in EAC. Confirming different pathways in BE and EAC, the number of T lymphocytes with p53 expression and downregulation of bcl2 expression (CD3+p53+Bcl2-NfkB-) was significantly increased in EAC compared to BE and controls. Interestingly, the number of precursor T cells (CD7+) was significantly elevated only in EAC. These cells lack Bax and caspase-8, suggesting impaired apoptosis in the early stages of T cell differentiation. CONCLUSION: Proteomic analysis showed for the first time that proteins, which are critically involved in the mucosal immune system of the esophagus, are distinctly expressed in BE and EAC, whereas others are comparably altered in both diseases, suggesting that many pathogenic events might be shared by both diseases. Topological proteomic analysis, therefore, helps us to understand the different pathogenic events in the underlying disease pathways.

The Hsp70 homolog Ssb is essential for glucose sensing via the SNF1 kinase network.

Yeast senses the availability of external energy sources via multiple interconnected signaling networks. One of the central components is SNF1, the homolog of mammalian AMP-activated protein kinase, which in yeast is essential for the expression of glucose-repressed genes. When glucose is available hyperphosphorylated SNF1 is rendered inactive by the type 1 protein phosphatase Glc7. Dephosphorylation requires Reg1, which physically targets Glc7 to SNF1. Here we show that the chaperone Ssb is required to keep SNF1 in the nonphosphorylated state in the presence of glucose. Using a proteome approach we found that the Deltassb1Deltassb2 strain displays alterations in protein expression and suffers from phenotypic characteristics reminiscent of glucose repression mutants. Microarray analysis revealed a correlation between deregulation on the protein and on the transcript level. Supporting studies uncovered that SSB1 was an effective multicopy suppressor of severe growth defects caused by the Deltareg1 mutation. Suppression of Deltareg1 by high levels of Ssb was coupled to a reduction of Snf1 hyperphosphorylation back to the wild-type phosphorylation level. The data are consistent with a model in which Ssb is crucial for efficient regulation within the SNF1 signaling network, thereby allowing an appropriate response to changing glucose levels.

A signal-anchor sequence stimulates signal recognition particle binding to ribosomes from inside the exit tunnel.

Sorting of eukaryotic membrane and secretory proteins depends on recognition of ribosome-bound nascent chain signal sequences by the signal recognition particle (SRP). The current model suggests that the SRP cycle is initiated when a signal sequence emerges from the ribosomal tunnel and binds to SRP. Then elongation is slowed until the SRP-bound ribosome-nascent chain complex (RNC) is targeted to the SRP receptor in the endoplasmic reticulum (ER) membrane. The RNC is then transferred to the translocon, SRP is released, and translation resumes. Because RNCs do not target to the translocon efficiently if nascent chains become too long, the window for SRP to identify its substrates is short. We now show that a transmembrane signal-anchor sequence (SA) significantly enhances binding of SRP to RNCs even before the SA emerges from the ribosomal tunnel. In this mode, SRP does not contact the SA directly but is in close proximity to the portion of the nascent polypeptide that has already left the ribosomal tunnel. Early recruitment of SRP provides a mechanism to expand the window for substrate identification. We suggest that the dynamics of the SRP-ribosome interaction is affected not only by the direct binding of SRP to an exposed signal sequence but also by properties of the translating ribosome that are triggered from within the tunnel.

Prognostic factors of long-term outcome in gastroenteropancreatic neuroendocrine tumours.

Neuroendocrine tumours (NET) of the gastroenteropancreatic system comprise a malignant entity with a low incidence. Only limited information is available on long-term clinical outcome and clinically applicable prognostic factors. We performed a retrospective analysis of a large, well-characterized centre-based patient cohort of 399 patients with histologically proven NET. Data were analysed according to epidemiological, clinical and histopathological characteristics. Detailed survival analyses using the Kaplan-Meier method were performed. Prognostic factors were tested by log-rank testing and independent risk factors were analysed using a Cox regression model. In the studied cohort, primary tumours originated in the fore-, mid- and hindgut in 46.1, 37.1 and 4.5% respectively. Extra-intestinal or unknown primary tumours were present in 8.4 and 10.5% respectively. Distant metastasis was present at initial diagnosis in 69.4%. Most frequent metastatic sites were liver (85%), peritoneal cavity (18%), bones (8%), other intra-abdominal sites (6%) and lungs (4%). Overall, 5- and 10-year survival rates were 78 and 63% respectively. Time to progression after initial diagnosis was significantly shorter in pancreatic as compared with ileal NET. Survival analysis revealed significantly better clinical outcome for primary tumours smaller than 25 mm, absence of metastasis, absence of any clinical symptoms, positive immunohistochemical staining for chromogranin A and a lower Ki67 index. These results were confirmed as independent by multivariate analysis. Therefore, this large retrospective analysis of a well-documented cohort of patients with NET demonstrates several prognostic factors of clinical relevance and wide availability, which should be considered for risk stratification in the management of NET.

Galectin-4 controls intestinal inflammation by selective regulation of peripheral and mucosal T cell apoptosis and cell cycle.

Galectin-4 is a carbohydrate-binding protein belonging to the galectin family. Here we provide novel evidence that galectin-4 is selectively expressed and secreted by intestinal epithelial cells and binds potently to activated peripheral and mucosal lamina propria T-cells at the CD3 epitope. The carbohydrate-dependent binding of galectin-4 at the CD3 epitope is fully functional and inhibited T cell activation, cycling and expansion. Galectin-4 induced apoptosis of activated peripheral and mucosal lamina propria T cells via calpain-, but not caspase-dependent, pathways. Providing further evidence for its important role in regulating T cell function, galectin-4 blockade by antisense oligonucleotides reduced TNF-alpha inhibitor induced T cell death. Furthermore, in T cells, galectin-4 reduced pro-inflammatory cytokine secretion including IL-17. In a model of experimental colitis, galectin-4 ameliorated mucosal inflammation, induced apoptosis of mucosal T-cells and decreased the secretion of pro-inflammatory cytokines. Our results show that galectin-4 plays a unique role in the intestine and assign a novel role of this protein in controlling intestinal inflammation by a selective induction of T cell apoptosis and cell cycle restriction. Conclusively, after defining its biological role, we propose Galectin-4 is a novel anti-inflammatory agent that could be therapeutically effective in diseases with a disturbed T cell expansion and apoptosis such as inflammatory bowel disease.

Prognostic relevance of a novel TNM classification system for upper gastroenteropancreatic neuroendocrine tumors.

BACKGROUND: Neuroendocrine tumors (NETs) of the gastroenteropancreatic (GEP) system comprise a rare but challenging group of malignant neoplasms and occur at virtually any site of the GEP system. In 2006, a new TNM classification system was proposed for the staging and grading of upper GEP NETs. METHODS: The prognostic relevance of the TNM classification system was analyzed retrospectively in 202 patients from a referral center with histologically proven foregut NET. Patients were classified according to previous classification systems and the TNM classification. Survival data were acquired and statistical analyses were performed by using log-rank and Cox regression testing. RESULTS: Primary tumors were gastric (n = 48), duodenal (n = 23), and pancreatic (n = 131). During the observation period, 21% of patients died. The overall 5- and 10-year survival rates were 75% and 64%, respectively. Previous classification systems discriminated between low-grade and high-grade malignant NETs but did not allow further prognostic differentiation. In contrast, the proposed TNM classification was able to differentiate significantly between different tumor stages (stages I-III vs stage IV; P < .01) and cellular proliferation rates according to Ki-67 labeling (grade 1 vs grade 2, P = .04; grade 1 vs grade 3 and grade 2 vs grade 3, P < .01). Cox regression analysis confirmed an increased risk of reduced survival for patients with stage III or IV NET and grade 2 or 3 NET. CONCLUSIONS: The current results demonstrated the prognostic relevance of the newly proposed TNM classification system for foregut NETs with statistical significance for the subgroups of both the staging classification and the grading system. Thus, the new classification system provides a valid and powerful tool for prognostic stratification of GEP NETs in clinical practice and research.

Somatostatin-producing neuroendocrine tumors of the duodenum and pancreas: incidence, types, biological behavior, association with inherited syndromes, and functional activity.

Somatostatin-producing neuroendocrine tumors (SOM-NETs) of the duodenum and pancreas appear to be heterogeneous. To determine their clinicopathological profiles, respective data were analyzed on a series of 82 duodenal and 541 pancreatic NETs. In addition, the clinical records of 821 patients with duodenal or pancreatic NETs were reviewed for evidence of a somatostatinoma syndrome. Predominant or exclusive expression of somatostatin was found in 21 (26%) duodenal and 21 (4%) pancreatic NETs. They were classified as sporadic (n=31) or neurofibromatosis type 1 (NF1)-associated duodenal NETs (n=3), gangliocytic paragangliomas (GCPGs; n=6), or poorly differentiated neuroendocrine carcinomas (pdNECs; n=2). In addition, five duodenal and four pancreatic SOM-NETs were found in five patients with multiple endocrine neoplasia type 1 (MEN1). Metastases occurred in 13 (43%) patients with sporadic or NF1-associated SOM-NETs, but in none of the duodenal or pancreatic MEN1-associated SOM-NETs or GCPGs. Sporadic advanced (stage IV) SOM-NETs were more commonly detected in the pancreas than in the duodenum. None of the patients (including the 821 patients for whom only the clinical records were reviewed) fulfilled the criteria of a somatostatinoma syndrome. Our data show that somatostatin expression is not only seen in sporadic NETs but may also occur in GCPGs, pdNECs, and hereditary NETs. Surgical treatment is effective in most duodenal and many pancreatic SOM-NETs. MEN1-associated SOM-NETs and GCPGs follow a benign course, while somatostatin-producing pdNECs are aggressive neoplasms. The occurrence of the so-called somatostatinoma syndrome appears to be extremely uncommon.

Systematic high-content proteomic analysis reveals substantial immunologic changes in colorectal cancer.

The immune system is a significant determinant of epithelial tumorigenesis, but its role in colorectal cancer pathogenesis is not well understood. The function of the immune system depends upon the integrity of the protein network environment, and thus, we performed MELC immunofluorescence microscopy focusing on the lamina propria. By analyzing structurally intact tissues from colorectal cancer, ulcerative colitis, and healthy colonic mucosa, we used this unique and novel highly multiplexed robotic-imaging technology, which allows visualizing dozens of proteins simultaneously, and explored the toponome in colorectal cancer mucosa for the first time. We identified 1,930 motifs that distinguish control from colorectal cancer tissue. In colorectal cancer, the number of activated T cells is increased, explained by a lack of bax, caspase-3, and caspase-8. Whereas CD4(+)CD25(+) T cells are decreased and are, other than in ulcerative colitis, not activated, cytotoxic T cells are significantly increased in colorectal cancer. Furthermore, the number of activated human lymphocyte antigen (HLA)-DR(+) T-cells is increased in colorectal cancer, pointing to an altered antigen presentation. In colorectal cancer, CD3(+)CD29(+) expression and assembly of the LFA-1 and LFA-3 receptor are differentially changed, indicating a distinct regulation of T-cell adhesion in colorectal cancer. We also identified increased numbers of natural killer and CD44(+) cells in the colorectal cancer mucosa and nuclear factor-kappaB as regulator of apoptosis in these cell populations. High-content proteomic analysis showed that colorectal cancer induces a tremendous modification of protein expression profiles in the lamina propria. Thus, topological proteomic analysis may help to unravel the role of the adaptive immune system in colorectal cancer and aid the development of new antitumor immunotherapy approaches.

Galectin-2 induces apoptosis of lamina propria T lymphocytes and ameliorates acute and chronic experimental colitis in mice.

Galectins have recently emerged as central regulators of the immune system. We have previously demonstrated that carbohydrate-dependent binding of galectin-2 induces apoptosis in activated T cells. Here, we investigate the potential therapeutic effect of galectin-2 in experimental colitis. Galectin-2 expression and its binding profile were determined by immunohistochemistry. Acute and chronic colitis was induced by dextran sodium sulfate administration and in a T-cell transfer colitis model. Apoptosis was assessed by TdT-mediated dUTP-biotin nick-end labeling, and cytokine secretion was determined by cytometric bead array. We show that galectin-2 was constitutively expressed mainly in the epithelial compartment of the mouse intestine and bind to lamina propria mononuclear cells. During colitis, galectin-2 expression was reduced, but could be restored to normal levels by immunosuppressive treatment. Galectin-2 treatment induced apoptosis of mucosal T cells and thus ameliorated acute and chronic dextran-sodium-sulfate-induced colitis and in a T-helper-cell 1-driven model of antigen-specific transfer colitis. Furthermore, the pro-inflammatory cytokine release was inhibited by galectin-2 treatment. In preliminary toxicity studies, galectin-2 was well tolerated. Our study provides evidence that galectin-2 induces apoptosis in vivo and ameliorates acute and chronic murine colitis. Furthermore, galectin-2 has no significant toxicity over a broad dose range, suggesting that it may serve as a new therapeutic agent in the treatment of inflammatory bowel disease.

Proteomic analysis of the inflamed intestinal mucosa reveals distinctive immune response profiles in Crohn's disease and ulcerative colitis.

Although Crohn's disease (CrD) and ulcerative colitis (UC) share several clinical features, the mechanisms of tissue injury differ. Because the global cellular function depends upon the protein network environment as a whole, we explored changes in the distribution and association of mucosal proteins to define key events involved in disease pathogenesis. Endoscopic biopsies were taken from CrD, UC, and control colonic mucosa, and Multi-Epitope-Ligand-Cartographie immunofluorescence microscopy with 32 different Abs was performed. Multi-Epitope-Ligand-Cartographie is a novel, highly multiplexed robotic imaging technology which allows integrating cell biology and biomathematical tools to visualize dozens of proteins simultaneously in a structurally intact cell or tissue. In CrD, the number of CD3+CD45RA+ naive T cells was markedly increased, but only activated memory, but not naive, T cells expressed decreased levels of Bax, active caspase-3 or -8. In UC, only CD4+ T cells coexpressing NF-kappaB were caspase-8 and poly(ADP-ribose)-polymerase positive. Furthermore, the number of CD4+CD25+ T cells was elevated only in UC, whereas in CrD and controls, the number of these cells was similar. By using hub analysis, we also identified that the colocalization pattern with NF-kappaB+ and poly(ADP-ribose)-polymerase+ as base motifs distinguished CrD from UC. High-content proteomic analysis of the intestinal mucosa demonstrated for the first time that different T cell populations within the intestinal mucosa express proteins translating distinct biological functions in each form of inflammatory bowel disease. Thus, topological proteomic analysis may help to unravel the pathogenesis of inflammatory bowel disease by defining distinct immunopathogenic profiles in CrD and UC.

Survival and clinical outcome of patients with neuroendocrine tumors of the gastroenteropancreatic tract in a german referral center.

Neuroendocrine tumors (NETs) are rare neoplasms. Approximately 75% of all cases manifest in the gastroenteropancreatic (GEP) system. Because of the low incidence of NETs, limited data about the clinical outcome and prognostic variables are available. In an attempt to identify prognostic parameters, we investigated the distribution of primary tumors, pattern of metastasis formation, clinical presentation, histological classification, and outcome of therapeutic interventions in a large patient cohort cared for in a German referral center. In 254 patients with GEP-NETs, the primary tumor was of foregut, midgut, or hindgut origin in 44.1% (28.7% pancreas), 43.7% (34.7% jejunoileum), and 4.3%, respectively. No primary tumor was found in 7.9%. Metastases occurred preferentially in lymph nodes and the liver. The overall 5-year survival rate was 57.1%. In the absence or presence of metastases at initial diagnosis the 5-year survival rate was 80.0% and 51.7%, respectively. The 5-year survival rate was related to the localization of the primary and was 75.0% and 42.9% for jejunoileal and pancreatic tumors, respectively. The size of the primary tumor (<2 cm) and histological grading as low-grade malignant were both associated with a significantly longer survival. Surgery with curative intent was attempted in 141 patients. However, an R(0) resection was achieved in only 66.0% of these patients. Five-year survival rate in the latter group was significantly higher (77.3%) as compared with all surgical patients (55.4%). Long-term tumor-free survival was obtained in only 53.7% of successfully resected patients. Palliative medical treatment, either with chemotherapy (i.e., especially for foregut NETs) or biotherapy (especially for midgut NETs), was only moderately effective for both therapeutic regimens.