MDS and AML show elevated fractions of CD34-positive blast cell populations with a high anti-apoptotic versus proliferation ratio.

This study investigates the intertwined processes of (anti-)apoptosis and cell proliferation in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Utilizing antibodies to Bcl-2 and Ki-67, the CD34-positive blast cell compartments in bone marrow aspirates from 50 non-malignant cases, 25 MDS patients, and 25 AML patients were analyzed for their anti-apoptotic and proliferative cell fractions through ten-color flow cytometry. MDS patients exhibited a significantly increased anti-apoptotic (p=0.0014) and reduced proliferative cell fraction (p=0.0030) in their blast cell population as compared to non-malignant cases. AML patients showed an even more exacerbated trend than MDS patients. The resulting Bcl-2:Ki-67 cell fraction ratios in MDS and AML were significantly increased as compared to the non-malignant cases (p=0.0004 and p<0.0001, respectively). AML patients displayed, however, a high degree of variability in their anti-apoptotic and proliferation index, attributed to heterogeneity in maturation stage and severity of the disease at diagnosis. Using double-labeling for Bcl-2 and Ki-67 it could be shown that besides blast cells with a mutually exclusive Ki-67 and Bcl-2 expression, also blast cells concurrently exhibiting anti-apoptotic and proliferative marker expression were found. Integrating these two dynamic markers into MDS and AML diagnostic workups may enable informed conclusions about their biological behavior, facilitating individualized therapy decisions for patients.

Impact of the gating strategy for Ki-67 and Bcl-2 on the determination of proliferation and anti-apoptosis data by flow cytometry in non-malignant bone marrow aspirates and aspirates from patients with myeloid malignancies.

This Data in Brief article displays a flow cytometric assay that was used for the acquisition and analyses of proliferative and anti-apoptotic activity in hematopoietic cells. This dataset includes analyses of the Ki-67 positive fraction (Ki-67 proliferation index) and Bcl-2 positive fraction (Bcl-2 anti-apoptotic index) of the different myeloid bone marrow (BM) cell populations in non-malignant BM, and in BM disorders, i.e. myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The present dataset comprises 1) the percentage of the CD34 positive blast cells, erythroid cells, myeloid cells and monocytic cells, and 2) the determined Ki-67 positive fraction and Bcl-2 positive fraction of these cell populations in tabular form. This allows the comparison and reproduction of the data when these analyses are repeated in a different setting. Because gating the Ki-67 positive and Bcl-2 positive cells is a critical step in this assay, different gating approaches were compared to determine the most sensitive and specific approach. BM cells from aspirates of 50 non-malignant, 25 MDS and 27 AML cases were stained with 7 different antibody panels and subjected to flow cytometry for determination of the Ki-67 positive cells and Bcl-2 positive cells of the different myeloid cell populations. The Ki-67 or Bcl-2 positive cells were then divided by the total number of cells of the respective cell population to generate the Ki-67 positive fraction (Ki-67 proliferation index) or the Bcl-2 positive fraction (Bcl-2 anti-apoptotic index). The presented data may facilitate the establishment and standardization of flow cytometric analyses of the Ki-67 proliferation index and Bcl-2 anti-apoptotic index of the different myeloid cell populations in non-malignant BM as well as MDS and AML patients in other laboratories. Directions for proper gating of the Ki-67 positive and Bcl-2 positive fraction are crucial for achieving standardization among different laboratories. In addition, the data and the presented assay allows application of Ki-67 and Bcl-2 in a research and clinical setting and this approach can serve as the basis for optimization of the gating strategy and subsequent investigation of other cell biological processes besides proliferation and anti-apoptosis. These data can also promote future research into the role of these parameters in diagnosis of myeloid malignancies, prognosis of myeloid malignancies and therapeutic resistance against anti-cancer therapies in these malignancies. As specific populations were identified based on cell biological characteristics, these data can be useful for evaluating gating algorithms in flow cytometry in general by confirming the outcome (e.g. MDS or AML diagnosis) with the respective proliferation and anti-apoptotic profile of these malignancies. The Ki-67 proliferation index and Bcl-2 anti-apoptotic index may potentially be used for classification of MDS and AML based on supervised machine learning algorithms, while unsupervised machine learning can be deployed at the level of single cells to potentially distinguish non-malignant from malignant cells in the identification of minimal residual disease. Therefore, the present dataset may be of interest for internist-hematologists, immunologists with affinity for hemato-oncology, clinical chemists with sub-specialization of hematology and researchers in the field of hemato-oncology.

Proliferative and anti-apoptotic fractions in maturing hematopoietic cell lineages and their role in homeostasis of normal bone marrow.

Recent developments in clinical flow cytometry allow the simultaneous assessment of proliferative and anti-apoptotic activity in the different hematopoietic cell lineages and during their maturation process. This can further advance the flow cytometric diagnosis of myeloid malignancies. In this study we established indicative reference values for the Ki-67 proliferation index and Bcl-2 anti-apoptotic index in blast cells, as well as maturing erythroid, myeloid, and monocytic cells from normal bone marrow (BM). Furthermore, the cell fractions co-expressing both proliferation and anti-apoptotic markers were quantified. Fifty BM aspirates from femoral heads of patients undergoing hip replacement were included in this study. Ten-color/twelve-parameter flow cytometry in combination with a software-based maturation tool was used for immunophenotypic analysis of Ki-67 and Bcl-2 positive fractions during the erythro-, myelo-, and monopoiesis. Indicative reference values for the Ki-67 and Bcl-2 positive fractions were established for different relevant hematopoietic cell populations in healthy BM. Ki-67 and Bcl-2 were equally expressed in the total CD34 positive blast cell compartment and 30% of Ki-67 positive blast cells also showed Bcl-2 positivity. The Ki-67 and Bcl-2 positive fractions were highest in the more immature erythroid, myeloid and monocytic cells. Both fractions then gradually declined during the subsequent maturation phases of these cell lineages. We present a novel application of an earlier developed assay that allows the simultaneous determination of the Ki-67 proliferative and Bcl-2 anti-apoptotic indices in maturing hematopoietic cell populations of the BM. Their differential expression levels during the maturation process were in accordance with the demand and lifespan of these cell populations. The indicative reference values established in this study can act as a baseline for further cell biological and biomedical studies involving hematological malignancies.

Application of bacteriophages EP75 and EP335 efficiently reduces viable cell counts of Escherichia coli O157 on beef and vegetables.

Shiga toxin producing Escherichia coli (STEC) are common etiological agents of food borne illnesses and outbreaks, most often caused by consuming contaminated beef products, followed by raw vegetables and dairy products. Patients infected with E. coli O157 are more likely hospitalized than patients infected with non-O157 STEC, making E. coli O157 an important target for microbiological interventions. We show that a cocktail of bacteriophages EP75 and EP335 effectively reduces E. coli O157 on beef, romaine lettuce, spinach, and zucchini. Treatment of contaminated beef with either 2 × 107 or 1 × 108 PFU/cm2 of bacteriophage cocktail EP75/EP335 resulted in reductions of 0.8-1.1 log10 CFU/cm2 and 0.9-1.3 log10 CFU/cm2, respectively (P < 0.0001). Similarly, bacteriophage treatments of contaminated romaine lettuce, zucchini, or spinach showed significant (P < 0.05) E. coli O157 reductions of 0.7-1.9 log10 CFU/cm2 (2 × 107 PFU/cm2), and 1.4-2.4 log10 CFU/cm2 (1 × 108 PFU/cm2). An E. coli O157 reduction of 0.9 log10 and 2.0 log10 was observed already 30 min after phage application of 1 × 108 PFU/cm2 on beef and romaine lettuce, respectively. These data show that bacteriophages EP75 and EP335 can be effectively used as a processing aid on beef and vegetables, and thereby can aid industry to reduce the risk of E. coli O157 food poisoning.

Protein Catalyzed Capture (PCC) Agents for Antigen Targeting.

The protein catalyzed capture agent (PCC) method is a powerful combinatorial screening strategy for discovering synthetic macrocyclic peptide ligands, called PCCs, to designated protein epitopes. The foundational concept of the PCC method is the use of in situ click chemistry to survey large combinatorial libraries of peptides for ligands to designated biological targets. State-of-the-art PCC screens integrate synthetic libraries of constrained macrocyclic peptides with epitope-specific targeting strategies to identify high-affinity (<100 nM) binders de novo. Automated instrumentation can accelerate PCC discovery to a rapid 2-week timeframe. Here, we describe methods to perform combinatorial screens that yield epitope-targeted PCCs.

Recombinant myelin oligodendrocyte glycoprotein quality modifies evolution of experimental autoimmune encephalitis in macaques.

Experimental autoimmune encephalitis (EAE) is a well-recognized model for the study of human acquired demyelinating diseases (ADD), a group of inflammatory disorders of the central nervous system (CNS) characterized by inflammation, myelin loss, and neurological impairment of variable severity. In rodents, EAE is typically induced by active immunization with a combination of myelin-derived antigen and a strong adjuvant as complete Freund's adjuvant (CFA), containing components of the mycobacterial wall, while myelin antigen alone or associated with other bacterial components, as lipopolysaccharides (LPS), often fails to induce EAE. In contrast to this, EAE can be efficiently induced in non-human primates by immunization with the recombinant human myelin oligodendrocyte glycoprotein (rhMOG), produced in Escherichia coli (E. coli), purified and formulated with incomplete Freund's adjuvant (IFA), which lacks bacterial elements. Here, we provide evidence indicating how trace amounts of bacterial contaminants within rhMOG may influence the course and severity of EAE in the cynomolgus macaque immunized with rhMOG/IFA. The residual amount of E. coli contaminants, as detected with mass spectrometry within rhMOG protein stocks, were found to significantly modulate the severity of clinical, radiological, and histologic hallmarks of EAE in macaques. Indeed, animals receiving the purest rhMOG showed milder disease severity, increased numbers of remissions, and reduced brain damage. Histologically, these animals presented a wider diversity of lesion types, including changes in normal-appearing white matter and prephagocytic lesions. Non-human primates EAE model with milder histologic lesions reflect more accurately ADD and permits to study of the pathogenesis of disease initiation and progression.

Positron Emission Tomography Tracer Design of Targeted Synthetic Peptides via 18F-Sydnone Alkyne Cycloaddition.

Chemically synthesized, small peptides that bind with high affinity and specificity to CD8-expressing (CD8+) tumor-infiltrating T cells, yet retain the desirable characteristics of small molecules, hold valuable potential for diagnostic molecular imaging of immune response. Here, we report the development of 18F-labeled peptides targeting human CD8α with nanomolar affinity via the strain-promoted sydnone-alkyne cycloaddition with 4-[18F]fluorophenyl sydnone. The 18F-sydnone is produced in one step, in high radiochemical yield, and the peptide labeling proceeds rapidly. A hydrophilic chemical linker results in a tracer with favorable pharmacokinetic properties and improved image contrast, as demonstrated by in vivo PET imaging studies.

Axon-Myelin Unit Blistering as Early Event in MS Normal Appearing White Matter.

OBJECTIVE: Multiple sclerosis (MS) is a chronic neuroinflammatory and neurodegenerative disease of unknown etiology. Although the prevalent view regards a CD4+ -lymphocyte autoimmune reaction against myelin at the root of the disease, recent studies propose autoimmunity as a secondary reaction to idiopathic brain damage. To gain knowledge about this possibility we investigated the presence of axonal and myelinic morphological alterations, which could implicate imbalance of axon-myelin units as primary event in MS pathogenesis. METHODS: Using high resolution imaging histological brain specimens from patients with MS and non-neurological/non-MS controls, we explored molecular changes underpinning imbalanced interaction between axon and myelin in normal appearing white matter (NAWM), a region characterized by normal myelination and absent inflammatory activity. RESULTS: In MS brains, we detected blister-like swellings formed by myelin detachment from axons, which were substantially less frequently retrieved in non-neurological/non-MS controls. Swellings in MS NAWM presented altered glutamate receptor expression, myelin associated glycoprotein (MAG) distribution, and lipid biochemical composition of myelin sheaths. Changes in tethering protein expression, widening of nodes of Ranvier and altered distribution of sodium channels in nodal regions of otherwise normally myelinated axons were also present in MS NAWM. Finally, we demonstrate a significant increase, compared with controls, in citrullinated proteins in myelin of MS cases, pointing toward biochemical modifications that may amplify the immunogenicity of MS myelin. INTERPRETATION: Collectively, the impaired interaction of myelin and axons potentially leads to myelin disintegration. Conceptually, the ensuing release of (post-translationally modified) myelin antigens may elicit a subsequent immune attack in MS. ANN NEUROL 2021;89:711-725.

Antibody-recruiting protein-catalyzed capture agents to combat antibiotic-resistant bacteria.

Antibiotic resistant infections are projected to cause over 10 million deaths by 2050, yet the development of new antibiotics has slowed. This points to an urgent need for methodologies for the rapid development of antibiotics against emerging drug resistant pathogens. We report on a generalizable combined computational and synthetic approach, called antibody-recruiting protein-catalyzed capture agents (AR-PCCs), to address this challenge. We applied the combinatorial protein catalyzed capture agent (PCC) technology to identify macrocyclic peptide ligands against highly conserved surface protein epitopes of carbapenem-resistant Klebsiella pneumoniae, an opportunistic Gram-negative pathogen with drug resistant strains. Multi-omic data combined with bioinformatic analyses identified epitopes of the highly expressed MrkA surface protein of K. pneumoniae for targeting in PCC screens. The top-performing ligand exhibited high-affinity (EC50 ∼50 nM) to full-length MrkA, and selectively bound to MrkA-expressing K. pneumoniae, but not to other pathogenic bacterial species. AR-PCCs that bear a hapten moiety promoted antibody recruitment to K. pneumoniae, leading to enhanced phagocytosis and phagocytic killing by macrophages. The rapid development of this highly targeted antibiotic implies that the integrated computational and synthetic toolkit described here can be used for the accelerated production of antibiotics against drug resistant bacteria.

Anti-MOG autoantibodies pathogenicity in children and macaques demyelinating diseases.

BACKGROUND: Autoantibodies against myelin oligodendrocyte glycoprotein (anti-MOG-Abs) occur in a majority of children with acquired demyelinating syndromes (ADS) and physiopathology is still under investigation. As cynomolgus macaques immunized with rhMOG, all develop an experimental autoimmune encephalomyelitis (EAE), we assessed relatedness between anti-MOG-Abs associated diseases in both species. METHODS: The study includes 27 children followed for ADS and nine macaques with rhMOG-induced EAE. MRI lesions, cytokines in blood, and CSF at onset of ADS or EAE, as well as histopathological features of brain lesions were compared. RESULTS: Twelve children with anti-MOG-Abs ADS (ADS MOG+) and nine macaques with EAE, presented increased IL-6 and G-CSF in the CSF, whereas no such signature was found in 15 ADS MOG-. Furthermore, IgG and C1q were associated to myelin and phagocytic cells in brains with EAE (n = 8) and in biopsies of ADS MOG+ (n = 2) but not ADS MOG- children (n = 1). Macaque brains also revealed prephagocytic lesions with IgG and C1q depositions but no leukocyte infiltration. CONCLUSIONS: Children with ADS MOG+ and macaques with EAE induced with rhMOG, present a similar cytokine signature in the CSF and a comparable aspect of brain lesions indicating analogous pathophysiological processes. In EAE, prephagocytic lesions points at IgG as an initial effector of myelin attack. These results support the pertinence of modeling ADS MOG+ in non-human primates to apprehend the natural development of anti-MOG-associated disease, find markers of evolution, and above all explore the efficacy of targeted therapies to test primate-restricted molecules.

Protein-Catalyzed Capture Agents.

Protein-catalyzed capture agents (PCCs) are synthetic and modular peptide-based affinity agents that are developed through the use of single-generation in situ click chemistry screens against large peptide libraries. In such screens, the target protein, or a synthetic epitope fragment of that protein, provides a template for selectively promoting the noncopper catalyzed azide-alkyne dipolar cycloaddition click reaction between either a library peptide and a known ligand or a library peptide and the synthetic epitope. The development of epitope-targeted PCCs was motivated by the desire to fully generalize pioneering work from the Sharpless and Finn groups in which in situ click screens were used to develop potent, divalent enzymatic inhibitors. In fact, a large degree of generality has now been achieved. Various PCCs have demonstrated utility for selective protein detection, as allosteric or direct inhibitors, as modulators of protein folding, and as tools for in vivo tumor imaging. We provide a historical context for PCCs and place them within the broader scope of biological and synthetic aptamers. The development of PCCs is presented as (i) Generation I PCCs, which are branched ligands engineered through an iterative, nonepitope-targeted process, and (ii) Generation II PCCs, which are typically developed from macrocyclic peptide libraries and are precisely epitope-targeted. We provide statistical comparisons of Generation II PCCs relative to monoclonal antibodies in which the protein target is the same. Finally, we discuss current challenges and future opportunities of PCCs.

Epitope-Targeted Macrocyclic Peptide Ligand with Picomolar Cooperative Binding to Interleukin-17F.

The IL-17 cytokine family is associated with multiple immune and autoimmune diseases and comprises important diagnostic and therapeutic targets. This work reports the development of epitope-targeted ligands designed for differential detection of human IL-17F and its closest homologue IL-17A. Non-overlapping and unique epitopes on IL-17F and IL-17A were identified by comparative sequence analysis of the two proteins. Synthetic variants of these epitopes were utilized as targets for in situ click screens against a comprehensive library of synthetic peptide macrocycles with 5-mer variable regions. Single generation screens yielded selective binders for IL-17F and IL-17A with low cross-reactivity. Macrocyclic peptide binders against two distinct IL-17F epitopes were coupled using variable length chemical linkers to explore the physical chemistry of cooperative binding. The optimized linker length yielded a picomolar affinity binder, while retaining high selectivity. The presented method provides a rational approach towards targeting discontinuous epitopes, similar to what is naturally achieved by many B cell receptors.

Lack of Evidence That Neural Empathic Responses Are Blunted in Excessive Users of Violent Video Games: An fMRI Study.

The use of violent video games has been often linked to increase of aggressive behavior. According to the General Aggression Model, one of the central mechanisms for this aggressiveness inducing impact is an emotional desensitization process resulting from long lasting repeated violent game playing. This desensitization should evidence itself in a lack of empathy. Recent research has focused primarily on acute, short term impact of violent media use but only little is known about long term effects. In this study 15 excessive users of violent games and control subjects matched for age and education viewed pictures depicting emotional and neutral situations with and without social interaction while fMRI activations were obtained. While the typical pattern of activations for empathy and theory of mind networks was seen, both groups showed no differences in brain responses. We interpret our results as evidence against the desensitization hypothesis and suggest that the impact of violent media on emotional processing may be rather acute and short-lived.

Excessive users of violent video games do not show emotional desensitization: an fMRI study.

Playing violent video games have been linked to long-term emotional desensitization. We hypothesized that desensitization effects in excessive users of violent video games should lead to decreased brain activations to highly salient emotional pictures in emotional sensitivity brain regions. Twenty-eight male adult subjects showing excessive long-term use of violent video games and age and education matched control participants were examined in two experiments using standardized emotional pictures of positive, negative and neutral valence. No group differences were revealed even at reduced statistical thresholds which speaks against desensitization of emotion sensitive brain regions as a result of excessive use of violent video games.

Protein catalyzed capture agents with tailored performance for in vitro and in vivo applications.

We report on peptide-based ligands matured through the protein catalyzed capture (PCC) agent method to tailor molecular binders for in vitro sensing/diagnostics and in vivo pharmacokinetics parameters. A vascular endothelial growth factor (VEGF) binding peptide and a peptide against the protective antigen (PA) protein of Bacillus anthracis discovered through phage and bacterial display panning technologies, respectively, were modified with click handles and subjected to iterative in situ click chemistry screens using synthetic peptide libraries. Each azide-alkyne cycloaddition iteration, promoted by the respective target proteins, yielded improvements in metrics for the application of interest. The anti-VEGF PCC was explored as a stable in vivo imaging probe. It exhibited excellent stability against proteases and a mean elimination in vivo half-life (T1/2 ) of 36 min. Intraperitoneal injection of the reagent results in slow clearance from the peritoneal cavity and kidney retention at extended times, while intravenous injection translates to rapid renal clearance. The ligand competed with the commercial antibody for binding to VEGF in vivo. The anti-PA ligand was developed for detection assays that perform in demanding physical environments. The matured anti-PA PCC exhibited no solution aggregation, no fragmentation when heated to 100°C, and > 81% binding activity for PA after heating at 90°C for 1 h. We discuss the potential of the PCC agent screening process for the discovery and enrichment of next generation antibody alternatives.

A single oral dose of a polyglucosamine influences the bioavailability of [9-(14)C]-Oleic acid in adult female Göttingen minipigs.

BACKGROUND: Worldwide obesity has nearly doubled since 1980 and is a leading risk for global deaths, profoundly affecting morbidity, mortality, health-care costs, and professional and personal quality of life. Treatment of obesity and its consequences include lifestyle intervention, pharmacotherapy, and bariatric surgery. Polyglucosamines have been proposed as an alternative strategy for treating obesity, by reducing the amount of absorbed fat through interaction with dietary fat through various mechanisms. The objective of this study is to investigate the influence of polyglucosamine on the bioavailability of the model compound [9-(14)C]-oleic acid in female Göttingen minipigs. METHOD: The study consisted of two treatment groups, each consisting of six adult female Göttingen minipigs with a catheterized vena jugularis to enable frequent blood sampling. One group served as the untreated group (control) and the other group was pre-treated with 2 tablets of 500 mg formoline L112. After 30 min, all animals were dosed orally with [9-(14)C]-oleic acid. Excreta and blood samples were collected for analysis of radioactivity from 48 h pre-dose up to 144 h post-dosing. At sacrifice, the liver and contents of the gastrointestinal tract were collected for radioanalysis. RESULTS: Upon treatment with polyglucosamine (formoline L112), the Tmax of [(14)C]-oleic acid in plasma was shifted from 4 to 16 h, and the Cmax decreased significantly from 14.1 µg/g to 3.3 µg/g. In addition, upon treatment with polyglucosamine the internal exposure to [(14)C]-oleic acid as reflected by the area under the curve during the 0-12 h post-dose time interval (AUC0-12h), is significantly decreased to 32.9 % of the plasma value of [(14)C]-oleic acid in untreated animals. Even up to 24 h post-dose, the AUC0-24h is significantly decreased to 50.7 % of the plasma value in untreated animals and this significant effect is prolonged up to 60 h post-dose. CONCLUSIONS: This study shows that treatment with polyglucosamine (formoline L112) reduces (as judged by Cmax & AUC) and delays (as judged by Tmax) fat absorption from the gastrointestinal tract into the systemic circulation and limits peak exposure to free fatty acids which may contribute to a more beneficial condition in overweight humans.

A cocktail of thermally stable, chemically synthesized capture agents for the efficient detection of anti-gp41 antibodies from human sera.

We report on a method to improve in vitro diagnostic assays that detect immune response, with specific application to HIV-1. The inherent polyclonal diversity of the humoral immune response was addressed by using sequential in situ click chemistry to develop a cocktail of peptide-based capture agents, the components of which were raised against different, representative anti-HIV antibodies that bind to a conserved epitope of the HIV-1 envelope protein gp41. The cocktail was used to detect anti-HIV-1 antibodies from a panel of sera collected from HIV-positive patients, with improved signal-to-noise ratio relative to the gold standard commercial recombinant protein antigen. The capture agents were stable when stored as a powder for two months at temperatures close to 60(o)C.

Pancreatic neuroendocrine neoplasms.

Pancreatic neuroendocrine tumors originate from the diffuse neuroendocrine system in the pancreatic region. These tumors exhibit a rising incidence despite their rareness and due to their benign behavior a considerable prevalence. Pathogenesis of pancreatic neuroendocrine tumors is characterized by common pathways of hereditary and sporadic tumors. Pancreatic neuroendocrine tumors may secrete peptide hormones or biogenic amines in an autonomous fashion as functional active tumors. Pathological grading and staging by TNM systems has been established in recent years classifying well and moderately differentiated pancreatice neuroendocrine tumors and poorly differentiated neuroendocrine carcinomas. Chromogranin A and less so pancreatic polypeptide are suitable tumor markers for pancreatic neuroendocrine tumors. Expression of receptors for somatostatin is the basis of treatment of pancreatic neuroendocrine tumors with somatostatin analogues as antisecretive and antiproliferative agents. In addition, somatostatin scintigraphy or PET/CT allows comprehensive diagnosis of pancreatic neuroendocrine tumors, which should be supported by (endoscopic and contrast enhanced) ultrasound, CT and MRI. Therapy of pancreatic neuroendocrine tumors consists of somatostatin analogues, chemotherapy, targeted therapy and peptide receptor radionuclide therapy. Two molecular substances hav been registered for pancreatic neuroendocrine tumors recently, sunitinib (Sutent®) and everolimus (Afinitor®). Predominant tumor load in the liver may be treated by local ablative therapy or liver transplantation. These treatment options have been included in guidelines of several professional societies and weighted for sequential therapy of patients with pancreatic neuroendocrine tumors according to effects and side effects.

Targeted delivery of CpG ODN to CD32 on human and monkey plasmacytoid dendritic cells augments IFNα secretion.

Atopic diseases are characterized by the presence of Th2 cells. Recent studies, in mice and man, demonstrated that allergen-specific Th2 responses can be shifted to Th0/Th1 responses. Plasmacytoid dendritic cells (pDCs) produce large amounts of type I interferons (IFNs) after stimulation of Toll Like Receptor 9 (TLR9) and are likely to play an important role in the reorientation of these Th2 cells. The expression of CD32a on the cell surface of pDCs makes this cell type attractive for targeted delivery of antigen and TLR agonists to revert Th2 responses. Therefore we sought to determine the efficacy of targeted delivery of CpG-C ODN to CD32a on the ability of human and monkey pDCs to secrete inflammatory cytokines. Here we demonstrate that targeted delivery of 3'-biotinylated CpG-C to CD32a on pDC induced phenotypical maturation as determined by CD80, CD83 and CD86 expression. Furthermore, targeting both monkey and human pDCs strongly augmented the secretion of IFNα compared to the delivery of CpG-C in an untargeted fashion (p<0.001). TLR9 induced activation hampers the ability of human pDCs to internalize CD32a. Therefore we opted for targeted delivery of CpG-ODNs to CD32a, which reduces the risk of undesired side effects of systemic TLR treatment and in addition delivers a superior signal for the activation of pDCs. This approach opens new treatment principles for allergic patients.

Animal models for arthritis: innovative tools for prevention and treatment.

The development of novel treatments for rheumatoid arthritis (RA) requires the interplay between clinical observations and studies in animal models. Given the complex molecular pathogenesis and highly heterogeneous clinical picture of RA, there is an urgent need to dissect its multifactorial nature and to propose new strategies for preventive, early and curative treatments. Research on animal models has generated new knowledge on RA pathophysiology and aetiology and has provided highly successful paradigms for innovative drug development. Recent focus has shifted towards the discovery of novel biomarkers, with emphasis on presymptomatic and emerging stages of human RA, and towards addressing the pathophysiological mechanisms and subsequent efficacy of interventions that underlie different disease variants. Shifts in the current paradigms underlying RA pathogenesis have also led to increased demand for new (including humanised) animal models. There is therefore an urgent need to integrate the knowledge on human and animal models with the ultimate goal of creating a comprehensive 'pathogenesis map' that will guide alignment of existing and new animal models to the subset of disease they mimic. This requires full and standardised characterisation of all models at the genotypic, phenotypic and biomarker level, exploiting recent technological developments in 'omics' profiling and computational biology as well as state of the art bioimaging. Efficient integration and dissemination of information and resources as well as outreach to the public will be necessary to manage the plethora of data accumulated and to increase community awareness and support for innovative animal model research in rheumatology.