VAF347, a novel low molecular weight immunomodulator, inhibits Interleukin-6 production in the human monocytic cell line Monomac1 via activation of the aryl hydrocarbon receptor

Interleukin (IL) -6 ist ein pleiotropes Zytokin das sowohl an der Regulation von Immunreaktionen, der Hämatopoese, dem Metabolismus des Knochens als auch an Entzündungsreaktionen beteiligt ist. Eine deregulierte Produktion von IL-6 ist bei mehreren Entzündungs- und Autoimmunerkrankungen gefunden worden, wie etwa bei Gelenkrheumatismus, Morbus Castleman oder Morbus Crohn. Deshalb stellt die Unterbrechung des IL-6 Signalweges ein interessantes therapeutisches Angriffsziel für die Behandlung dieser Krankheiten dar. Es wurde bereits gezeigt, dass VAF347 als ein neuer niedermolekularer Immunmodulator sowohl spezifisch die Immunglobulin-E Synthese von humanen B-Zellen, als auch die IL-6 Produktion von humanen Dendritischen Zellen monozytären Ursprungs inhibiert. Diese Studie wurde gestaltet, um das (die) molekulare(n) Ziel(e) von VAF347, das zur Inhibierung der IL-6 Produktion führt, zu identifizieren. Ähnlich wie bei primären Zellen inhibiert VAF347 die von IL-4 und GM-CSF als auch die von PMA induzierte IL-6 Produktion in der humanen monozytären Zelllinie Monomac1. Es wird gezeigt, dass der Aryl Hydrocarbon Rezeptor (AhR) das funktionell bedeutende Target für die Inhibition der IL-6 Produktion durch VAF347 ist. Der AhR ist für die biologische Aktivität von VAF347 in Monomac1 Zellen verantwortlich, da 1) andere AhR Liganden ein ähnliches Aktivitätsprofil zeigen, 2) ektopische Überexpression eines transdominant negativen AhR Proteins die biologische Aktivität von VAF347 aufhebt und 3) die Stilllegung des AhR Gens Monomac1 Zellen resistent gegen VAF347 macht. Darüber hinaus inhibiert VAF347 die Expression eines IL-6 Promoter Reporter-Gen Konstrukts und blockiert die Bindung des Transkriptionsfaktors NF-IL6, welcher als einer der Hauptfaktoren für die Regulation der IL-6 Expression angesehen wird, an den IL-6 Promoter. Die Überexpression des Wildtyp NF-IL6 Proteins hebt teilweise die Aktivität von VAF347 auf. Diese Ergebnisse demonstrieren, dass VAF347 seinen inhibitorischen Effekt auf transkriptioneller Ebene ausübt, da die Interaktion von NF-IL6 mit dessen Bindungsstelle im IL-6 Promoter verhindert wurde. Zusammenfassend identifizieren diese Daten AhR als das funktionell entscheidende Target von VAF347. Da die Deregulation von IL-6 am Krankheitsbild mehrerer inflammatorischer- und Autoimmunerkrankungen beteiligt ist, könnte das Targeting von AhR eine neue Möglichkeit zur Behandlung dieser Krankheiten darstellen.Interleukin (IL) -6 is a pleiotropic cytokine that is involved in the regulation of immune reactions, hematopoiesis, bone metabolism and inflammation. Deregulation of IL-6 has been shown to be involved in several inflammatory and autoimmune disorders, including rheumatoid arthritis, Castleman’s disease and Crohn’s disease. Therefore, interception of the IL-6 signaling pathway represents an interesting therapeutic target for the treatment of these diseases. VAF347 is a novel low molecular weight immunomodulator which has been shown to selectively block IgE synthesis in human B cells and to inhibit IL-6 production in human monocyte-derived dendritic cells. This study was designed to identify the molecular target(s) of VAF347 that lead to inhibition of IL-6 synthesis. Similar to primary cells, VAF347 inhibits IL-4 and GM-CSF as well as PMA induced IL-6 production in the human monocytic cell line Monomac1. We demonstrate, that the aryl hydrocarbon receptor (AhR) is the functionally relevant target for inhibition of IL-6 response by VAF347. The AhR is responsible for mediating the biological activity of VAF347 in Monomac1, since 1) other AhR ligands display an identical activity profile, 2) ectopic over-expression of a trans-dominant negative AhR protein abolishes VAF347 activity, and 3) silencing of AhR gene expression renders Monomac1 resistant to VAF347. Furthermore, VAF347 inhibits the activation of an IL-6 promoter reporter gene construct and blocks the binding of the transcription factor NF-IL6, which is thought to be one of the main factors involved in IL-6 regulation, to the IL-6 promoter. Over-expression of wild type NF-IL6 partially abolishes VAF347 activity. These findings showed that VAF347 mediates its inhibitory effect at the transcriptional level by inhibiting the interaction of NF-IL6 with its binding site on the IL-6 promoter. Collectively, these data identify the AhR as the functional target of VAF347. Given the fact that IL-6 deregulation is involved in the pathology of several inflammatory and autoimmune diseases, targeting the AhR may provide a new tool for the treatment of these diseases

Intra-molecular cross-linking of acidic residues for protein structure studies.

Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would be useful in developing structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine selective reagents that can cross-link lysine residues to other lysine residues or the amino terminus. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution, and solvent accessibility of the lysines in the protein sequence. To overcome these limitations we have investigated the use of cross-linking reagents that can react with other reactive sidechains in proteins. We used 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E), and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO sidechains can react to form 'zero-length' cross-links with nearby primary amine containing resides, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO sidechains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker ann of variable length. Using these reagents, we have found three new 'zero-length' cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18, and K63-E64). Using the dihydrazide cross-linkers, we have identified 2 new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 angstroms. These results show that additional structural information can be obtained by exploiting new cross-linker chemistry, increasing the probability that the protein target of choice will yield sufficient distance constraints to develop a structural model

An insulin hypersecretion phenotype precedes pancreatic β cell failure in MODY3 patient-specific cells

MODY3 is a monogenic hereditary form of diabetes caused by mutations in the transcription factor HNF1A. The patients progressively develop hyperglycemia due to perturbed insulin secretion, but the pathogenesis is unknown. Using patient-specific hiPSCs, we recapitulate the insulin secretion sensitivity to the membrane depolarizing agent sulfonylurea commonly observed in MODY3 patients. Unexpectedly, MODY3 patient-specific HNF1A+/R272C β cells hypersecrete insulin both in vitro and in vivo after transplantation into mice. Consistently, we identified a trend of increased birth weight in human HNF1A mutation carriers compared with healthy siblings. Reduced expression of potassium channels, specifically the KATP channel, in MODY3 β cells, increased calcium signaling, and rescue of the insulin hypersecretion phenotype by pharmacological targeting ATP-sensitive potassium channels or low-voltage-activated calcium channels suggest that more efficient membrane depolarization underlies the hypersecretion of insulin in MODY3 β cells. Our findings identify a pathogenic mechanism leading to β cell failure in MODY3.Peer reviewe

Chemical crosslinking and mass spectrometry studies of the structure and dynamics of membrane proteins and receptors.

Membrane proteins make up a diverse and important subset of proteins for which structural information is limited. In this study, chemical cross-linking and mass spectrometry were used to explore the structure of the G-protein-coupled photoreceptor bovine rhodopsin in the dark-state conformation. All experiments were performed in rod outer segment membranes using amino acid 'handles' in the native protein sequence and thus minimizing perturbations to the native protein structure. Cysteine and lysine residues were covalently cross-linked using commercially available reagents with a range of linker arm lengths. Following chemical digestion of cross-linked protein, cross-linked peptides were identified by accurate mass measurement using liquid chromatography-fourier transform mass spectrometry and an automated data analysis pipeline. Assignments were confirmed and, if necessary, resolved, by tandem MS. The relative reactivity of lysine residues participating in cross-links was evaluated by labeling with NHS-esters. A distinct pattern of cross-link formation within the C-terminal domain, and between loop I and the C-terminal domain, emerged. Theoretical distances based on cross-linking were compared to inter-atomic distances determined from the energy-minimized X-ray crystal structure and Monte Carlo conformational search procedures. In general, the observed cross-links can be explained by re-positioning participating side-chains without significantly altering backbone structure. One exception, between C3 16 and K325, requires backbone motion to bring the reactive atoms into sufficient proximity for cross-linking. Evidence from other studies suggests that residues around K325 for a region of high backbone mobility. These findings show that cross-linking studies can provide insight into the structural dynamics of membrane proteins in their native environment

The nuclear receptor PPARγ selectively inhibits Th17 differentiation in a T cell–intrinsic fashion and suppresses CNS autoimmunity

T helper cells secreting interleukin (IL)-17 (Th17 cells) play a crucial role in autoimmune diseases like multiple sclerosis (MS). Th17 differentiation, which is induced by a combination of transforming growth factor (TGF)-β/IL-6 or IL-21, requires expression of the transcription factor retinoic acid receptor–related orphan receptor γt (RORγt). We identify the nuclear receptor peroxisome proliferator–activated receptor γ (PPARγ) as a key negative regulator of human and mouse Th17 differentiation. PPARγ activation in CD4+ T cells selectively suppressed Th17 differentiation, but not differentiation into Th1, Th2, or regulatory T cells. Control of Th17 differentiation by PPARγ involved inhibition of TGF-β/IL-6–induced expression of RORγt in T cells. Pharmacologic activation of PPARγ prevented removal of the silencing mediator for retinoid and thyroid hormone receptors corepressor from the RORγt promoter in T cells, thus interfering with RORγt transcription. Both T cell–specific PPARγ knockout and endogenous ligand activation revealed the physiological role of PPARγ for continuous T cell–intrinsic control of Th17 differentiation and development of autoimmunity. Importantly, human CD4+ T cells from healthy controls and MS patients were strongly susceptible to PPARγ-mediated suppression of Th17 differentiation. In summary, we report a PPARγ-mediated T cell–intrinsic molecular mechanism that selectively controls Th17 differentiation in mice and in humans and that is amenable to pharmacologic modulation. We therefore propose that PPARγ represents a promising molecular target for specific immunointervention in Th17-mediated autoimmune diseases such as MS

The interfacial bioscience grand challenge.

This report is broken down into the following 3 sections: (1) Chemical Cross-linking and Mass Spectrometry Applied to Determination of Protein Structure and Dynamics; (2) Computational Modeling of Membrane Protein Structure and Dynamics; and (3) Studies of Toxin-Membrane Interactions using Single Molecule Biophysical Methods

Insulin gene VNTR genotype associates with frequency and phenotype of the autoimmune response to proinsulin

Immune responses to autoantigens are in part controlled by deletion of autoreactive cells through genetically regulated selection mechanisms. We have directly analyzed peripheral CD4+ proinsulin (PI) 76–90 (SLQPLALEGSLQKRG)-specific T cells using soluble fluorescent major histocompatibility complex class II tetramers. Subjects with type I diabetes and healthy controls with high levels of peripheral proinsulin-specific T cells were characterized by the presence of a disease-susceptible polymorphism in the insulin variable number of tandem repeats (INS-VNTR) gene. Conversely, subjects with a ‘protective' polymorphism in the INS-VNTR gene had nearly undetectable levels of proinsulin tetramer-positive T cells. These results strongly imply a direct relationship between genetic control of autoantigen expression and peripheral autoreactivity, in which proinsulin genotype restricts the quantity and quality of the potential T-cell response. Using a modified tetramer to isolate low-avidity proinsulin-specific T cells from subjects with the susceptible genotype, transcript arrays identified several induced pro-apoptotic genes in the control, but not diabetic subjects, likely representing a second peripheral mechanism for maintenance of tolerance to self antigens

Koštani morfogenetski proteini (BMP): Od otkrića do razvoja nove autologne koštane naprave koja se sastoji od rekombinantnog humanog BMP6 u autolognom krvnom ugrušku kao nosaču

Bone Morphogenetic Proteins (BMPs) are growth and differentiation factors within the TGFβ superfam- ily of proteins. They induce ectopic and orthotopic endochondral bone formation and are involved in the regulation of cell proliferation, differentiation, apoptosis and mesenchymal-epithelial interactions in critical morphogenetic processes of tissues beyond bone. BMP2 and BMP7 osteogenic devices have been approved for enhancing healing in patients with long bone defects and anterior spinal fusion proce- dures. However, due to a high price and various serious adverse events including heterotopic ossifica- tion, retrograde ejaculation and pain their clinical use have been limited. In this review we discuss the BMP discovery, biology and their use in clinical studies with particular reference to the newly developed BMP6 based autologous bone graft substitute (ABGS). A novel ABGS consisting of an autologous bone coagulum (ABC) carrier with dispersed BMP6 to initiate the differentiation of mesenchymal cells into endochondral bone. The ABC met the conditions for an optimal delivery system for BMP6 due to han- dling simplicity, without an immunogenic and inflammatory response at the implantation site. Addition of allograft or synthetic ceramics to ABGS demonstrated in animal models significantly increased volume and better microarchitecture of the newly formed bone. The first clinical study was conducted in patients with distal radial fractures (Phase I study) and the second in patients undergoing high tibial osteotomy (Phase I/II study) and no serious adverse events have been observed. Finally, in the ongoing OSTEO- proSPINE study ABGS enforced with allograft bone is evaluated in patients with chronic back pain due to degenerative disc diseases. The novel ABGS bone mimetic is a major breakthrough and contribution to bone biology and regenerative medicine of skeletal repair.Koštani morfogenetski proteini (BMP) čine grupu čimbenika rasta i diferencijacije unutar TGFβ nado- bitelji. Oni induciraju stvaranje ektopične i ortotopične endohondralne kosti te su uključeni u regulaciju stanične proliferacije, diferencijacije, apoptoze i mezenhimalno-epitelne interakcije u važnim tkivnim morfogenetskim procesima izvan koštanog sustava. Koštane naprave koje sadrže BMP2 i BMP7 pro- tein odobrene su za poboljšanje koštanog cijeljenja kod pacijenata s defektima dugih cjevastih kostiju i kod prednje spinalne fuzije kralježnice. Međutim, zbog visoke cijene i mnogobrojnih nuspojava koje su uključivale pojavu heterotopičnih osifikacija, retrogradnu ejakulaciju i bol, njihova je klinička prim- jena ograničena. U ovom smo preglednom radu raspravili otkriće BMP molekula, njihovu biologiju i primjenu u kliničkim studijama s posebnim osvrtom na nedavno otkrivenu novu autolognu koštanu napravu (ABGS) koja sadrži BMP6. Novi ABGS sastoji se od nosača autolognog koaguluma (ABC) s otopljenim BMP6 koji je ključan za pokretanje diferencijacije mezenhimalnih stanica u smjeru stvaranja endohondralne kosti. ABC je ispunio sve potrebne uvjete za formulaciju optimalnog nosača za BMP6 isključivo zbog jednostavnosti priprave i primjene te odsustva imunogenog i upalnog odgovora na mjestu implantacije. Uz dodatak alografta ili sintetičke keramike što je potvrđeno na životinjskim modelima došlo je do značajnog povećanja volumena te poboljšanja mikroarhitekture novonastale kosti. Prvo kliničko ispitivanje provedeno je na pacijentima s distalnim prijelomima radijusa (faza I studije), a drugo na pacijentima koji su podvrgnuti visokoj osteotomiji tibije (faza I/II studije) bez uočenih ozbiljnih nuspojava. Trenutno je u tijeku studija OSTEOproSPINE u kojoj se testira učinkovitost ABGS u kom- binaciji s koštanim alograftom u bolesnika s kroničnim bolovima u leđima uzrokovanim degenerativnim promjenama intervertebralnog diska. Nova ABGS koštana naprava značajna je prekretnica i napredak u području koštane biologije te regenerativne medicine koštanog sustava

Teaching the Process of Science: Faculty Perceptions and an Effective Methodology

Most scientific endeavors require science process skills such as data interpretation, problem solving, experimental design, scientific writing, oral communication, collaborative work, and critical analysis of primary literature. These are the fundamental skills upon which the conceptual framework of scientific expertise is built. Unfortunately, most college science departments lack a formalized curriculum for teaching undergraduates science process skills. However, evidence strongly suggests that explicitly teaching undergraduates skills early in their education may enhance their understanding of science content. Our research reveals that faculty overwhelming support teaching undergraduates science process skills but typically do not spend enough time teaching skills due to the perceived need to cover content. To encourage faculty to address this issue, we provide our pedagogical philosophies, methods, and materials for teaching science process skills to freshman pursuing life science majors. We build upon previous work, showing student learning gains in both reading primary literature and scientific writing, and share student perspectives about a course where teaching the process of science, not content, was the focus. We recommend a wider implementation of courses that teach undergraduates science process skills early in their studies with the goals of improving student success and retention in the sciences and enhancing general science literacy