Production and Evaluation of Ligand Sneaking-Antibody Fusion Proteins

Ligand Sneaking-Proteine sind Antikörperfusionsproteine, mit denen intrazelluläre Signaltransduktionswege moduliert werden sollen. Sie bestehen aus drei Komponenten: Der erste Teil besteht aus einem scFv-Antikörperfragment, mit dem das Protein an einen internalisierenden Zelloberflächenrezeptor bindet. Der zweite Teil besteht aus einer Translokationsdomäne, welche die Translokation des dritten Teils, der Effektordomäne, aus dem Endosom in das Zytoplasma vermittelt. Die Effektordomäne interagiert im Zytoplasma mit Komponenten des Signaltransduktionsweges, um diesen zu modulieren. In einer vorangegangenen Arbeit wurde ein Ligand Sneaking-Fusionsprotein aus einem gegen den Transferrinrezeptor CD71 gerichteten scFv-Antikörperfragment, der Translokationsdomäne ETA II aus dem Pseudomonas Exotoxin A und einer NEMO binding domain (NBD) aus dem humanen IkappaB Kinase 2 (IKK-2)-Protein als Effektordomäne entwickelt. NBD bindet intrazellulär an NEMO (NF-kappaB essential modulator), die regulatorische Untereinheit des IKK-Komplexes, wodurch die Aktivierung des Transkriptionsfaktors NF-kappaB durch proinflammatorische Stimuli unterbrochen wird. Die Ausbeute bei der Produktion des Ligand Sneaking-Antikörperfusionsproteins durch Sekretion in das Periplasma von E. coli war sehr gering. In dieser Arbeit wurden die Ursachen dafür untersucht und mehrere Produktionsmethoden auf ihre Eignung für die Produktion des Fusionsproteins überprüft. Es konnten grundlegende Voraussetzungen für die Produktion von Ligand Sneaking-Antikörperfusionsproteinen geklärt werden. Es stellte sich heraus, dass sowohl der gewählte scFv-Klon als auch die verwendete NBD-Variante die Sekretion massiv behinderten. Durch Austausch des scFv-Antikörperfragmentes und der NBD-Variante im LS-Fusionsprotein gegen andere Varianten ließen sich Fusionsproteine durch Sekretion ins Periplasma von E. coli erfolgreich produzieren und aufreinigen. Das Protein band spezifisch an Zielzellen. Eine Reduktion der NF-kappaB-Aktivität konnte über einen zellbasierten Reporterassay allerdings nicht nachgewiesen werden, wofür eine geringe Affinität des alternativen scFv-Klons und die mangelnde Sensitivität des Assaysystems verantwortlich waren.The Ligand Sneaking concept aims at the manipulation of intracellular signal transduction pathways using antibody fusion proteins that consist of three parts. The first part is an scFv antibody fragment directed against an internalizing receptor on the surface of a target cell. The second part of the fusion protein is a translocation domain that is capable of translocating the third part of the fusion protein, an effector domain, from the endosome into the cytoplasm of the target cell. In the cytoplasm the effector domain interacts with components of the signal transduction pathway, thereby leading to its disruption or down-regulation. In a previous work a Ligand Sneaking fusion protein was developed that consisted of an scFv antibody fragment directed against the transferrin receptor CD71, the translocation domain ETA II derived from the Pseudomonas Exotoxin A and the NEMO binding domain (NBD) derived from the human IkappaB kinase (IKK) 2 protein as the effector domain that binds to the intracellular protein NEMO (NF-kappaB essential modulator). NEMO represents the regulatory subunit of the IKK complex. By binding of NBD to NEMO the formation of the native IKK complex is inhibited. The yield of Ligand Sneaking fusion proteins produced by secretion into the periplasm of E. coli was very low. In this work the potential of several production methods to yield functional fusion protein was evaluated and basic requirements for the production of Ligand Sneaking fusion proteins were identified. It was shown that the scFv antibody fragment and the NBD variant used in the original fusion protein were detrimental to secretion. New anti-CD71 scFv antibody fragments were isolated from phage display libraries und characterised. After exchange of both the scFv antibody fragment and the NBD variant originally used in the Ligand Sneaking fusion protein modified fusion proteins were successfully produced in E. coli and purified. The material specifically bound to target cells. However, using a cell based NF-kappaB activity reporter assay it was not possible to show a reduction of NF-kappaB-activity because of the low affinity of the alternative scFv antibody fragment and the low sensitivity of the assay system

Single chain Fab (scFab) fragment

BACKGROUND: The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display. RESULTS: Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd) and the light chain (LC), resulting in the formation of a single chain Fab fragment (scFab), can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabΔC) connecting the constant part 1 of the heavy chain (CH1) and the constant part of the light chain (CL) were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies) as well as multimers were characterised. CONCLUSION: A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of common standard sera for detection

Mass spectrometry imaging identifies palmitoylcarnitine as an immunological mediator during Salmonella Typhimurium infection

Salmonella Typhimurium causes a self-limiting gastroenteritis that may lead to systemic disease. Bacteria invade the small intestine, crossing the intestinal epithelium from where they are transported to the mesenteric lymph nodes (MLNs) within migrating immune cells. MLNs are an important site at which the innate and adaptive immune responses converge but their architecture and function is severely disrupted during S. Typhimurium infection. To further understand host-pathogen interactions at this site, we used mass spectrometry imaging (MSI) to analyse MLN tissue from a murine model of S. Typhimurium infection. A molecule, identified as palmitoylcarnitine (PalC), was of particular interest due to its high abundance at loci of S. Typhimurium infection and MLN disruption. High levels of PalC localised to sites within the MLNs where B and T cells were absent and where the perimeter of CD169+ sub capsular sinus macrophages was disrupted. MLN cells cultured ex vivo and treated with PalC had reduced CD4+CD25+ T cells and an increased number of B220+CD19+ B cells. The reduction in CD4+CD25+ T cells was likely due to apoptosis driven by increased caspase-3/7 activity. These data indicate that PalC significantly alters the host response in the MLNs, acting as a decisive factor in infection outcome

Nature and consequences of interactions between Salmonella enterica serovar Dublin and host cells in cattle

International audienceAbstractSalmonella enterica is a veterinary and zoonotic pathogen of global importance. While murine and cell-based models of infection have provided considerable knowledge about the molecular basis of virulence of Salmonella, relatively little is known about salmonellosis in naturally-affected large animal hosts such as cattle, which are a reservoir of human salmonellosis. As in humans, Salmonella causes bovine disease ranging from self-limiting enteritis to systemic typhoid-like disease and exerts significant economic and welfare costs. Understanding the nature and consequences of Salmonella interactions with bovine cells will inform the design of effective vaccines and interventions to control animal and zoonotic infections. In calves challenged orally with S. Dublin expressing green fluorescent protein (GFP) we observed that the bacteria were predominantly extracellular in the distal ileal mucosa and within gut-associated lymph nodes 48 h post-infection. Intracellular bacteria, identified by flow cytometry using the GFP signal, were predominantly within MHCII+ macrophage-like cells. In contrast to observations from murine models, these S. Dublin-infected cells had elevated levels of MHCII and CD40 compared to both uninfected cells from the same tissue and cells from the cognate tissue of uninfected animals. Moreover, no gross changes of the architecture of infected lymph nodes were observed as was described previously in a mouse model. In order to further investigate Salmonella-macrophage interactions, net replication of S. enterica serovars that differ in virulence in cattle was measured in bovine blood-derived macrophages by enumeration of gentamicin-protected bacteria and fluorescence dilution, but did not correlate with host-specificity

Compartment-specific immunity in the human gut: Properties and functions of dendritic cells in the colon versus the ileum

© 2015 The Authors. Published by BMJ. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: http://dx.doi.org/10.1136/gutjnl-2014-307916Objective Dendritic cells (DC) mediate intestinal immune tolerance. Despite striking differences between the colon and the ileum both in function and bacterial load, few studies distinguish between properties of immune cells in these compartments. Furthermore, information of gut DC in humans is scarce. We aimed to characterise human colonic versus ileal DC. Design Human DC from paired colonic and ileal samples were characterised by flow cytometry, electron microscopy or used to stimulate T cell responses in a mixed leucocyte reaction. Results A lower proportion of colonic DC produced pro-inflammatory cytokines (tumour necrosis factor-a and interleukin (IL)-1ß) compared with their ileal counterparts and exhibited an enhanced ability to generate CD4+FoxP3+IL-10+ (regulatory) T cells. There were enhanced proportions of CD103+Sirpa- DC in the colon, with increased proportions of CD103+Sirpa+ DC in the ileum. A greater proportion of colonic DC subsets analysed expressed the lymph-node-homing marker CCR7, alongside enhanced endocytic capacity, which was most striking in CD103+Sirpa+ DC. Expression of the inhibitory receptor ILT3 was enhanced on colonic DC. Interestingly, endocytic capacity was associated with CD103+ DC, in particular CD103+Sirpa+ DC. However, expression of ILT3 was associated with CD103- DC. Colonic and ileal DC differentially expressed skin-homing marker CCR4 and small-bowel-homing marker CCR9, respectively, and this corresponded to their ability to imprint these homing markers on T cells. Conclusions The regulatory properties of colonic DC may represent an evolutionary adaptation to the greater bacterial load in the colon. The colon and the ileum should be regarded as separate entities, each comprising DC with distinct roles in mucosal immunity and imprinting.This research was funded by St. Mark's Foundation (Harrow, UK), The Biotechnology and Biological Sciences Research Council (BBSRC; BB/J004529/1) and The National Institutes of Health (NIH; US) including The National Institute of Diabetes and Digestive and Kidney Diseases (NIH/NIDDK; T32-DK07632 and P01-DK072084) and The National Institute of Allergy and Infectious Disease (NIH/NIAID; R21-AI094033). We also gratefully acknowledge funding support from The Harvey M. and Lyn P. Meyerhoff Inflammatory Bowel Disease Centre at The Johns Hopkins Hospital, Baltimore, US.Published versio

Peripheral nerve injury associated with a subdermal contraceptive implant: illustrative cases and systematic review of literature

BACKGROUND: Despite demonstrable safety and efficacy of subdermal contraceptive implants (SCIs), both insertion and removal of SCIs in the arm have been associated with neurovascular complications. The aim of this study was to investigate type and prognosis of nerve injuries associated with SCIs. METHODS: We performed a comprehensive search of 4 electronic databases for studies pertaining to patients with nerve injury and concurrent SCI. Studies published between January 1987 and June 2017 were included. Implant location, damaged nerves, clinical presentation, preoperative imaging (x-ray, ultrasound, magnetic resonance imaging), neurologic evaluation (nerve conduction studies, electromyography), and treatment methods were reviewed. To outline management strategies, 2 illustrative cases of major nerve injury caused by SCI removal were presented. RESULTS: We analyzed 10 studies including 12 patients. Fourteen nerve injuries in 12 patients were reported during SCI insertion (n = 1) and removal (n = 11). Medial antebrachial cutaneous (n = 5) and median (n = 5) nerves were primarily affected. Neuropathic pain was the main symptom. Primary reasons for nerve injury were pulling or grasping of the nerve (n = 9) after mistaking it for the implant. Neurapraxia (n = 7) was the most common lesion and was treated with implant removal and clinical surveillance (n = 6). Five patients completely recovered; the remaining patients continued to have motor and/or sensory deficit at mean follow-up of 0.7 year (range, 0-2 years). CONCLUSIONS: Nerve injuries related to SCIs are rare but potentially serious. For nonpalpable SCIs, a multidisciplinary approach, including practitioners with experience treating peripheral nerve injuries, is invaluable

Retrospective application of transposon-directed insertion-site sequencing to investigate niche-specific virulence of Salmonella Typhimurium in cattle.

Background: Salmonella enterica subspecies enterica is an animal and zoonotic pathogen of global importance. Cattle are a significant reservoir of human non-typhoidal salmonellosis and can suffer enteric and systemic disease owing to the ability of Salmonella to survive within the bovine lymphatic system and intestines. Contamination of food can occur due to the incorporation of contaminated peripheral lymph nodes or by direct contamination of carcasses with gut contents. It is essential to understand the mechanisms used by Salmonella to enter and persist within the bovine lymphatic system and how they differ from those required for intestinal colonization to minimize zoonotic infections. Results: Transposon-directed insertion site sequencing (TraDIS) was applied to pools of mutants recovered from mesenteric lymph nodes (MLNs) draining the distal ileum of calves after oral inoculation with a library of 8550 random S. Typhimurium mini-Tn5Km2 mutants in pools of 475 mutants per calf. A total of 8315 mutants representing 2852 different genes were detected in MLNs and their in vivo fitness was calculated. Using the same improved algorithm for analysis of transposon-flanking sequences, the identity and phenotype of mutants recovered from the distal ileal mucosa of the same calves was also defined, enabling comparison with previously published data and of mutant phenotypes across the tissues. Phenotypes observed for the majority of mutants were highly significantly correlated in the two tissues. However, 32 genes were identified in which transposon insertions consistently resulted in differential fitness in the ileal wall and MLNs, suggesting niche-specific roles for these genes in pathogenesis. Defined null mutations affecting ptsN and spvC were confirmed to result in tissue-specific phenotypes in calves, thus validating the TraDIS dataset. Conclusions: This validation of the role of thousands of Salmonella genes and identification of genes with niche-specific roles in a key target species will inform the design of control strategies for bovine salmonellosis and zoonotic infections, for which efficacious and cross-protective vaccines are currently lacking

Testosterone Therapy in Women: A Clinical Challenge.

The physiology of testosterone as a normal female hormone in reproductive years and beyond is poorly taught and understood. This has led to unregulated and dangerous prescribing practices by physicians and other health care professionals. There are data for safe use, and as women\u27s health care practitioners, we owe it to our patients to follow these guidelines and practices, as well as advocate for more research and safer, regulated products to prescribe

Neuropeptides influence airway dendritic cell behavior

The airway mucosal epithelium permanently faces airborne particles. A network of immune cells patrols at this frontier to the environmental surface. The interplay of immune cells is orchestrated by different mediators. In the current study we investigated whether neuropeptides can alter key features of dendritic cells (DC) such as movement behavior and phagocytosis capacity. With a two-photon microscopic time-lapse analysis of DC in the airways of ex vivo vital lung sections of CD11c-EYFP transgenic mice we focused on the influence of neuropeptides on DC. Additionally, with a confocal microscopic approach and by means of particles becoming fluorescent in the phagolysosomal milieu we determined the phagocytosis capacity of CD11c+ cells. Neuronal irritation here mimicked by electrical field stimulation (EFS) leads to an unspecific release of several neuropeptides in nerves. After EFS of vital lung slices, airway DC showed an increased motility. In subsequent experiments this effect could be reproduced by specific application of the neuropeptide calcitonin gene-related peptide (CGRP). The EFS-mediated effect could partially be blocked by pre-treatment with the neuropeptide receptor antagonist CGRP8-37. In contrast, the application of the neuropeptide vasoactive intestinal peptide (VIP) led to a decrease of airway DC motility. Additionally, phagocytosis capacity of bone marrow-derived and whole lung CD11c+ cells could be negatively affected by neuropeptides CGRP, VIP, and Substance P. We then correlated these data with the in vivo situation by analyzing DC motility in two different OVA asthma models. Both in the acute and prolonged OVA asthma model we could determine altered neuropeptide amounts in the airways and DC motility. In summary, our data suggest that neuropeptides alter key features motility and phagocytosis of mouse airway DC and therefore might contribute to the pathophysiology of asthma. This work was funded by the SFB 587 B