The SARS-coronavirus-host interactome

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock

Immunogene therapy with fusogenic nanoparticles modulates macrophage response to Staphylococcus aureus.

The incidence of adverse effects and pathogen resistance encountered with small molecule antibiotics is increasing. As such, there is mounting focus on immunogene therapy to augment the immune system's response to infection and accelerate healing. A major obstacle to in vivo gene delivery is that the primary uptake pathway, cellular endocytosis, results in extracellular excretion and lysosomal degradation of genetic material. Here we show a nanosystem that bypasses endocytosis and achieves potent gene knockdown efficacy. Porous silicon nanoparticles containing an outer sheath of homing peptides and fusogenic liposome selectively target macrophages and directly introduce an oligonucleotide payload into the cytosol. Highly effective knockdown of the proinflammatory macrophage marker IRF5 enhances the clearance capability of macrophages and improves survival in a mouse model of Staphyloccocus aureus pneumonia

Methods of Transfection with Messenger RNA Gene Vectors

Non-viral gene delivery vectors with messenger RNA (mRNA) as a carrier of genetic information are among the staple gene transfer vectors for research in gene therapy, gene vaccination and cell fate reprogramming. As no passage of genetic cargo in and out of the nucleus is required, mRNA-based vectors typically offer the following five advantages: 1) fast start of transgene expression; 2) ability to express genes in non-dividing cells with an intact nuclear envelope; 3) insensitivity to the major gene silencing mechanisms, which operate in the nucleus; 4) absence of potentially mutagenic genomic insertions; 5) high cell survival rate after transfection procedures, which do not need to disturb nuclear envelope. In addition, mRNA-based vectors offer a simple combination of various transgenes through mixing of several mRNAs in a single multi-gene cocktail or expression of a number of proteins from a single mRNA molecule using internal ribosome entry sites (IRESes), ribosome skipping sequences and proteolytic signals. However, on the downside, uncontrolled extracellular and intracellular decay of mRNA can be a substantial hurdle for mRNA-mediated gene transfer. Procedures for mRNA delivery are analogous to DNA transfer methods, which are well-established. In general, there are three actors in the gene delivery play, namely, the vector, the cell and the transfer environment. The desired outcome, that is, the efficient delivery of a gene to a target cell population, depends on the efficient interaction of all three parties. Thus, the vector should be customised for the target cell population and presented in a form that is resistant to the aggressive factors in the delivery milieu. At the same time, the delivery environment should be adjusted to be more vector-friendly and more cell-friendly. The recipient cells should be subjected to a specific regimen or artificially modified to become receptive to gene transfer with a particular vector and resistant to the environment. As a rule, barriers outside tissues (e.g. mucus) and an aggressive intercellular environment complicate gene delivery in vivo, which, therefore, requires more complex gene transfer procedures than transfection of tissue culture cells. This review is focused on transfection methods for mRNA vectors, which rely either on the forceful propulsion of mRNA inside the target cells (e.g. by electroporation or gene gun) or on the complexing of mRNA with other substances (e.g. polycationic transfection reagents) for delivery via endocytic pathways

Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and b1-integrin activation

Lymphatic invasion and lymph node metastasis correlate with poor clinical outcome in melanoma. However, the mechanisms of lymphatic dissemination in distant metastasis remain incompletely understood. We show here that exposure of expansively growing human WM852 melanoma cells, but not singly invasive Bowes cells, to lymphatic endothelial cells (LEC) in 3D co-culture facilitates melanoma distant organ metastasis in mice. To dissect the underlying molecular mechanisms, we established LEC co-cultures with different melanoma cells originating from primary tumors or metastases. Notably, the expansively growing metastatic melanoma cells adopted an invasively sprouting phenotype in 3D matrix that was dependent on MMP14, Notch3 and β1-integrin. Unexpectedly, MMP14 was necessary for LEC-induced Notch3 induction and coincident β1-integrin activation. Moreover, MMP14 and Notch3 were required for LEC-mediated metastasis of zebrafish xenografts. This study uncovers a unique mechanism whereby LEC contact promotes melanoma metastasis by inducing a reversible switch from 3D growth to invasively sprouting cell phenotype

Calcium Phosphate as a Key Material for Socially Responsible Tissue Engineering

Socially responsible technologies are designed while taking into consideration the socioeconomic, geopolitical and environmental limitations of regions in which they will be implemented. In the medical context, this involves making therapeutic platforms more accessible and affordable to patients in poor regions of the world wherein a given disease is endemic. This often necessitates going against the reigning trend of making therapeutic nanoparticles ever more structurally complex and expensive. However, studies aimed at simplifying materials and formulations while maintaining the functionality and therapeutic response of their more complex counterparts seldom provoke a significant interest in the scientific community. In this review we demonstrate that such compositional simplifications are meaningful when it comes to the design of a solution for osteomyelitis, a disease that is in its natural, non-postoperative form particularly prevalent in the underdeveloped parts of the world wherein poverty, poor sanitary conditions, and chronically compromised defense lines of the immune system are the norm. We show that calcium phosphate nanoparticles, which are inexpensive to make, could be chemically designed to possess the same functionality as a hypothetic mixture additionally composed of: (a) a bone growth factor; (b) an antibiotic for prophylactic or anti-infective purposes; (c) a bisphosphonate as an antiresorptive compound; (d) a viral vector to enable the intracellular delivery of therapeutics; (e) a luminescent dye; (f) a radiographic component; (g) an imaging contrast agent; (h) a magnetic domain; and (i) polymers as viscous components enabling the injectability of the material and acting as carriers for the sustained release of a drug. In particular, calcium phosphates could: (a) produce tunable drug release profiles; (b) take the form of viscous and injectable, self-setting pastes; (c) be naturally osteo-inductive and inhibitory for osteoclastogenesis; (d) intracellularly deliver bioactive compounds; (e) accommodate an array of functional ions; (f) be processed into macroporous constructs for tissue engineering; and (g) be naturally antimicrobial. All in all, we see in calcium phosphates the presence of a protean nature whose therapeutic potentials have been barely tapped into

A review of emerging physical transfection methods for CRISPR/Cas9-mediated gene editing

Gene editing is a versatile technique in biomedicine that promotes fundamental research as well as clinical therapy. The development of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) as a genome editing machinery has accelerated the application of gene editing. However, the delivery of CRISPR components often suffers when using conventional transfection methods, such as viral transduction and chemical vectors, due to limited packaging size and inefficiency toward certain cell types. In this review, we discuss physical transfection methods for CRISPR gene editing which can overcome these limitations. We outline different types of physical transfection methods, highlight novel techniques to deliver CRISPR components, and emphasize the role of micro and nanotechnology to improve transfection performance. We present our perspectives on the limitations of current technology and provide insights on the future developments of physical transfection methods

Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma

The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma and atopic dermatitis. The emergence of human TSLP as a clinical target against asthma calls for maximally harnessing its therapeutic potential via structural and mechanistic considerations. Here we employ an integrative experimental approach focusing on productive and antagonized TSLP complexes and free cytokine. We reveal how cognate receptor TSLPR allosterically activates TSLP to potentiate the recruitment of the shared interleukin 7 receptor a-chain (IL-7Ra) by leveraging the flexibility, conformational heterogeneity and electrostatics of the cytokine. We further show that the monoclonal antibody Tezepelumab partly exploits these principles to neutralize TSLP activity. Finally, we introduce a fusion protein comprising a tandem of the TSLPR and IL-7Ra extracellular domains, which harnesses the mechanistic intricacies of the TSLP-driven receptor complex to manifest high antagonistic potency

Statische und dynamische Magnetfelder für die Nanopartikel-basierte zielgerichtete Wirkstofffreisetzung

Although medicine has made great progress in the last centuries and decades, it is still facing basic challenges that make doctors fail to efficiently and successfully treat the continuously emerging diseases and ailments due to ageing, industrialization, pollution and resulting biological mutations. In this context, the systemic chemotherapeutic treatment of cancer seems to be one of the most fitting examples for the wide gap between the usually followed medical approach and the theoretically optimal solution. Extrapolating from in vitro experiments and mouse models to humans, treating children as “miniaturized” adults when analyzing therapeutic effects, estimating drug doses based on relatively coarse processes like up scaling on weight, volume or area, and flooding the human body with drugs to solely achieve a minimal effect at the ailment site are just few examples for improvement needs in medical methods. One of the most promising approaches intended to bring more specificity and precision into the therapeutic toolbox is the directed delivery of drugs, already prophesized and described one hundred years ago by the German immunologist and Nobel Laureate in Medicine (1908) Paul Ehrlich (1854-1915) as the “magic bullet” principle. It is a visionary medical method in which active agents -such as drugs or antibodies- are guided within the human body and brought to bind directly and exclusively to their biological target. This approach was triggered and has been remarkably promoted by the introduction and continuous development of nano-sized medical systems since the 1950s, and is expected to experience a real breakthrough by the clinical validation of the so called “Magnetic Drug Targeting”. According to this technique, magnetically active nanoparticles are coated with a therapeutically active biomaterial and guided through external magnetic fields in the natural transport pathways of the body, then retained and concentrated at target sites where the biologically active load is set free. The delivered dose is augmented, side effects are lowered and the overall therapeutic efficiency is enhanced. Especially for cancer treatment, the magnetically guided drug delivery represents a huge potential. In fact, conventional chemotherapy methods are used systemically and succeed in best cases in delivering only a fractional amount of the drug to the target sites, while the rest is absorbed by the healthy tissue of the treated body. This is so inefficient that dose levels of about 50 to 100-fold those of conventional doses need to be administered to achieve cures of cancer cells (T. A. Connors 1995). As a result, blood filtering and trafficking organs, such as the liver, the kidneys, the spleen and most importantly the heart, are the direct victims of the highly toxic substances used in chemotherapy. Even the apparently more gentle approach of applying the maximum tolerated dose at defined intervals -in order to avoid toxicity- can unintentionally lead to a chemoresistance of the tumor (C. Damyanov 2009). These shortcomings of the chemical therapy further aggravate the fact that cancer is still the worldwide deadliest disease, with an upward trend. For instance, around 25 % of all registered death cases in the European Union are reported by the World Health Organization to be caused by tumors. Despite the development of advanced anti-cancer medicine, it still remains a difficult challenge to keep costs at an affordable level. For that reason, new and more efficient cancer treatment methods with higher success rates and lower side effects and costs are urgently needed and would help physicians cope with an ever ageing world population. In this work, we report improvements achieved in the understanding and control of the magnetically targeted drug delivery, mainly realized by the consideration of time issues and the investigation of dynamic magnetic fields. New approaches to assess the magnetic behavior of nanoparticles in suspensions as well as an advanced examination of the lung drug targeting and the mechanisms of cellular drug uptake after successful localized delivery represent the major achievements compiled in this manuscript. The registered improvements are an important contribution to the further development of the idea of directed therapies promoted by the emerging nanomedicine. This modern medicine is expected to provide techniques that can act on a cellular and even sub-cellular level, treating ailments with considerably more accuracy. Gradually, modern diagnostic and therapeutic techniques should elevate us slowly to the point where we can start thinking more in terms of real “regenerative” medicine. That means, we should be able to precisely and directly address pathologic tissues, save cells and organs, repair and heal them, rather than extinguish them.Mehr als hundert Jahre nach dem Tod von Paul Ehrlich, dem bedeutendsten deutschen Immunologen, verfolgt die "Nachwelt" noch mit großen Schritten eine seiner wichtigsten Visionen, die er während seiner Arbeiten zur Behandlung der Syphilis entwickelte: eine „Zauberkugel“ (magic bullet), die einen gegebenen krankmachenden Erreger gezielt abtöten kann. Ganz nach diesem noch -mehr denn je- aktuellen Prinzip, entwickeln Forscher heutzutage weltweit neue Methoden, um nicht nur Krankheitserreger, sondern auch befallene Gewebe, spezifisch zu behandeln. In den letzten Jahren entwickelte sich dadurch die Medizin von der konventionellen Anwendung, über die personalisierte Behandlung, wo die genetische Information eines jeden Patienten präventiv untersucht werden kann und die Ergebnisse zur Auswahl und Anpassung der Therapie-Art herangezogen werden, bis hin zur "Nanomedizin", einer neuen Ära der Arzneimittel-Konzipierung, -Synthese, -Dosierung und -Verabreichung, die Therapien auf zellulärer und sub-zellulärer Ebene ermöglichen sollte. Mediziner sind heutzutage weit entfernt von der Darstellung von Christian Friedrich Hebbel (18.03.1813 - 13.12.1863), dass "ein Arzt eine Aufgabe hat, als ob ein Mensch in einem dunklen Zimmer in einem Buche lesen sollte". Sie sind in der Lage, durch die Integration der Nanotechnologie im biomedizinischen Bereich, Gewebe und Zellen, die durchschnittliche Dimensionen von 10 µm haben, mit Nanosystemen im Submikrometer-Bereich zu adressieren und gezielt zu behandeln. In diesem Rahmen präsentiert sich das Magnetic Drug Targeting (MDT) als besonders wirksamer Therapie-Ansatz. Dabei werden Wirkstoff-beladene magnetische Nanopartikel über externe Magnetfelder im Körper geführt und an einem gegebenen Krankheitsort lokal angereichert. Die verabreichte Wirkdosis wird dadurch erhöht, Nebeneffekte minimiert. Besonders in der Krebsbekämpfung verspricht dieser Ansatz hohe Erfolgsquoten und eine Reduzierung der ohnehin enormen Chemo- und Radiotherapie-Kosten, die meistens einen bremsenden Effekt auf die Entwicklung und Verbreitung zahlreicher Behandlungsmethoden haben. An dieser Stelle sei daran erinnert, dass Krebs nach wie vor die weltweit wichtigste Todesursache ist, an der schätzungsweise 11.5 Millionen Weltbewohner im Jahre 2030 sterben werden, was einem Anstieg von 45% zum Jahre 2007 darstellt. Die zielgerichtete Arzneimittel-Applikation, zu Englisch "Directed Drug Delivery", soll hierfür Lösungen anbieten, die Tumore spezifisch angreifen und ausschalten können. Durch eine magnetische Lenkung und Anreicherung wird dieses Verfahren weiter optimiert. Die somit entstehende MDT-Methode eignet sich für Anwendungen in der Blutbahn, sowie in den Atemwegen von Patienten, mit entsprechenden Anpassungen. Entscheidend ist hierbei vor Allem das eingesetzte Magnetfeld, in Bezug auf Amplitude, Homogenität und Dynamik. In zahlreichen wissenschaftlichen Arbeiten, wurden bisher Erfolg versprechende Ergebnisse präsentiert, die überwiegend durch die Manipulation und Aufkonzentrierung von Nanopartikel-Wirkstoff-Komplexen mit statischen Magnetfeldern realisiert wurden. Eine hierzu komplementäre Betrachtung mit dynamischen Magnetfeldern wird in dieser Arbeit untersucht. Im Rahmen dieses Forschungsprojekts wurden Ansätze mit statischen und dynamischen Magnetfeldern zur Verbesserung des Magnetic Drug Targeting theoretisch überprüft, simulativ validiert und systemtechnisch umgesetzt. Nach einer ausführlichen Untersuchung der Nanopartikel-Eigenschaften, die den MDT-Effekt überhaupt ermöglichen und besonders beeinflussen, wurde der Anreicherungsprozess unter Magnetkraftwirkung modelliert und ein für Anwendungen in der Blutbahn optimiertes Magnetsystem simuliert, konstruiert und bei in-vivo-Versuchen eingesetzt. Dadurch konnte eine aktive und vor Allem reproduzierbare Retention von beladenen Nanopartikel-Komplexen in den Arterien und Venen der Rückenhaut einer Maus verzeichnet werden