Direct Potential Modulation of Neurogenic Differentiation Markers by Granulocyte-Colony Stimulating Factor (G-CSF) in the Rodent Brain

The hematopoietic granulocyte-colony stimulating growth factor (G-CSF, filgrastim) is an approved drug in hematology and oncology. Filgrastim’s potential in neurodegenerative disorders is gaining increasingly more attention, as preclinical and early clinical studies suggest it could be a promising treatment option. G-CSF has had a tremendous record as a safe drug for more than three decades; however, its effects upon the central nervous system (CNS) are still not fully understood. In contrast to conceptual long-term clinical application with lower dosing, our present pilot study intends to give a first insight into the molecular effects of a single subcutaneous (s.c.) high-dose G-CSF application upon different regions of the rodent brain. We analyzed mRNA—and in some instances—protein data of neurogenic and non-neurogenic differentiation markers in different regions of rat brains five days after G-CSF (1.3 mg/kg) or physiological saline. We found a continuous downregulation of several markers in most brain regions. Remarkably, cerebellum and hypothalamus showed an upregulation of different markers. In conclusion, our study reveals minor suppressive or stimulatory effects of a single exceptional high G-CSF dose upon neurogenic and non-neurogenic differentiation markers in relevant brain regions, excluding unregulated responses or unexpected patterns of marker expression

Targeting TGF-ß in the Central Nervous System: Assessment of Cynomolgus Monkey—Toxicity and Pharmacokinetics for an LNA-Antisense Oligonucleotide

Increasingly antisense oligonucleotides (ASOs) are developed for potential treatment of CNS disorders, and due to the inability to cross the blood brain barrier, they require direct administration into the cerebrospinal fluid (CSF). In this regard, intrathecal (i.th.) administration in cynomolgus monkeys (Macaca fascicularis) is a well-established approach for preclinical safety studies. Here, we present an innovative preclinical approach that is intended to support rapid entry into clinical development with ASOs targeting the CNS. The preclinical approach comprises one non-GLP study in 26 non-human primates, followed by a pivotal GLP repeated dose toxicity study in the same species. No pivotal rodent studies were conducted, and regulatory guidance to initiate this study was met by in vitro work. The non-GLP study consists of three separate phases: Phase A determines toxicity after i.th. administrations with five escalating dose levels in a single male and female animal, respectively. Dosing is conducted on days 1, 8, 15, 22, and 29 and the experiment is terminated 36 days after start of the study. The second phase (Phase B) investigates pharmacokinetics over a 2- or 4-week period at two dose levels following single administrations in eight (8) animals (4 females, 4 males). Finally, a third phase (Phase C) investigates toxicity and pharmacokinetics after repeated (9×) dosing over a 13-week period at two dose levels in sixteen (8 females, 8 males) animals. In each phase, clinical observations and physical/neurological parameters are investigated directly pre-dose, 4 h and 24 h post-dose, respectively. In all phases, CSF and blood samples are taken pre-dose and after each dosing, for determination of test article concentration, biomarkers of tolerability and biomarkers of pharmacology. In all phases, tissue samples from the liver, kidney, spinal cord, and brain are collected for determination of NVP-13 tissue concentrations. The above concept has successfully supported first-in-human clinical trials. The entire non-GLP program is completed within less than six months and requires fewer animals in comparison to the conduct of three independent studies

Safe and Effective Cynomolgus Monkey GLP—Tox Study with Repetitive Intrathecal Application of a TGFBR2 Targeting LNA-Gapmer Antisense Oligonucleotide as Treatment Candidate for Neurodegenerative Disorders

The capability of the adult central nervous system to self-repair/regenerate was demonstrated repeatedly throughout the last decades but remains in debate. Reduced neurogenic niche activity paralleled by a profound neuronal loss represents fundamental hallmarks in the disease course of neurodegenerative disorders. We and others have demonstrated the endogenous TGFβ system to represent a potential pathogenic participant in disease progression, of amyotrophic lateral sclerosis (ALS) in particular, by generating and promoting a disequilibrium of neurodegenerative and neuroregenerative processes. The novel human/primate specific LNA Gapmer Antisense Oligonucleotide “NVP-13”, targeting TGFBR2, effectively reduced its expression and lowered TGFβ signal transduction in vitro and in vivo, paralleled by boosting neurogenic niche activity in human neuronal progenitor cells and nonhuman primate central nervous system. Here, we investigated NVP-13 in vivo pharmacology, safety, and tolerability following repeated intrathecal injections in nonhuman primate cynomolgus monkeys for 13 weeks in a GLP-toxicology study approach. NVP-13 was administered intrathecally with 1, 2, or 4 mg NVP-13/animal within 3 months on days 1, 15, 29, 43, 57, 71, and 85 in the initial 13 weeks. We were able to demonstrate an excellent local and systemic tolerability, and no adverse events in physiological, hematological, clinical chemistry, and microscopic findings in female and male Cynomolgus Monkeys. Under the conditions of this study, the no observed adverse effect level (NOAEL) is at least 4 mg/animal NVP-13

Biomarker Supervised G-CSF (Filgrastim) Response in ALS Patients

Objective: To evaluate safety, tolerability and feasibility of long-term treatment with Granulocyte-colony stimulating factor (G-CSF), a well-known hematopoietic stem cell factor, guided by assessment of mobilized bone marrow derived stem cells and cytokines in the serum of patients with amyotrophic lateral sclerosis (ALS) treated on a named patient basis.Methods: 36 ALS patients were treated with subcutaneous injections of G-CSF on a named patient basis and in an outpatient setting. Drug was dosed by individual application schemes (mean 464 Mio IU/month, range 90-2160 Mio IU/month) over a median of 13.7 months (range from 2.7 to 73.8 months). Safety, tolerability, survival and change in ALSFRS-R were observed. Hematopoietic stem cells were monitored by flow cytometry analysis of circulating CD34+ and CD34+CD38− cells, and peripheral cytokines were assessed by electrochemoluminescence throughout the intervention period. Analysis of immunological and hematological markers was conducted.Results: Long term and individually adapted treatment with G-CSF was well tolerated and safe. G-CSF led to a significant mobilization of hematopoietic stem cells into the peripheral blood. Higher mobilization capacity was associated with prolonged survival. Initial levels of serum cytokines, such as MDC, TNF-beta, IL-7, IL-16, and Tie-2 were significantly associated with survival. Continued application of G-CSF led to persistent alterations in serum cytokines and ongoing measurements revealed the multifaceted effects of G-CSF.Conclusions: G-CSF treatment is feasible and safe for ALS patients. It may exert its beneficial effects through neuroprotective and -regenerative activities, mobilization of hematopoietic stem cells and regulation of pro- and anti-inflammatory cytokines as well as angiogenic factors. These cytokines may serve as prognostic markers when measured at the time of diagnosis. Hematopoietic stem cell numbers and cytokine levels are altered by ongoing G-CSF application and may potentially serve as treatment biomarkers for early monitoring of G-CSF treatment efficacy in ALS in future clinical trials

Modeling and Bioinformatics Identify Responders to G-CSF in Patients With Amyotrophic Lateral Sclerosis

Objective: Developing an integrative approach to early treatment response classification using survival modeling and bioinformatics with various biomarkers for early assessment of filgrastim (granulocyte colony stimulating factor) treatment effects in amyotrophic lateral sclerosis (ALS) patients. Filgrastim, a hematopoietic growth factor with excellent safety, routinely applied in oncology and stem cell mobilization, had shown preliminary efficacy in ALS. Methods: We conducted individualized long-term filgrastim treatment in 36 ALS patients. The PRO-ACT database, with outcome data from 23 international clinical ALS trials, served as historical control and mathematical reference for survival modeling. Imaging data as well as cytokine and cellular data from stem cell analysis were processed as biomarkers in a non-linear principal component analysis (NLPCA) to identify individual response. Results: Cox proportional hazard and matched-pair analyses revealed a significant survival benefit for filgrastim-treated patients over PRO-ACT comparators. We generated a model for survival estimation based on patients in the PRO-ACT database and then applied the model to filgrastim-treated patients. Model-identified filgrastim responders displayed less functional decline and impressively longer survival than non-responders. Multimodal biomarkers were then analyzed by PCA in the context of model-defined treatment response, allowing identification of subsequent treatment response as early as within 3 months of therapy. Strong treatment response with a median survival of 3.8 years after start of therapy was associated with younger age, increased hematopoietic stem cell mobilization, less aggressive inflammatory cytokine plasma profiles, and preserved pattern of fractional anisotropy as determined by magnetic resonance diffusion tensor imaging (DTI-MRI). Conclusion: Long-term filgrastim is safe, is well-tolerated, and has significant positive effects on disease progression and survival in a small cohort of ALS patients. Developing and applying a model-based biomarker response classification allows use of multimodal biomarker patterns in full potential. This can identify strong individual treatment responders (here: filgrastim) at a very early stage of therapy and may pave the way to an effective individualized treatment option

A mammalian cell based FACS-panning platform for the selection of HIV-1 envelopes for vaccine development

An increasing number of broadly neutralizing monoclonal antibodies (bnMAb) against the HIV-1 envelope (Env) protein has been discovered recently. Despite this progress, vaccination efforts with the aim to re-elicit bnMAbs that provide protective immunity have failed so far. Herein, we describe the development of a mammalian cell based FACS-panning method in which bnMAbs are used as tools to select surface-exposed envelope variants according to their binding affinity. For that purpose, an HIV-1 derived lentiviral vector was developed to infect HEK293T cells at low multiplicity of infection (MOI) in order to link Env phenotype and genotype. For proof of principle, a gp145 Env model-library was established in which the complete V3 domain was substituted by five strain specific V3 loop sequences with known binding affinities to nMAb 447-52D, respectively. Env genes were recovered from selected cells by PCR, subcloned into a lentiviral vector (i) to determine and quantify the enrichment nMAb binders and (ii) to generate a new batch of transduction competent particles. After 2 selection cycles the Env variant with highest affinity was enriched 20-fold and represented 80% of the remaining Env population. Exploiting the recently described bnMAbs, this procedure might prove useful in selecting Env proteins from large Env libraries with the potential to elicit bnMAbs when used as vaccine candidates

Entwicklung von Expressions- und Anreicherungsmethoden zur Selektion von HIV-1 Vakzinekandidaten in Säugerzellen

An ever-increasing number of broadly neutralizing antibodies (bNAb) against the HIV-1 envelope (Env) have been discovered so far. Unfortunately, most antibody responses against HIV-1 in an acute infection are only strain-specific and quickly bypassed by escape mutations. Vaccination trials designed to re-elicit bNAbs that mediate protective immunity have remained futile (reverse vaccinology). The neutralization activity of the discovered bNAbs has been attributed to their affinity to functional Env trimers, although each cross-reacts with non- trimeric, immature or otherwise non-functional Env molecules. Therefore, a discovery platform was developed in order to select bNAb-bound, native, trimeric envelope variants that are displayed on the surface of mammalian cells. Conclusively, an HIV-1 derived lentiviral vector was developed to infect HEK293T cells by a low multiplicity of infection (MOI), in order to link phenotype and genotype. Hence, infected cells expressed and displayed membrane-associated Env proteins on their surface (lenti-cellular display). Subsequently to staining with an appropriate monoclonal antibody, Env-displaying cells were selected for high affinity binding via FACS sorting (Panning). A small model-library of Env variants with distinct binding capacities against the bNAb 447-52D was generated for evaluation. In a proof-of-concept study, it was demonstrated that the Env variant with the highest affinity to the applied bNAb was enriched in two rounds of selection (up to 10-fold). Hence, the demonstrated technique provides the possibility to screen membrane-bound Env variants for high binding capacities against the bNAb applied. Nevertheless, the presented lenti-cellular display and FACS-mediated panning platform still needs further optimizations. Prospectively, it needs to be challenged by libraries of larger diversity and selected candidates should be evaluated in vaccination trials.Die Entwicklung eines wirksamen Impfstoffs gegen das humane Immundefizienz-Virus (HIV-1) gilt bis heute als ein ungelöstes Problem. Bei fast allen zugelassenen Impfstoffen beruht die Wirksamkeit auf der Bildung von Pathogen-neutralisierenden Antikörpern. Als zwingende Eigenschaft für ein erfolgreiches HIV-1-Vakzin gilt daher, dass es eine Antikörper-Immunantwort gegen essentielle Bestandteile des HI-Virus auslöst und ein breites Wirkspektrum aufweist. Einige sogenannte breit neutralisierende Antikörper (bNAb) sind bereits länger bekannt und wurden ausgiebig charakterisiert. In jüngster Zeit wurden in steigendem Maße weitere vielversprechende bNAbs isoliert und Ihre Eigenschaften sind derzeitig Bestandteil vieler Forschungsbemühungen. Eine Vielzahl von Versuchen, Antikörper mit ähnlichen Eigenschaften durch Impfungen zu induzieren, blieb jedoch bisher erfolglos (Reverse Vakzinologie). In der vorliegenden Arbeit wurde eine Selektionsmethode entwickelt, bei der Varianten trimerer Env-Proteine auf der Oberfläche von Säugetierzellen präsentiert werden. Die unterschiedlichen Bindungsstärken der verwendeten Env-Varianten zu einem gegebenen bNAb sollten es hierbei ermöglichen, auf Env-Varianten mit der höchsten Affinität zu selektionieren. Dies mündete in einer sogenannten lenti-zellulären Expressions- und FACS-basierten Anreicherungsmethode (Panning). Die entwickelte Methode wurde mit einer Modell-Bibliothek aus Env-Varianten mit bekannten Affinitäten zu bNAb 447-52D evaluiert. In dieser Studie konnte die Anreicherung der Env-Variante mit der höchsten Affinität zum verwendeten 447-52D Antikörper gezeigt werden (bis zu 10-fache Anreicherung). Nach weiterer Optimierung sollen zukünftig Env-Bibliotheken mit höherer Variabilität eingesetzt werden, um Env-Varianten zu selektionieren, die in präklinischen und klinischen Studien auf ihr Potential zur bNAb-Induktion untersucht werden sollen

Soil disturbance as a grassland restoration measure-effects on plant species composition and plant functional traits.

Soil disturbance is recognized as an important driver of biodiversity in dry grasslands, and can therefore be implemented as a restoration measure. However, because community re-assembly following disturbance includes stochastic processes, a focus only on species richness or establishment success of particular species will not inform on how plant communities respond ecologically to disturbance. We therefore evaluated vegetation development following disturbance by quantifying species richness, species composition and functional trait composition. Degraded calcareous sandy grassland was subjected to experimental disturbance treatments (ploughing or rotavation), and the vegetation was surveyed during four subsequent years of succession. Treated plots were compared with control plots representing untreated grassland, as well as nearby plots characterized by plant communities representing the restoration target. Species richness and functional diversity both increased in response to soil disturbance, and rotavation, but not ploughing, had a persistent positive effect on the occurrence of specialist species of calcareous sandy grassland. However, no type of soil disturbance caused the plant species composition to develop towards the target vegetation. The disturbance had an immediate and large impact on the vegetation, but the vegetation developed rapidly back towards the control sites. Plant functional composition analysis indicated that the treatments created habitats different both from control sites and target sites. Community-weighted mean Ellenberg indicator values suggested that the observed plant community response was at least partially due to an increase in nitrogen and water availability following disturbance. This study shows that a mild type of disturbance, such as rotavation, may be most successful in promoting specialist species in calcareous sandy grassland, but that further treatments are needed to reduce nutrient availability. We conclude that a functional trait based analysis provides additional information of the vegetation response and the abiotic conditions created, complementing the information from the species composition

The measured functions and a short description of which functions they are related to.

<p>Footnotes indicate from where the trait data used in the study was collected.</p><p>^a Collected at site</p><p>^b [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123698#pone.0123698.ref072" target="_blank">72</a>]</p><p>^c [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123698#pone.0123698.ref073" target="_blank">73</a>]</p><p>^dLEDA traitbase [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123698#pone.0123698.ref074" target="_blank">74</a>]</p><p>The measured functions and a short description of which functions they are related to.</p