Preclinical Validation Research Articles

Preclinical Validation of MIN-T: A Novel Controlled-Released Formulation for the Adjunctive Local Application of Minocycline in Periodontitis

Background: Adjunctive treatment of periodontitis lacks solutions which allow for enough time for wound healing in the periodontal pockets by avoiding fast re-colonization. Such a solution might be an antibiotic-containing formulation with a controlled release over a period of weeks. Here, a recently described minocycline-containing approach is qualified for further clinical development by focusing on proof-of-concept, systemic burden, resistance development, and degradation studies. Methods: Animal studies were done in two different (mouse-chamber, rat Porphyromonas gingivalis challenging) models, including effects on inflammation markers, bone loss, and bone structure. Also, serum concentrations of minocycline after local application were determined by HPLC-MS/MS. The resistance status of bacterial clinical isolates against minocycline was investigated and the degradation of the formulation was characterized by laser scanning and scanning electron microscopy. Results: Animal studies clearly demonstrated the applicability of the new formulation in the investigated models. Inflammation markers decreased in a dose-dependent manner and reduced bone loss compared to non-treated group was observed. Therefore, the systemic burden of the antibiotic was neglectable. Minocycline is still effective against oral pathogens; resistance development was not seen. The biodegradable thread was first swollen and subsequently degraded over a period of weeks. Conclusions: The results support the continued clinical development of this new formulation. A phase I clinical trial is planned to further evaluate its safety and efficacy.

GLP-1 Receptor Agonists Models for Type 1 Diabetes: A Narrative Review.

Glucagon-like peptide 1 (GLP-1) is a hormone that promotes insulin secretion, delays gastric emptying, and inhibits glucagon secretion. The GLP-1 receptor agonists have been developed as adjunctive therapies for type 2 diabetes to improve glucose control. Recently, there has been an interest in introducing GLP-1 receptor agonists as adjunctive therapies in type 1 diabetes alongside automatic insulin delivery systems. The preclinical validation of these systems often relies on mathematical simulators that replicate the glucose dynamics of a person with diabetes. This review aims to explore mathematical models available in the literature to describe GLP-1 effects to be used in a type 1 diabetes simulator. Three databases were examined in the search for GLP-1 mathematical models. More than 1500 works were found after searching for specific keywords that were narrowed down to 39 works for full-text assessment. A total of 23 works were selected describing GLP-1 pharmacokinetics and pharmacodynamics. However, none of the found models was designed for type 1 diabetes. An analysis is included of the available models' features that could be translated into a GLP-1 receptor agonist model for type 1 diabetes. There is a gap in research in GLP-1 receptor agonists mathematical models for type 1 diabetes, which could be incorporated into type 1 diabetes simulators, providing a safe and inexpensive tool to carry out preclinical validations using these therapies.

Augmented and virtual reality imaging for collaborative planning of structural cardiovascular interventions: a proof-of-concept and validation study.

Virtual reality (VR) and augmented reality (AR) have led to significant advancements in cardiac preoperative planning, shaping the world in profound ways. A noticeable gap exists in the availability of a comprehensive multi-user, multi-device mixed reality application that can be used in a multidisciplinary team meeting. A multi-user, multi-device mixed reality application was developed, supporting AR and VR implementations. Technical validation involved a standardized testing protocol and comparison of AR and VR measurements regarding absolute error and time. Preclinical validation engaged experts in interventional cardiology, evaluating the clinical applicability prior to clinical validation. Clinical validation included patient-specific measurements for five patients in VR compared with standard computed tomography (CT) for preoperative planning. Questionnaires were used at all stages for subjective evaluation. Technical validation, including 106 size measurements, demonstrated an absolute median error of 0.69mm (0.25 to 1.18mm) compared with ground truth. The time to complete the entire task was on average, with VR measurements being faster than AR ( versus , ). On clinical validation of five preoperative patients, there was no statistically significant difference between paired CT and VR measurements (0.58 [95% CI, to 2.74], ). Questionnaires showcased unanimous agreement on the user-friendly nature, effectiveness, and clinical value. The mixed reality application, validated through technical, preclinical, and clinical assessments, demonstrates precision and user-friendliness. Further research of our application is needed to validate the generalizability and impact on patient outcomes.

Self-adaptive epidermal blood flow sensor for high-flux vascular access monitoring of hemodialysis patients

Well-functioning vascular access (VA) is essential for hemodialysis treatment in patients with end-stage renal disease (ESRD). However, continuous and accurate monitoring of blood flow to assess high-flux VA during hospitalization or at home is not feasible for either clinical instruments or wearable sensors. Here, we report the design and preclinical validation of a high-precision, long-term, epidermal blood flow sensor that self-adapts to unavoidable sensor-mounting deviations on the skin and is compatible with individual tissue differences. Specifically, the technology is based on thermal dissipation of the skin, and improves the signal-to-error ratio surpassing 4 times when measuring high-flux blood (100–600 mL/min). In preclinical validation, the sensor is compared with the Doppler ultrasound and demonstrate a blood flow resolution of 10–50 mL/min. Furthermore, it is highly-integrated and wearable, measuring 36 × 50 mm2. The sensor paves the way for accurate, convenient, high-flux blood monitoring, offering significant potential to extend the lives of patients with ESRD.

SpinDoctor-IVIM: A virtual imaging framework for intravoxel incoherent motion MRI

Intravoxel incoherent motion (IVIM) imaging is increasingly recognised as an important tool in clinical MRI, where tissue perfusion and diffusion information can aid disease diagnosis, monitoring of patient recovery, and treatment outcome assessment. Currently, the discovery of biomarkers based on IVIM imaging, similar to other medical imaging modalities, is dependent on long preclinical and clinical validation pathways to link observable markers derived from images with the underlying pathophysiological mechanisms. To speed up this process, virtual IVIM imaging is proposed. This approach provides an efficient virtual imaging tool to design, evaluate, and optimise novel approaches for IVIM imaging. In this work, virtual IVIM imaging is developed through a new finite element solver, SpinDoctor-IVIM, which extends SpinDoctor, a diffusion MRI simulation toolbox. SpinDoctor-IVIM simulates IVIM imaging signals by solving the generalised Bloch-Torrey partial differential equation. The input velocity to SpinDoctor-IVIM is computed using HemeLB, an established Lattice Boltzmann blood flow simulator. Contrary to previous approaches, SpinDoctor-IVIM accounts for volumetric microvasculature during blood flow simulations, incorporates diffusion phenomena in the intravascular space, and accounts for the permeability between the intravascular and extravascular spaces. The above-mentioned features of the proposed framework are illustrated with simulations on a realistic microvasculature model.

Preclinical validation of human recombinant glutamate-oxaloacetate transaminase for the treatment of acute ischemic stroke

Preclinical validation of human recombinant glutamate-oxaloacetate transaminase for the treatment of acute ischemic stroke

Internally-crosslinked alginate dialdehyde/alginate/gelatin-based hydrogels as bioprinting inks for prospective cardiac tissue engineering applications

Cardiovascular diseases represent a worldwide social and economic challenge, due to heart tissue limited regenerative capabilities. 3D bioprinted cell-laden constructs are a promising approach to develop patches for cardiac regeneration or in vitro models for new drug preclinical discovery and validation. However, the development of bioinks with optimal mechanical, rheological, and biological properties is still a challenge. Although alginate (Alg)-based bioinks have been extensively explored, such hydrogels lack cell adhesion properties and degradability. Additionally, 3D Alg structures are usually obtained by micro-extrusion bioprinting exploiting conventional external cross-linking methods, which introduce inhomogeneities and unpredictability in construct formation. This work exploits Alg internal ionic gelation mechanism to obtain homogeneous self-standing multilayered 3D printed constructs without employing support baths or post-printing crosslinking treatments, combining an insoluble calcium salt (calcium carbonate, CaCO3) with an acidifying agent (D-(+)-Glucono-1,5-lactone, GDL) to promote controlled release of calcium ions. Alg was blended with oxidized alginate (ADA) and gelatin (Gel) to achieve degradable and cell adhesive hydrogels for cardiac tissue engineering. Firstly, ADA/Alg bioink composition was tailored by varying polymer weight ratio (keeping ADA as the main component) and calcium ions concentration to achieve cardiac tissue-like viscoelastic properties. Then, the amount of Gel in ADA/Alg hydrogels was optimized to support adult human cardiac fibroblasts (AHCFs) adhesion, producing shear thinning inks with tunable viscoelastic properties (G’ 650-1300 Pa) and degradation profile (40-80% weight loss after 21 days in phosphate buffered saline, PBS) by varying Gel concentration. ADA/Alg/Gel hydrogels showed a shear thinning behavior suitable for 3D bioprinting and dependent on ink stabilization time, due to the gradual pH-triggered release of calcium ions over time. AHCFs-laden ADA/Alg/Gel bioinks could be successfully printed producing scaffolds with high shape fidelity and good cell viability post-printing. Finally, 3D AHCF-laden and H9C2-laden ADA/Alg/Gel bioinks with the highest gelatin content (25% w/w) allowed cell adhesion after 24 hours of incubation, showing potential application for cardiac tissue modelling. This research presented a comprehensive framework for advancing the design of bioink formulations enabling the printing of cell-adhesive and self-supporting constructs.

Ultra-high-resolution mapping of myelin and g-ratio in a panel of Mbp enhancer-edited mouse strains using microstructural MRI

Non-invasive myelin water fraction (MWF) and g-ratio mapping using microstructural MRI have the potential to offer critical insights into brain microstructure and our understanding of neuroplasticity and neuroinflammation. By leveraging a unique panel of variably hypomyelinating mouse strains, we validated a high-resolution, model-free image reconstruction method for whole-brain MWF mapping. Further, by employing a bipolar gradient echo MRI sequence, we achieved high spatial resolution and robust mapping of MWF and g-ratio across the whole mouse brain. Our regional white matter-tract specific analyses demonstrated a graded decrease in MWF in white matter tracts which correlated strongly with myelin basic protein gene (Mbp) mRNA levels. Using these measures, we derived the first sensitive calibrations between MWF and Mbp mRNA in the mouse. Minimal changes in axonal density supported our hypothesis that observed MWF alterations stem from hypomyelination. Overall, our work strongly emphasizes the potential of non-invasive, MRI-derived MWF and g-ratio modeling for both preclinical model validation and ultimately translation to humans.

Advanced Nanotechnological Approaches for Biofilm Prevention and Control

Biofilm-associated infections present a significant challenge in modern medicine, primarily due to their resilience and resistance to conventional treatments. These infections occur when bacteria form biofilms, protective layers formed by bacterial communities, which are notoriously resistant to traditional antibiotics on surfaces such as medical implants and biological surfaces, making eradication with standard antibiotics difficult. This resilience leads to persistent infections, imposing a substantial economic burden on healthcare systems. The urgency to find alternative treatments is critical as current methods are insufficient and costly. Innovative approaches, such as nanotechnology-based therapies, offer promising alternatives by targeting biofilms more effectively and reducing the need for invasive procedures. Nanocarriers hold significant promise in the fight against biofilm-associated infections. Nanocarriers can penetrate biofilms more effectively than conventional treatments, delivering higher concentrations of antibiotics or other antimicrobial agents precisely where they are needed. This targeted approach not only enhances the efficacy of treatments but also minimizes potential side effects. The development of nanocarrier-based therapies is crucial for overcoming the limitations of current treatments and ultimately improving patient outcomes and reducing the economic burden of biofilm-associated infections on healthcare systems. In this review, nanotechnology-based systems, their characteristics, limitations, and potential benefits are explored to address biofilms-related infections. Additionally, biofilm evaluation models and the tests necessary for the preclinical validation of these nanosystems to facilitate their clinical application are addressed.

Exploiting a tumor softening targeted bomb for mechanical gene therapy of chemoresistant Triple-Negative breast cancer

Triple-negative breast cancer (TNBC)-the most aggressive and refractory subtype of breast cancer-plagues many patients. The tumor physical microenvironment (TPME) is characterized by altered stromal composition and extracellular matrix (ECM), which may contribute to tissue stiffness and compromise therapeutic efficacy. Here, based on clinical data analysis and preclinical model validation, we discover that chemoresistant TNBC tends to have a stiffer TPME with a more aggressive genomic landscape, in which lysyl oxidase (LOX) exerts pivotal effects. Anlotinib (An), a multi-targeted tyrosine kinase inhibitor, has great promising potential in the treatment of TNBC. In this regard, we developed a tumor-softening nanobomb (An&siLOX@NPs) that remodels TPME by co-delivering small interfering siRNAs silencing LOX and the anti-angiogenic drug, anlotinib (An). The improved cytosolic delivery of siLOX enables TPME remodeling via reducing deposition and cross-linking between collagen and fibronectin, thereby promoting enrichment of An to improve its therapeutic outcomes. In chemoresistant TNBC, we confirm the An&siLOX@NPs nanobomb-mediated mechanical gene therapy results in obvious inhibition of local tumor outgrowth and prolongs survival. Additionally, softened TPME reactivates intratumoral immune milieu to bolster sensitivity to anti-PD-L1 immunotherapy, completely curing 33.3% of the metastatic tumors, further generating a synergistic anti-tumor efficacy. Collectively, our mechanical gene therapy strategy represents a highly efficient, safe, and versatile anti-drug resistance modality, which not only can solve the snag problem of TNBC treatment but also provides a new demonstration for other tumor treatments.

Next-generation BCMA-targeted chimeric antigen receptor CARTemis-1: the impact of manufacturing procedure on CAR T-cell features.

CAR therapy targeting BCMA is under investigation as treatment for multiple myeloma. However, given the lack of plateau in most studies, pursuing more effective alternatives is imperative. We present the preclinical and clinical validation of a new optimized anti-BCMA CAR (CARTemis-1). In addition, we explored how the manufacturing process could impact CAR-T cell product quality and fitness. CARTemis-1 optimizations were evaluated at the preclinical level both, in vitro and in vivo. CARTemis-1 generation was validated under GMP conditions, studying the dynamics of the immunophenotype from leukapheresis to final product. Here, we studied the impact of the manufacturing process on CAR-T cells to define optimal cell culture protocol and expansion time to increase product fitness. Two different versions of CARTemis-1 with different spacers were compared. The longer version showed increased cytotoxicity. The incorporation of the safety-gene EGFRt into the CARTemis-1 structure can be used as a monitoring marker. CARTemis-1 showed no inhibition by soluble BCMA and presents potent antitumor effects both in vitro and in vivo. Expansion with IL-2 or IL-7/IL-15 was compared, revealing greater proliferation, less differentiation, and less exhaustion with IL-7/IL-15. Three consecutive batches of CARTemis-1 were produced under GMP guidelines meeting all the required specifications. CARTemis-1 cells manufactured under GMP conditions showed increased memory subpopulations, reduced exhaustion markers and selective antitumor efficacy against MM cell lines and primary myeloma cells. The optimal release time points for obtaining the best fit product were > 6 and < 10 days (days 8-10). CARTemis-1 has been rationally designed to increase antitumor efficacy, overcome sBCMA inhibition, and incorporate the expression of a safety-gene. The generation of CARTemis-1 was successfully validated under GMP standards. A phase I/II clinical trial for patients with multiple myeloma will be conducted (EuCT number 2022-503063-15-00).

The Effectiveness of Hydroalcoholic Extract and Fraction of Cucumis melo on Amelioration of High Fat Diet Induced hepatotoxicity: A Comprehensive Approach Integrating Computational and Preclinical Validation

The Effectiveness of Hydroalcoholic Extract and Fraction of Cucumis melo on Amelioration of High Fat Diet Induced hepatotoxicity: A Comprehensive Approach Integrating Computational and Preclinical Validation

Development and preclinical validation of 2-deoxy 2-[18F]fluorocellobiose as an Aspergillus-specific PET tracer.

The global incidence of invasive fungal infections (IFIs) has increased over the past few decades, mainly in immunocompromised patients, and is associated with high mortality and morbidity. Aspergillus fumigatus is one of the most common and deadliest IFI pathogens. Major hurdles to treating fungal infections remain the lack of rapid and definitive diagnosis, including the frequent need for invasive procedures to provide microbiological confirmation, and the lack of specificity of structural imaging methods. To develop an Aspergillus-specific positron emission tomography (PET) imaging agent, we focused on fungal-specific sugar metabolism. We radiolabeled cellobiose, a disaccharide known to be metabolized by Aspergillus species, and synthesized 2-deoxy-2-[18F]fluorocellobiose ([18F]FCB) by enzymatic conversion of 2-deoxy-2-[18F]fluoroglucose ([18F]FDG) with a radiochemical yield of 60 to 70%, a radiochemical purity of >98%, and 1.5 hours of synthesis time. Two hours after [18F]FCB injection in A. fumigatus pneumonia as well as A. fumigatus, bacterial, and sterile inflammation myositis mouse models, retained radioactivity was only seen in foci with live A. fumigatus infection. In vitro testing confirmed production of β-glucosidase enzyme by A. fumigatus and not by bacteria, resulting in hydrolysis of [18F]FCB into glucose and [18F]FDG, the latter being retained by the live fungus. The parent molecule was otherwise promptly excreted through the kidneys, resulting in low background radioactivity and high target-to-nontarget ratios at A. fumigatus infectious sites. We conclude that [18F]FCB is a promising and clinically translatable Aspergillus-specific PET tracer.

Isolation of Mouse Retinal Capillaries and Subendothelial Matrix for Stiffness Measurement Using Atomic Force Microscopy.

Retinal capillary degeneration is a clinical hallmark of the early stages of diabetic retinopathy (DR). Our recent studies have revealed that diabetes-induced retinal capillary stiffening plays a crucial and previously unrecognized causal role in inflammation-mediated degeneration of retinal capillaries. The increase in retinal capillary stiffness results from the overexpression of lysyl oxidase, an enzyme that crosslinks and stiffens the subendothelial matrix. Since tackling DR at the early stage is expected to prevent or slow down DR progression and associated vision loss, subendothelial matrix, and capillary stiffness represent relevant and novel therapeutic targets for early DR management. Further, direct measurement of retinal capillary stiffness can serve as a crucial preclinical validation step for the development of new imaging techniques for non-invasive assessment of retinal capillary stiffness in animal and human subjects. With this view in mind, we here provide a detailed protocol for the isolation and stiffness measurement of mouse retinal capillaries and subendothelial matrix using atomic force microscopy.

Preclinical prototype validation and characterization of a thermobrachytherapy system for interstitial hyperthermia and high-dose-rate brachytherapy

Background and purposeIntegrating simultaneous interstitial hyperthermia in high-dose-rate brachytherapy treatments (HDR-BT) is expected to lead to enhanced therapeutic effect. However, there is currently no device available for such an integration. In this study, we presented and validated the thermobrachytherapy (TBT) preclinical prototype system that is able to seamlessly integrate into the HDR-BT workflow. Materials and methodsThe TBT system consisted of an advanced radiofrequency power delivery and control system, dual-function interstitial applicators, and integrated connection and impedance matching system. The efficiency and minimum heating ability of the system was calculated performing calorimetric experiments. The effective-heating-length and heating pattern was evaluated using single-applicator split phantom experiments. The heating independence between applicators, the ability of the system to adaptable and predictable temperature steering was evaluated using multi-applicator split phantom experiments. ResultsThe system satisfied interstitial hyperthermia requirements. It demonstrated 50 % efficiency and ability to reach 6 °C temperature increase in 6 min. Effective-heating-length of the applicator was 43.7 mm, following the initial design. Heating pattern interference between applicators was lower than recommended. The system showed its ability to generate diverse heating patterns by adjusting the phase and amplitude settings of each electrode, aligning well with simulations (minimum agreement of 88 %). ConclusionsThe TBT preclinical prototype system complied with IHT requirements, and agreed well with design criteria and simulations, hence performing as expected. The preclinical prototype TBT system can now be scaled to an in-vivo validation prototype, including an adaptable impedance matching solution, appropriate number of channels, and ensuring biocompatibility and regulatory compliance.

Distributable, metabolic PET reporting of tuberculosis

Tuberculosis remains a large global disease burden for which treatment regimens are protracted and monitoring of disease activity difficult. Existing detection methods rely almost exclusively on bacterial culture from sputum which limits sampling to organisms on the pulmonary surface. Advances in monitoring tuberculous lesions have utilized the common glucoside [18F]FDG, yet lack specificity to the causative pathogen Mycobacterium tuberculosis (Mtb) and so do not directly correlate with pathogen viability. Here we show that a close mimic that is also positron-emitting of the non-mammalian Mtb disaccharide trehalose – 2-[18F]fluoro-2-deoxytrehalose ([18F]FDT) – is a mechanism-based reporter of Mycobacteria-selective enzyme activity in vivo. Use of [18F]FDT in the imaging of Mtb in diverse models of disease, including non-human primates, successfully co-opts Mtb-mediated processing of trehalose to allow the specific imaging of TB-associated lesions and to monitor the effects of treatment. A pyrogen-free, direct enzyme-catalyzed process for its radiochemical synthesis allows the ready production of [18F]FDT from the most globally-abundant organic 18F-containing molecule, [18F]FDG. The full, pre-clinical validation of both production method and [18F]FDT now creates a new, bacterium-selective candidate for clinical evaluation. We anticipate that this distributable technology to generate clinical-grade [18F]FDT directly from the widely-available clinical reagent [18F]FDG, without need for either custom-made radioisotope generation or specialist chemical methods and/or facilities, could now usher in global, democratized access to a TB-specific PET tracer.

Harmonic Imaging of Stem Cells in Whole Blood at GHz Pixel Rate.

The pre-clinical validation of cell therapies requires monitoring the biodistribution of transplanted cells in tissues of host organisms. Real-time detection of these cells in the circulatory system and identification of their aggregation state is a crucial piece of information, but necessitates deep penetration and fast imaging with high selectivity, subcellular resolution, and high throughput. In this study, multiphoton-based in-flow detection of human stem cells in whole, unfiltered blood is demonstrated in a microfluidic channel. The approach relies on a multiphoton microscope with diffractive scanning in the direction perpendicular to the flow via a rapidly wavelength-swept laser. Stem cells are labeled with metal oxide harmonic nanoparticles. Thanks to their strong and quasi-instantaneous second harmonic generation (SHG), an imaging rate in excess of 10 000 frames per second is achieved with pixel dwell times of 1 ns, a duration shorter than typical fluorescence lifetimes yet compatible with SHG. Through automated cell identification and segmentation, morphological features of each individual detected event are extracted and cell aggregates are distinguished from isolated cells. This combination of high-speed multiphoton microscopy and high-sensitivity SHG nanoparticle labeling in turbid media promises the detection of rare cells in the bloodstream for assessing novel cell-basedtherapies.

The USP10/13 inhibitor, spautin-1, attenuates the progression of glioblastoma by independently regulating RAF-ERK mediated glycolysis and SKP2.

Glioblastoma is a malignant brain tumor with poor prognosis. Though several dysregulated pathways were found to mediate the tumor progression, hyperactivation of RAS-RAF-ERK pathway, enhanced glycolysis and SKP2 are associated with several glioblastomas. Recent findings on the role of USP10 in the transition from pro-neural to mesenchymal subtype of glioblastoma and, USP13 in the stabilization of RAF1 in mouse embryonic stem cells prompted us to examine their role in the mechanisms mediating the progression of glioblastoma. In the present study, we have examined the role of spautin-1, a pharmacological inhibitor of USP10 and USP13 in the mechanisms mediating glioblastoma. Our results indicate that spautin-1 as well as knockdown of its downstream targets, USP10 and USP13, reduced the proliferation and migration of glioblastoma cells. Also, spautin-1 mediated inhibition of RAF-ERK pathway or inhibition of RAF1 and MEK1 per se reduced the glycolytic function via PKM2/Glut-1 and inhibited the progression of glioblastoma. Further, the protooncogene, SKP2, which was shown to be a direct target of USP10 /USP13 was also reduced by spautin-1. While inhibition of SKP2 enhanced its downstream target p21, no apparent changes in the RAF-ERK levels or glycolytic function were evident. Also, inhibition of MEK1 did not affect SKP2 levels, indicating that these two pathways act independent of each other. Overall, our findings indicate that spautin-1 by virtue of its inhibitory effects on USP10/13 counteracts RAS-RAF-ERK mediated glycolysis and SKP2 that are critical in the progression of glioblastoma. Hence, further preclinical validation is warranted for taking the present observations forward.

Chemogenetic inhibition of the lateral hypothalamus effectively reduces food intake in rats in a translational proof-of-concept study

Despite the therapeutic potential of chemogenetics, the method lacks comprehensive preclinical validation, hindering its progression to human clinical trials. We aimed to validate a robust but simple in vivo efficacy assay in rats which could support chemogenetic drug discovery by providing a quick, simple and reliable animal model. Key methodological parameters such as adeno-associated virus (AAV) serotype, actuator drug, dose, and application routes were investigated by measuring the food-intake-reducing effect of chemogenetic inhibition of the lateral hypothalamus (LH) by hM4D(Gi) designer receptor stimulation. Subcutaneous deschloroclozapine in rats transfected with AAV9 resulted in a substantial reduction of food-intake, comparable to the efficacy of exenatide. We estimated that the effect of deschloroclozapine lasts 1–3 h post-administration. AAV5, oral administration of deschloroclozapine, and clozapine-N-oxide were also effective but with slightly less potency. The strongest effect on food-intake occurred within the first 30 min after re-feeding, suggesting this as the optimal experimental endpoint. This study demonstrates that general chemogenetic silencing of the LH can be utilized as an optimal, fast and reliable in vivo experimental model for conducting preclinical proof-of-concept studies in order to validate the in vivo effectiveness of novel chemogenetic treatments. We also hypothesize based on our results that universal LH silencing with existing and human translatable genetic neuroengineering techniques might be a viable strategy to affect food intake and influence obesity.

P16 Fibroblast signalling crosstalk drives junctional epidermolysis bullosa disease progression

Abstract Introduction and aims Junctional epidermolysis bullosa (JEB) is a devastating hereditary skin disorder characterized by severe fragility of skin and mucus membrane, emerges as blistering with little or no trauma. The major classification of JEB includes JEB intermediate and JEB severe; the latter is the most common subtype, which is the result of loss of function mutations in the skin basement membrane protein laminin-332. Lacking this protein impairs basement membrane adhesion of epidermal keratinocytes and leads to deregulated epidermal–dermal crosstalk. The skin dermis is composed of multiple fibroblast subpopulations with different functions in skin homeostasis and repair; however, their specific involvement in skin diseases is largely unknown. Here, we investigate how fibroblast subpopulations, namely papillary and reticular fibroblasts, impact JEB disease progression and skin regeneration. Methods Three-dimensional (3D) organotypic (OT) combining healthy (control) and diseased (JEB) keratinocytes cells with distinct fibroblast subpopulations from different anatomical sites were developed and analysed by immunohistochemistry and RNA-Seq. Results JEB OTs with papillary fibroblasts showed increased epidermal growth, invagination of basal layer and deregulated differentiation compared with reticular fibroblasts. RNA-Seq analysis revealed altered activity in key signalling including immune, cytokine, gap junction, mitogen-activated protein kinase and remodelling of keratinization and ECM proteins, particularly in JEB OT cultured with papillary fibroblast. JEB OTs and cells also demonstrated variation in cell proliferation and activation of transforming growth factor-β1 and immune cell signalling. Currently, we are targeting identified signalling pathways in our two-dimensional and 3D cell culture models and identifying druggable targets, before testing selected candidates in our JEB mouse disease model for further preclinical validation. Conclusions By dissecting the pathological signalling interactions between keratinocytes and distinct fibroblast subpopulations, our goal is to identify new therapeutic target to improve the skin health and regeneration in patients with JEB.