Behavioral comorbidities treatment by fecal microbiota transplantation in canine epilepsy: a pilot study of a novel therapeutic approach.

Introduction: Anxiety and cognitive dysfunction are frequent, difficult to treat and burdensome comorbidities in human and canine epilepsy. Fecal microbiota transplantation (FMT) has been shown to modulate behavior in rodent models by altering the gastrointestinal microbiota (GIM). This study aims to investigate the beneficial effects of FMT on behavioral comorbidities in a canine translational model of epilepsy. Methods: Nine dogs with drug-resistant epilepsy (DRE) and behavioral comorbidities were recruited. The fecal donor had epilepsy with unremarkable behavior, which exhibited a complete response to phenobarbital, resulting in it being seizure-free long term. FMTs were performed three times, two weeks apart, and the dogs had follow-up visits at three and six months after FMTs. Comprehensive behavioral analysis, including formerly validated questionnaires and behavioral tests for attention deficit hyperactivity disorder (ADHD)- and fear- and anxiety-like behavior, as well as cognitive dysfunction, were conducted, followed by objective computational analysis. Blood samples were taken for the analysis of antiseizure drug (ASD) concentrations, hematology, and biochemistry. Urine neurotransmitter concentrations were measured. Fecal samples were subjected to analysis using shallow DNA shotgun sequencing, real-time polymerase chain reaction (qPCR)-based Dysbiosis Index (DI) assessment, and short-chain fatty acid (SCFA) quantification. Results: Following FMT, the patients showed improvement in ADHD-like behavior, fear- and anxiety-like behavior, and quality of life. The excitatory neurotransmitters aspartate and glutamate were decreased, while the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and GABA/glutamate ratio were increased compared to baseline. Only minor taxonomic changes were observed, with a decrease in Firmicutes and a Blautia_A species, while a Ruminococcus species increased. Functional gene analysis, SCFA concentration, blood parameters, and ASD concentrations remained unchanged. Discussion: Behavioral comorbidities in canine IE could be alleviated by FMT. This study highlights FMT's potential as a novel approach to improving behavioral comorbidities and enhancing the quality of life in canine patients with epilepsy.

Prevalence of Clostridioides difficile in Canine Feces and Its Association with Intestinal Dysbiosis.

The role of Clostridioides (C.) difficile as an enteropathogen in dogs is controversial. In humans, intestinal bile acid-dysmetabolism is associated with C. difficile prevalence. The relationship between fecal qPCR-based dysbiosis index (DI) and especially the abundance of bile acid-converting Clostridium hiranonis with the presence of C. difficile in dogs was explored across the following 4 cohorts: 358 fecal samples submitted for routine diagnostic work-up, 33 dogs with chronic enteropathy, 14 dogs with acute diarrhea, and 116 healthy dogs. Dogs that tested positive for C. difficile had significantly higher DI (median, 4.4 (range from 0.4 to 8.6)) and lower C. hiranonis (median, 0.1 (range from 0.0 to 7.5) logDNA/g) than dogs that tested negative for C. difficile (median DI, -1 (range from -7.2 to 8.9); median C. hiranonis abundance, 6.2 (range from 0.1 to 7.5) logDNA/g; p < 0.0001, respectively). In 33 dogs with CE and 14 dogs with acute diarrhea, the treatment response did not differ between C. difficile-positive and -negative dogs. In the group of clinically healthy dogs, 9/116 tested positive for C. difficile, and 6/9 of these had also an abnormal DI. In conclusion, C. difficile is strongly linked to intestinal dysbiosis and lower C. hiranonis levels in dogs, but its presence does not necessitate targeted treatment.

Systemic sclerosis-associated myositis features minimal inflammation and characteristic capillary pathology.

Systemic sclerosis represents a chronic connective tissue disease featuring fibrosis, vasculopathy and autoimmunity, affecting skin, multiple internal organs, and skeletal muscles. The vasculopathy is considered obliterative, but its pathogenesis is still poorly understood. This may partially be due to limitations of conventional transmission electron microscopy previously being conducted only in single patients. The aim of our study was therefore to precisely characterize immune inflammatory features and capillary morphology of systemic sclerosis patients suffering from muscle weakness. In this study, we identified 18 individuals who underwent muscle biopsy because of muscle weakness and myalgia in a cohort of 367 systemic sclerosis patients. We performed detailed conventional and immunohistochemical analysis and large-scale electron microscopy by digitizing entire sections for in-depth ultrastructural analysis. Muscle biopsies of 12 of these 18 patients (67%) presented minimal features of myositis but clear capillary alteration, which we termed minimal myositis with capillary pathology (MMCP). Our study provides novel findings in systemic sclerosis-associated myositis. First, we identified a characteristic and specific morphological pattern termed MMCP in 67% of the cases, while the other 33% feature alterations characteristic of other overlap syndromes. This is also reflected by a relatively homogeneous clinical picture among MMCP patients. They have milder disease with little muscle weakness and a low prevalence of interstitial lung disease (20%) and diffuse skin involvement (10%) and no cases of either pulmonary arterial hypertension or renal crisis. Second, large-scale electron microscopy, introducing a new level of precision in ultrastructural analysis, revealed a characteristic capillary morphology with basement membrane thickening and reduplications, endothelial activation and pericyte proliferation. We provide open-access pan-and-zoom analysis to our datasets, enabling critical discussion and data mining. We clearly highlight characteristic capillary pathology in skeletal muscles of systemic sclerosis patients.

German guidelines on the diagnosis and treatment of neurosyphilis.

INTRODUCTION: In view of the importance of neurosyphilis and the difficulties encountered in diagnosing it, the S1 guideline "Neurosyphilis" has been published by the German Society for Neurology (DGN) in accordance with the stipulations of the Association of the Scientific Medical Societies in Germany (AWMF). The present article is an abridged translation of that German guideline. MAIN RECOMMENDATIONS: (a) Neurosyphilis can manifest as early neurosyphilis (meningitis, meningovascular neurosyphilis or syphilitic gummas) or late neurosyphilis (tabes dorsalis, general paresis). (b) The following diagnostic criteria help to establish the presence of probable neurosyphilis (always point iv, accompanied by any two of points i to iii): (i) subacute or chronic neuro-psychiatric symptoms; (ii) increased cerebrospinal fluid (CSF) cell count or signs of blood-CSF barrier disruption; (iii) positive effect of anti-neurosyphilis antibiotic therapy on clinical course and CSF findings; (iv) positive TPHA/TPPA or FTA test in serum. (c) The diagnosis of neurosyphilis is confirmed by the subsequent detection of intrathecal production of antibodies against Treponema pallidum. (d) In neurosyphilis, treatment with intravenous penicillin or ceftriaxone for 14 days is recommended. (e) The following parameters can be used to assess a therapeutic effect: clinical findings, serum VDRL, and CSF cell count. CONCLUSION: The German guideline on the diagnosis and treatment of neurosyphilis is a practical tool to support clinicians in diagnosing and treating patients with neurosyphilis. This article is an abridged translation of this guideline (Klein MW, J.; Angstwurm, K.; Esser, S.; Hahn, K.; Matschke, M.; Scheithauer, S.; Schoefer, H.; Sturzenegger, M.; Wildemann, B. Neurosyphilis, S1-Leitlinie. Deutsche Gesellschaft für Neurologie, Leitlinien für Diagnostik und Thearpie in der Neurologie 2020).

Isolated atrial amyloidosis suspected by electrophysiological voltage mapping and diagnosed by 99m Tc-DPD scintigraphy.

We present not-yet-seen multimodal images of a 55-year-old female patient with isolated atrial amyloidosis (IAA) who clinically suffered from multiple atrial arrhythmias and heart failure symptoms with preserved left ventricular ejection fraction. We aim to show structural and functional abnormalities detected by electrophysiological voltage mapping, cardiac magnetic resonance imaging (MRI) [cMRI; atrial strain measurements, late gadolinium enhancement (LGE) visualization], and 99m Tc-DPD scintigraphy. Bipolar voltage mapping performed during two electrophysiological procedures showed diffuse left atrial low-voltage areas (bipolar < 0.5 mV) and also a moderately diseased right atrium suspected of infiltrative cardiomyopathy. Catheter ablation did successfully treat a left atrial and two right atrial focal tachycardias. For further diagnostics, a 3T cMRI was performed, revealing a subendocardial circumferential left atrial LGE and pathological atrial strain measurements, especially during conduit and reservoir phase. Afterwards, nuclear imaging with 559 MBq of 99m Tc-DPD was performed. The scan revealed amyloid infiltration of the left atrium. Neither an uptake in the ventricular myocardium nor an extra-cardiac uptake of DPD was seen. Genetic testing for transthyretin amyloidosis mutations in this patient was negative, and peripheral neuropathy was ruled out by electromyogram analysis. The synopsis of these findings reveals IAA as the most possible diagnosis and showed isolated atrial nuclear tracer uptake with 99m Tc-DPD scintigraphy for the first time. Non-invasive imaging techniques might help in suggesting IAA but need further investigation.

Hydroxyethylstarch (130/0.4) tightens the blood-brain barrier in vitro.

In order to prevent cerebral vasospasm after a subarachnoid hemorrhage (SAH), the so-called triple H-therapy (hypertension, hypervolemia, hemodilution) could be applied. In these cases, colloidal solutions containing Hydroxyethylstarch (HES) are used to induce hypervolemia. The administration of HES is very much under debate for the mentioned use, because in general the application of HES for the treatment of critical ill patients has been reduced tremendously in the last years due to its nephrotoxic effects. In this context, there are limited data investigating the influence of HES on the blood-brain barrier. These data might help to assess if a transient administration of HES is possibly justifiable to prevent cerebral ischemia during vasospasm despite the risk of an acute kidney injury. To address this question, a mouse blood-brain barrier in vitro model based on cell line cerebEND was exposed to different HES concentrations and compared to NaCl-containing control solutions. In order to assess the effects of HES on blood-brain barrier properties, cell viability, transendothelial electrical resistance, permeability of carboxyfluorescein, mRNA and protein expression and localization of tight junction proteins were determined. In summary, 1.5-4% HES attenuated cell viability in a mild, concentration dependent manner compared to the NaCl control solution (0% HES). At the mRNA level 1% and 4% HES significantly increased the expression of tight junction associated proteins (ZO-1 and occludin) and the glucose transporter Glut-1 (Slc2a1). In correspondence to this, 4% HES inhibited breakdown of the paracellular barrier in comparison to the control NaCl group (0% HES) shown by transendothelial electrical resistance values and the permeability of the paracellular marker carboxyfluorescein. These effects at the functional level were confirmed by immunofluorescence microscopic images of junctional proteins. The obtained in vitro data showed a potential for HES to counteract blood-brain barrier damage. Future studies are needed to reveal the applicability of HES as a blood-brain barrier stabilizing agent.

An electrochemiluminescence based assay for quantitative detection of endogenous and exogenously applied MeCP2 protein variants.

Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional chromosomal protein that plays a key role in the central nervous system. Its levels need to be tightly regulated, as both deficiency and excess of the protein can lead to severe neuronal dysfunction. Loss-of-function mutations affecting MeCP2 are the primary cause of Rett syndrome (RTT), a severe neurological disorder that is thought to result from absence of functional protein in the brain. Several therapeutic strategies for the treatment of RTT are currently being developed. One of them is the use of stable and native TAT-MeCP2 fusion proteins to replenish its levels in neurons after permeation across the blood-brain barrier (BBB). Here we describe the expression and purification of various transactivator of transcription (TAT)-MeCP2 variants and the development of an electrochemiluminescence based assay (ECLIA) that is able to measure endogenous MeCP2 and recombinant TAT-MeCP2 fusion protein levels in a 96-well plate format. The MeCP2 ECLIA produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error throughout a wide working range. To underline its broad applicability, this assay was used to analyze brain tissue and study the transport of TAT-MeCP2 variants across an in vitro model of the blood-brain barrier.

Synergistic estrogenic effects of Fusarium and Alternaria mycotoxins in vitro.

Mycotoxins are toxic secondary metabolites formed by various fungal species that are found as natural contaminants in food. This very heterogeneous group of compounds triggers multiple toxic mechanisms, including endocrine disruptive potential. Current risk assessment of mycotoxins, as for most chemical substances, is based on the effects of single compounds. However, concern on a potential enhancement of risks by interactions of single substances in naturally occurring mixtures has greatly increased recently. In this study, the combinatory effects of three mycoestrogens were investigated in detail. This includes the endocrine disruptors zearalenone (ZEN) and α-zearalenol (α-ZEL) produced by Fusarium fungi and alternariol (AOH), a cytotoxic and estrogenic mycotoxin formed by Alternaria species. For evaluation of effects, estrogen-dependent activation of alkaline phosphatase (AlP) and cell proliferation were tested in the adenocarcinoma cell line Ishikawa. The estrogenic potential varied among the single substances. Half maximum effect concentrations (EC50) for AlP activation were evaluated for α-ZEL, ZEN and AOH as 37 pM, 562 pM and 995 nM, respectively. All three mycotoxins were found to act as partial agonists. The majority of binary combinations, even at very low concentrations in the case of α-ZEL, showed strong synergism in the AlP assay. These potentiating phenomena of mycotoxin mixtures highlight the urgent need to incorporate combinatory effects into future risk assessment, especially when endocrine disruptors are involved. To the best of our knowledge, this study presents the first investigation on synergistic effects of mycoestrogens.

Additive manufacturing of scaffolds with sub-micron filaments via melt electrospinning writing.

The aim of this study was to explore the lower resolution limits of an electrohydrodynamic process combined with direct writing technology of polymer melts. Termed melt electrospinning writing, filaments are deposited layer-by-layer to produce discrete three-dimensional scaffolds for in vitro research. Through optimization of the parameters (flow rate, spinneret diameter, voltage, collector distance) for poly-ϵ-caprolactone, we could direct-write coherent scaffolds with ultrafine filaments, the smallest being 817 ± 165 nm. These low diameter filaments were deposited to form box-structures with a periodicity of 100.6 ± 5.1 µm and a height of 80 µm (50 stacked filaments; 100 overlap at intersections). We also observed oriented crystalline regions within such ultrafine filaments after annealing at 55 °C. The scaffolds were printed upon NCO-sP(EO-stat-PO)-coated glass slide surfaces and withstood frequent liquid exchanges with negligible scaffold detachment for at least 10 days in vitro.

Quantifying ligand-cell interactions and determination of the surface concentrations of ligands on hydrogel films: The measurement challenge.

Hydrogels are extensively studied for biomaterials application as they provide water swollen noninteracting matrices in which specific binding motifs and enzyme-sensitive degradation sites can be incorporated to tailor cell adhesion, proliferation, and migration. Hydrogels also serve as excellent basis for surface modification of biomaterials where interfacial characteristics are decisive for implant success or failure. However, the three-dimensional nature of hydrogels makes it hard to distinguish between the bioactive ligand density at the hydrogel-cell interface that is able to interact with cells and the ligands that are immobilized inside the hydrogel and not accessible for cells. Here, the authors compare x-ray photoelectron spectrometry (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), enzyme linked immunosorbent assay (ELISA), and the correlation with quantitative cell adhesion using primary human dermal fibroblasts (HDF) to gain insight into ligand distribution. The authors show that although XPS provides the most useful quantitative analysis, it lacks the sensitivity to measure biologically meaningful concentrations of ligands. However, ToF-SIMS is able to access this range provided that there are clearly distinguishable secondary ions and a calibration method is found. Detection by ELISA appears to be sensitive to the ligand density on the surface that is necessary to mediate cell adhesion, but the upper limit of detection coincides closely with the minimal ligand spacing required to support cell proliferation. Radioactive measurements and ELISAs were performed on amine reactive well plates as true 2D surfaces to estimate the ligand density necessary to allow cell adhesion onto hydrogel films. Optimal ligand spacing for HDF adhesion and proliferation on ultrathin hydrogel films was determined as 6.5 ± 1.5 nm.

Hydrogel-fibre composites with independent control over cell adhesion to gel and fibres as an integral approach towards a biomimetic artificial ECM.

In the body, cells are surrounded by an interconnected mesh of insoluble, bioactive protein fibres to which they adhere in a well-controlled manner, embedded in a hydrogel-like highly hydrated matrix. True morphological and biochemical mimicry of this so-called extracellular matrix (ECM) remains a challenge but appears decisive for a successful design of biomimetic three-dimensional in vitro cell culture systems. Herein, an approach is presented which describes the fabrication and in vitro assessment of an artificial ECM which contains two major components, i.e. specifically biofunctionalized fibres and a semi-synthetic hyaluronic acid-based hydrogel, which allows control over cell adhesion towards both components. As proof of principle for the control of cell adhesion, RGD as well-known cell adhesive cue and the control sequence RGE are immobilized in the system. In vitro studies with primary human dermal fibroblasts were conducted to evaluate the specificity of cell adhesion and the potential of the composite system to support cell growth. Finally, one possible application example for guided cell growth is shown by the use of oriented fibres in a hydrogel matrix.

A hydrogel-based versatile screening platform for specific biomolecular recognition in a well plate format.

Precise determination of biomolecular interactions in high throughput crucially depends on a surface coating technique that allows immobilization of a variety of interaction partners in a non-interacting environment. We present a one-step hydrogel coating system based on isocyanate functional six-arm poly(ethylene oxide)-based star polymers for commercially available 96-well microtiter plates that combines a straightforward and robust coating application with versatile bio-functionalization. This system generates resistance to unspecific protein adsorption and cell adhesion, as demonstrated with fluorescently labeled bovine serum albumin and primary human dermal fibroblasts (HDF), and high specificity for the assessment of biomolecular recognition processes when ligands are immobilized on this surface. One particular advantage is the wide range of biomolecules that can be immobilized and convert the per se inert coating into a specifically interacting surface. We here demonstrate the immobilization and quantification of a broad range of biochemically important ligands, such as peptide sequences GRGDS and GRGDSK-biotin, the broadly applicable coupler molecule biocytin, the protein fibronectin, and the carbohydrates N-acetylglucosamine and N-acetyllactosamine. A simplified protocol for an enzyme-linked immunosorbent assay was established for the detection and quantification of ligands on the coating surface. Cell adhesion on the peptide and protein-modified surfaces was assessed using HDF. All coatings were applied using a one-step preparation technique, including bioactivation, which makes the system suitable for high-throughput screening in a format that is compatible with the most routinely used testing systems.

Minicircle performance depending on S/MAR-nuclear matrix interactions.

The ideal vector for cell and tissue modification does not depend on integration but rather behaves as an independent functional unit that replicates as an episome. Based on a scaffold/matrix attachment region (S/MAR), we have introduced, in 2006, an approximately 4-kb replicating nonviral minicircle able to exploit the cellular replication machinery in a way reminiscent of ARS vectors. Consisting of only one active transcription unit and the S/MAR, it resists silencing as it is free of prokaryotic vector parts and drug selection markers. The rate of final establishment in the nuclear architecture is moderate but comparable to Epstein-Barr virus-based episomes (<5%). Here, we demonstrate that this parameter can be improved if the host cell chromatin is opened by histone hyperacetylation prior to transfection. It remains unaffected, however, by cell cycle position. Still, this class of episomes revealed intrinsic instability and integration after 5 months of continuous culture. In vivo evolution enabled the effective reduction of S/MAR size from 2 kb to 733 bp (resulting in a minicircle of approximately 3 kb) with largely improved stability and cloning capacity. Investigation of individual clones served to prove persistent and homogenous expression, which is ascribed to stable association with nuclear attachment sites. Optimum expression levels were shown to depend on the authentic usage of a polyadenylation site 3' from the S/MAR as anticipated by the stress-induced duplex destabilization algorithm, which finds increasing use to predict the functional parameters of these systems.