The emotional well-being of parents with children at genetic risk for type 1 diabetes before and during participation in the POInT-study.

INTRODUCTION: This study examined the emotional impact that parents experience when confronted with an increased genetic risk of type 1 diabetes (T1D) in their child. Population-based screening of neonates for genetic risk of chronic disease carries the risk of increased emotional burden for parents. METHODS: Information was collected using a well-being questionnaire for parents of infants identified as having an increased risk for T1D in a multinational research study. Parents were asked to complete this questionnaire after they were told their child had an increased risk for T1D (Freder1k-study) and at several time points during an intervention study (POInT-study), where oral insulin was administered daily. RESULTS: Data were collected from 2595 parents of 1371 children across five countries. Panic-related anxiety symptoms were reported by only 4.9% after hearing about their child having an increased risk. Symptoms of depression were limited to 19.4% of the parents at the result-communication visit and declined over time during the intervention study. When thinking about their child's risk for developing T1D (disease-specific anxiety), 47.2% worried, felt nervous and tense. Mothers and parents with a first-degree relative (FDR) with T1D reported more symptoms of depression and disease-specific anxiety (p < 0.001) than fathers and parents without a FDR. CONCLUSION: Overall, symptoms of depression and panic-related anxiety are comparable with the German population. When asked about their child's risk for T1D during the intervention study, some parents reported disease-specific anxiety, which should be kept in mind when considering population-based screening. As certain subgroups are more prone, it will be important to continue psychological screening and, when necessary, to provide support by an experienced, multidisciplinary team.

Identification of infants with increased type 1 diabetes genetic risk for enrollment into Primary Prevention Trials-GPPAD-02 study design and first results.

Primary prevention of type 1 diabetes (T1D) requires intervention in genetically at-risk infants. The Global Platform for the Prevention of Autoimmune Diabetes (GPPAD) has established a screening program, GPPAD-02, that identifies infants with a genetic high risk of T1D, enrolls these into primary prevention trials, and follows the children for beta-cell autoantibodies and diabetes. Genetic testing is offered either at delivery, together with the regular newborn testing, or at a newborn health care visits before the age of 5 months in regions of Germany (Bavaria, Saxony, Lower Saxony), UK (Oxford), Poland (Warsaw), Belgium (Leuven), and Sweden (Region Skåne). Seven clinical centers will screen around 330 000 infants. Using a genetic score based on 46 T1D susceptibility single-nucleotide polymorphisms (SNPs) or three SNPS and a first-degree family history for T1D, infants with a high (>10%) genetic risk for developing multiple beta-cell autoantibodies by the age of 6 years are identified. Screening from October 2017 to December 2018 was performed in 50 669 infants. The prevalence of high genetic risk for T1D in these infants was 1.1%. Infants with high genetic risk for T1D are followed up and offered to participate in a randomized controlled trial aiming to prevent beta-cell autoimmunity and T1D by tolerance induction with oral insulin. The GPPAD-02 study provides a unique path to primary prevention of beta-cell autoimmunity in the general population. The eventual benefit to the community, if successful, will be a reduction in the number of children developing beta-cell autoimmunity and T1D.

New approach for detection of LDL-hypercholesterolemia in the pediatric population: The Fr1dolin-Trial in Lower Saxony, Germany.

BACKGROUND AND AIMS: Lipid disorders are often detected very late, particularly in affected young children. We evaluated the feasibility of a screening for LDL-hypercholesterolemia (highLDL) among toddlers and preschoolers. METHODS: Population-based screening has been offered to all children (2-6 years) living in the State of Lower Saxony, Germany, with capillary blood sampling for detection of elevated LDL-cholesterol (LDL-C ≥ 135 mg/dL). Positive results were confirmed by a second measurement. Follow-up in specialized centers, including disease specific counselling and extended diagnostics, as well as evaluation of psychological distress of the parents, is carried out longitudinally. RESULTS: Up to March 2018, 5656 children have participated in the screening program. 5069/5656 children have completed the screening for highLDL (52.0% boys; median age: 4.0 years [Interquartile range, IQR 3.0-5.1]; mother age: 35 years [IQR 31-38]; father's age: 37 years; [IQR 33-42]). HighLDL was identified in 112 children (2.2%; 40.2% boys; LDL-C 157.6 ±â€¯29.5 mg/dL, mean ±â€¯SD). In the total cohort, parents stated in 40.9% of the cases a positive family history for hyperlipidemia and in 29.9% a premature cardiovascular event. Children with highLDL had more often both risk factors in their family history; however, in 37% of them none of these factors were reported. CONCLUSIONS: The first results of the screening program showed its feasibility and revealed high prevalence of highLDL in the general population. Furthermore, a large proportion of families of affected children were not aware about their lipid disorders.

Enhanced neurite outgrowth of human model (NT2) neurons by small-molecule inhibitors of Rho/ROCK signaling.

Axonal injury in the adult human central nervous system often results in loss of sensation and motor functions. Promoting regeneration of severed axons requires the inactivation of growth inhibitory influences from the tissue environment and stimulation of the neuron intrinsic growth potential. Especially glial cell derived factors, such as chondroitin sulfate proteoglycans, Nogo-A, myelin-associated glycoprotein, and myelin in general, prevent axon regeneration. Most of the glial growth inhibiting factors converge onto the Rho/ROCK signaling pathway in neurons. Although conditions in the injured nervous system are clearly different from those during neurite outgrowth in vitro, here we use a chemical approach to manipulate Rho/ROCK signalling with small-molecule agents to encourage neurite outgrowth in cell culture. The development of therapeutic treatments requires drug testing not only on neurons of experimental animals, but also on human neurons. Using human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA (Ras homolog gene family, member A GTPase) activation and promotes neurite growth. Inhibition of the downstream effector Rho kinase by the drug Y-27632 results in a strong increase in neurite outgrowth. Conversely, activation of the Rho pathway by lysophosphatidic acid results in growth cone collapse and eventually to neurite retraction. Finally, we show that blocking of Rho kinase, but not RhoA results in an increase in neurons bearing neurites. Due to its anti-inflammatory and neurite growth promoting action, the use of a pharmacological treatment of damaged neural tissue with Ibuprofen should be explored.

Spider silk as guiding biomaterial for human model neurons.

Over the last years, a number of therapeutic strategies have emerged to promote axonal regeneration. An attractive strategy is the implantation of biodegradable and nonimmunogenic artificial scaffolds into injured peripheral nerves. In previous studies, transplantation of decellularized veins filled with spider silk for bridging critical size nerve defects resulted in axonal regeneration and remyelination by invading endogenous Schwann cells. Detailed interaction of elongating neurons and the spider silk as guidance material is unknown. To visualize direct cellular interactions between spider silk and neurons in vitro, we developed an in vitro crossed silk fiber array. Here, we describe in detail for the first time that human (NT2) model neurons attach to silk scaffolds. Extending neurites can bridge gaps between single silk fibers and elongate afterwards on the neighboring fiber. Culturing human neurons on the silk arrays led to an increasing migration and adhesion of neuronal cell bodies to the spider silk fibers. Within three to four weeks, clustered somata and extending neurites formed ganglion-like cell structures. Microscopic imaging of human neurons on the crossed fiber arrays in vitro will allow for a more efficient development of methods to maximize cell adhesion and neurite growth on spider silk prior to transplantation studies.

Nitric oxide/cyclic GMP signaling regulates motility of a microglial cell line and primary microglia in vitro.

Microglia are the resident immune cells of the brain, which become rapidly activated and migrate to the site of insult in brain infection and disease. Activated microglia generate large amounts of the highly reactive messenger molecule nitric oxide (NO). NO is able to raise cyclic GMP levels via binding to soluble guanylyl cyclase. We investigated potential mechanistic links between inflammation, NO signaling, and microglial migration. To monitor cell migration, we used a scratch wound assay and compared results obtained in the BV-2 microglial line to primary microglia. Incubation with lipopolysaccharide (LPS) as stimulator of acute inflammatory processes enhanced migration of both microglial cell types. LPS activated NO production in BV-2 cells and application of an NO donor increased BV-2 cell migration while an NO scavenger reduced motility. Pharmacological inhibition of soluble guanylyl cyclase and the resulting decrease in motility can be rescued by a membrane permeant analog of cGMP. Despite differences in the threshold towards stimulation with the chemical agents, both BV-2 cells and primary microglia react in a similar way. The important role of NO/cGMP as positive regulator of microglial migration, the downstream targets of the signaling cascade, and resulting cytoskeletal changes can be conveniently investigated in a microglial cell line.

Schwann cell-free adult canine olfactory ensheathing cell preparations from olfactory bulb and mucosa display differential migratory and neurite growth-promoting properties in vitro.

BACKGROUND: Transplantation of olfactory ensheathing cells (OEC) and Schwann cells (SC) is a promising therapeutic strategy to promote axonal growth and remyelination after spinal cord injury. Previous studies mainly focused on the rat model though results from primate and porcine models differed from those in the rat model. Interestingly, canine OECs show primate-like in vitro characteristics, such as absence of early senescence and abundance of stable p75NTR expression indicating that this species represents a valuable translational species for further studies. So far, few investigations have tested different glial cell types within the same study under identical conditions. This makes it very difficult to evaluate contradictory or confirmatory findings reported in various studies. Moreover, potential contamination of OEC preparations with Schwann cells was difficult to exclude. Thus, it remains rather controversial whether the different glial types display distinct cellular properties. RESULTS: Here, we established cultures of Schwann cell-free OECs from olfactory bulb (OB-OECs) and mucosa (OM-OECs) and compared them in assays to Schwann cells. These glial cultures were obtained from a canine large animal model and used for monitoring migration, phagocytosis and the effects on in vitro neurite growth. OB-OECs and Schwann cells migrated faster than OM-OECs in a scratch wound assay. Glial cell migration was not modulated by cGMP and cAMP signaling, but activating protein kinase C enhanced motility. All three glial cell types displayed phagocytic activity in a microbead assay. In co-cultures with of human model (NT2) neurons neurite growth was maximal on OB-OECs. CONCLUSIONS: These data provide evidence that OB- and OM-OECs display distinct migratory behavior and interaction with neurites. OB-OECs migrate faster and enhance neurite growth of human model neurons better than Schwann cells, suggesting distinct and inherent properties of these closely-related cell types. Future studies will have to address whether, and how, these cellular properties correlate with the in vivo behavior after transplantation.

The erythropoietin-derived peptide mimetic pHBSP affects cellular and cognitive consequences in a rat post-status epilepticus model.

PURPOSE: The selection of a minimal active sequence of erythropoietin allowed the design of peptide mimetics that exert beneficial effects in the central nervous system but lack an erythropoietic effect. Erythropoietin has been suggested as a promising therapeutic and prophylactic for epilepsies based on its neuroprotective, neuroregenerative, and antiinflammatory potency. Therefore, it is of particular interest to evaluate whether the nonerythropoietic erythropoietin-derived peptide pHBSP can affect epileptogenesis. METHODS: In a post-status epilepticus model in rats, we determined the effects of pHBSP and of recombinant human erythropoietin with short-term administration following status epilepticus. KEY FINDINGS: Both pHBSP and erythropoietin further enhanced the status epilepticus-associated increase in hippocampal cell proliferation. Thereby, pHBSP seemed to promote neuronal differentiation and survival resulting in a significant increase in neurogenesis. Neither pHBSP nor erythropoietin affected the number of animals exhibiting spontaneous recurrent seizures as well as the seizure frequency in the chronic phase. In the Morris water maze, pHBSP attenuated cognitive deficits in epileptic animals. SIGNIFICANCE: In conclusion, the helix B-derived erythropoietin peptide pHBSP can modulate the cellular and cognitive consequences of a status epilepticus. The impact of pHBSP on spatial learning might indicate that the peptide allows beneficial effects on epileptogenesis-associated cognitive deficits. However, it needs to be considered that learning deficits were not abolished by pHBSP and that the effects were not observed consistently until the end of the study. Therefore, adjustment of timing, duration, and dose of peptide administration might be necessary to further evaluate the efficacy of pHBSP.

Rapid differentiation of human embryonal carcinoma stem cells (NT2) into neurons for neurite outgrowth analysis.

Human neurons derived from stem cells can be employed as in vitro models to predict the potential of neurochemicals affecting neurodevelopmental cellular processes including proliferation, migration, and differentiation. Here, we developed a model of differentiating human neurons from well characterized human embryonal carcinoma stem cells (NT2). NT2 cells were induced to differentiate into neuronal phenotypes after 2 weeks of treatment with retinoic acid in aggregate culture. Nestin positive progenitor cells migrate out of NT2 aggregates and differentiate into ßIII-tubulin expressing neuronal cells. Culturing the NT2 cells for an additional 7-14 days resulted in increased percentage of ßIII-tubulin expressing cells, elaborating a long neurite that positively stained for axonal marker (Tau) and presynaptic protein (synapsin). We then asked whether neurite outgrowth from NT2 cells is modulated by bioactive chemicals. Since the cAMP/PKA pathway has been widely investigated as a regulator of neurite outgrowth/regeneration in several experimental systems, we used chemical activators and inhibitors of cAMP/PKA pathway in the culture. The adenylyl cyclase activator, forskolin, and cell-permeable analog of cAMP, 8-Br-cAMP increased the percentage of neurite bearing cells and neurite extension. Application of the protein kinase A inhibitors, H-89 and Rp-cAMP, blocked neurite formation. Taken together, NT2 aggregates undergo migration, differentiation, and neurite elaboration and can be used as a model of differentiating human neurons to screen neurochemicals and to understand cellular mechanisms of human nerve cell development.