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Fabry disease (FD) is a lysosomal storage disorder of X-linked inheritance. Mutations in the α-galactosidase A gene lead to cellular globotriaosylceramide (Gb3) depositions and triggerable acral burning pain in both sexes as an early FD symptom of unknown pathophysiology. We aimed at elucidating the link between skin cells and nociceptor sensitization contributing to FD pain in a sex-associated manner. We used cultured keratinocytes and fibroblasts of 27 adult FD patients and 20 healthy controls. Epidermal keratinocytes and dermal fibroblasts were cultured and immunoreacted to evaluate Gb3 load. Gene expression analysis of pain-related ion channels and pro-inflammatory cytokines was performed in dermal fibroblasts. We further investigated electrophysiological properties of induced pluripotent stem cell (iPSC) derived sensory-like neurons of a man with FD and a healthy man and incubated the cells with interleukin 8 (IL-8) or fibroblast supernatant as an in vitro model system. Keratinocytes displayed no intracellular, but membrane-bound Gb3 deposits. In contrast, fibroblasts showed intracellular Gb3 and revealed higher gene expression of potassium intermediate/small conductance calcium-activated potassium channel 3.1 (KCa 3.1, KCNN4 ) in both, men and women with FD compared to controls. Additionally, cytokine expression analysis showed increased IL-8 RNA levels only in female FD fibroblasts. Patch-clamp studies revealed reduced rheobase currents for both iPSC neuron cell lines incubated with IL-8 or fibroblast supernatant of women with FD. We conclude that Gb3 deposition in female FD patient skin fibroblasts may lead to increased KCa3.1 activity and IL-8 secretion. This may result in cutaneous nociceptor sensitization as a potential mechanism contributing to a sex-associated FD pain phenotype.

IgG4 autoantibodies against paranodal proteins are known to induce acute-onset and often severe sensorimotor autoimmune neuropathies. How autoantibodies reach their antigens at the paranode in spite of the myelin barrier is still unclear. We performed in vitro incubation experiments with patient sera on unfixed and unpermeabilized nerve fibers and in vivo intraneural and intrathecal passive transfer of patient IgG to rats, to explore the access of IgG autoantibodies directed against neurofascin-155 and contactin-1 to the paranodes and their pathogenic effect. We found that in vitro incubation resulted in weak paranodal binding of anti-contactin-1 autoantibodies whereas anti-neurofascin-155 autoantibodies bound to the nodes more than to the paranodes. After short-term intraneural injection, no nodal or paranodal binding was detectable when using anti-neurofascin-155 antibodies. After repeated intrathecal injections, nodal more than paranodal binding could be detected in animals treated with anti-neurofascin-155, accompanied by sensorimotor neuropathy. In contrast, no paranodal binding was visible in rats intrathecally injected with anti-contactin-1 antibodies, and animals remained unaffected. These data support the notion of different pathogenic mechanisms of anti-neurofascin-155 and anti-contactin-1 autoantibodies and different accessibility of paranodal and nodal structures.

Reprogramming of human-induced pluripotent stem cells (iPSCs) and their differentiation into specific cell types, such as induced sensory-like neurons (iSNs), are critical for disease modeling and drug testing. However, the variability of cell populations challenges reliability and reproducibility. While various protocols for iSN differentiation exist, the development of non-iSN cells in these cultures remains an issue. Therefore, standardization of protocols is essential. This study aimed to improve iSN culture conditions by reducing the number of non-iSN cells while preserving the survival and quality of iSNs. iSNs were differentiated from a healthy control iPSC line using an established protocol. Interventions for protocol optimization included floxuridine (FdU) or 1-β-D-arabinofuranosyl-cytosine hydrochloride (AraC) treatment, magnetic-activated cell sorting (MACS), early cell passaging, and replating. Cell viability and iSN-to-total-cell-count ratio were assessed using a luminescent assay and immunocytochemistry, respectively. Passaging of cells during differentiation did not increase the iSN-to-total-cell-count ratio, and MACS of immature iSNs led to neuronal blebbing and reduced the iSN-to-total-cell-count ratio. Treatment with high concentrations and prolonged incubation of FdU or AraC resulted in excessive cell death. However, treatment with 10 μM FdU for 24 h post-differentiation showed the most selective targeting of non-iSN cells, leading to an increase in the iSN-to-total-cell count ratio without compromising the viability or functionality of the iSN population. Replating of iSNs shortly after seeding also helped to reduce non-iSN cells. In direct comparison with other methods, treatment with 10 μM FdU for 24 h after differentiation shows promise for improving iSN culture purity, which could benefit downstream applications in disease modeling and drug discovery. However, further investigations involving multiple iPSC lines and optimization of protocol parameters are warranted to fully exploit the potential of this method and enhance its reproducibility and applicability. Overall, this study provides valuable insights into optimizing culture conditions for iSN differentiation and highlights the importance of standardized protocols in iPSC-based research.

Reprogramming of human induced pluripotent stem cells (iPSC) and their differentiation into specific cell types, such as induced sensory-like neurons (iSN), are critical for disease modeling and drug testing. However, the variability of cell populations challenges reliability and reproducibility. While various protocols for iSN differentiation exist, development of non-iSN cells in these cultures remains an issue. Therefore, standardization of protocols is essential. This study aimed to improve iSN culture conditions by reducing the number of non-iSN cells while preserving survival and quality of iSN. iSN were differentiated from a healthy control iPSC line using an established protocol. Interventions for protocol optimization included floxuridine (FdU) or 1-β-D-arabinofuranosyl-cytosin-hydrochlorid (AraC) treatment, Magnetic-Activated Cell Sorting (MACS), early cell passaging, and replating. Cell viability and iSN-to-total-cell-count ratio were assessed using a luminescent assay and immunocytochemistry, respectively. Passaging of cells during differentiation did not increase the iSN-to-total-cell-count ratio, and MACS of immature iSN led to neuronal blebbing and reduced the iSN-to-total-cell-count ratio. Treatment with high concentrations and prolonged incubation of FdU or AraC resulted in excessive cell death. However, treatment with 10 μM FdU for 24 h post-differentiation showed the most selective targeting of non-iSN cells, leading to an increase in the iSN-to-total-cell count ratio without compromising the viability or functionality of the iSN population. Replating of iSN shortly after seeding also helped to reduce non-iSN cells. In direct comparison to other methods, treatment with 10 μM FdU for 24 h after differentiation represents a promising approach to improve iSN culture purity, offering a potential benefit for downstream applications in disease modeling and drug discovery. However, further investigations involving multiple iPSC lines and optimization of protocol parameters are warranted to fully exploit the potential of this method and enhance its reproducibility and applicability. Overall, this study provides valuable insights into optimizing culture conditions for iSN differentiation and highlights the importance of standardized protocols in iPSC-based research.

Acral burning pain triggered by fever, thermal hyposensitivity, and skin denervation are hallmarks of small fibre neuropathy in Fabry disease, a life-threatening X-linked lysosomal storage disorder. Variants in the gene encoding alpha-galactosidase A may lead to impaired enzyme activity with cellular accumulation of globotriaosylceramide (Gb3). To study the underlying pathomechanism of Fabry-associated small fibre neuropathy, we generated a neuronal in vitro disease model using patient-derived induced pluripotent stem cells from three Fabry patients and one healthy control. We further generated an isogenic control line via CRISPR/Cas9 gene editing. We subjected iPSC to targeted peripheral neuronal differentiation and observed intra-lysosomal Gb3 accumulations in somas and neurites of Fabry sensory neurons using super-resolution microscopy. At functional level, patch-clamp analysis revealed a hyperpolarizing shift of voltage-gated sodium channel steady-state inactivation kinetics in Fabry cell lines as compared to the healthy control. Moreover, we demonstrate a drastic increase in Fabry sensory neuron Ca2+ levels at 39°C mimicking clinical fever (p < 0.001). This pathophysiological phenotype was accompanied by thinning of neurite calibres in sensory neurons obtained from Fabry patients compared to healthy control cells (p < 0.001). Linear-Nonlinear cascade models fit to spiking responses revealed that Fabry cell lines exhibit altered single neuron encoding properties relative to control. We further observed jam of mitochondrial trafficking at sphingolipid accumulations within Fabry sensory neurites utilizing a click-chemistry approach together with mitochondrial dysmorphism compared to healthy control cells. We pioneer insights into the cellular mechanisms contributing to pain, thermal hyposensitivity, and denervation in Fabry small fibre neuropathy, and pave the way for further mechanistic in vitro studies in Fabry disease and the development of novel treatment approaches.

Infection-related diabetes can arise as a result of virus-associated β-cell destruction. Clinical data suggest that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the coronavirus disease 2019 (COVID-19), impairs glucose homoeostasis, but experimental evidence that SARS-CoV-2 can infect pancreatic tissue has been lacking. In the present study, we show that SARS-CoV-2 infects cells of the human exocrine and endocrine pancreas ex vivo and in vivo. We demonstrate that human β-cells express viral entry proteins, and SARS-CoV-2 infects and replicates in cultured human islets. Infection is associated with morphological, transcriptional and functional changes, including reduced numbers of insulin-secretory granules in β-cells and impaired glucose-stimulated insulin secretion. In COVID-19 full-body postmortem examinations, we detected SARS-CoV-2 nucleocapsid protein in pancreatic exocrine cells, and in cells that stain positive for the β-cell marker NKX6.1 and are in close proximity to the islets of Langerhans in all four patients investigated. Our data identify the human pancreas as a target of SARS-CoV-2 infection and suggest that β-cell infection could contribute to the metabolic dysregulation observed in patients with COVID-19. SARS-CoV-2 is shown to infect and replicate in human pancreatic tissue, including in β-cells, which is associated with morphological, transcriptomic and functional changes.

PurposeKnowledge regarding patients’ clinical condition at severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection is sparse. Data in the international, multicenter Lean European Open Survey on SARS-CoV-2-Infected Patients (LEOSS) cohort study may enhance the understanding of COVID-19.MethodsSociodemographic and clinical characteristics of SARS-CoV-2-infected patients, enrolled in the LEOSS cohort study between March 16, 2020, and May 14, 2020, were analyzed. Associations between baseline characteristics and clinical stages at diagnosis (uncomplicated vs. complicated) were assessed using logistic regression models.ResultsWe included 2155 patients, 59.7% (1,287/2,155) were male; the most common age category was 66–85 years (39.6%; 500/2,155). The primary COVID-19 diagnosis was made in 35.0% (755/2,155) during complicated clinical stages. A significant univariate association between age; sex; body mass index; smoking; diabetes; cardiovascular, pulmonary, neurological, and kidney diseases; ACE inhibitor therapy; statin intake and an increased risk for complicated clinical stages of COVID-19 at diagnosis was found. Multivariable analysis revealed that advanced age [46–65 years: adjusted odds ratio (aOR): 1.73, 95% CI 1.25–2.42, p = 0.001; 66–85 years: aOR 1.93, 95% CI 1.36–2.74, p < 0.001; > 85 years: aOR 2.38, 95% CI 1.49–3.81, p < 0.001 vs. individuals aged 26–45 years], male sex (aOR 1.23, 95% CI 1.01–1.50, p = 0.040), cardiovascular disease (aOR 1.37, 95% CI 1.09–1.72, p = 0.007), and diabetes (aOR 1.33, 95% CI 1.04–1.69, p = 0.023) were associated with complicated stages of COVID-19 at diagnosis.ConclusionThe LEOSS cohort identified age, cardiovascular disease, diabetes and male sex as risk factors for complicated disease stages at SARS-CoV-2 diagnosis, thus confirming previous data. Further data regarding outcomes of the natural course of COVID-19 and the influence of treatment are required.

The progression of diabetic nephropathy is associated with an infiltration of macrophages expressing different phenotypes. As classically activated chemokine receptor CCR2+ macrophages are thought to drive tissue inflammation and remodeling, we tested whether blocking CCR2 could reduce intrarenal inflammation and prevent glomerulosclerosis in type 2 diabetes. This was achieved with RO5234444, an orally active small-molecule CCR2 antagonist that blocks ligand binding, its internalization, and monocyte chemotaxis. Male type 2 diabetic db/db mice were uninephrectomized to increase glomerular hyperfiltration to accelerate the development of glomerulosclerosis. From 16 weeks until killing at 24 weeks of age, mice were chow fed with or without admixed antagonist to achieve a trough plasma concentration above IC50 for binding in the mouse. CCR2 blockade reduced circulating monocyte levels, but did not affect total leukocyte or neutrophil numbers, and was associated with a reduction in the number of macrophages and apoptotic podocytes in the glomerulus. This treatment resulted in a higher total number of podocytes, less glomerulosclerosis, reduced albuminuria, and a significantly improved glomerular filtration rate. This successful pre-clinical trial suggests that this antagonist may now be ready for testing in humans with the nephropathy of diabetes mellitus.

Contamination of plastic particles in environmental and biological systems raises concerns regarding their potential negative impacts. Human exposure to microplastics (MPs) and nanoplastics (NPs) is increasing; however, some potential adverse health effects might remain unclear, due to analytical challenges in detecting trace concentrations. To address this, we propose a workflow for NPs assessment in biological samples combining three complimentary methods, namely inductively coupled plasma mass spectrometry (ICP-MS), X-ray fluorescence imaging (XFI), and imaging mass cytometry (IMC) to detect palladium-doped NPs (Pd-NPs). This approach was used to quantify the temporal distribution and accumulation of Pd-NPs in mouse models under different experimental conditions, dosages, and time frames. Acute exposure showed a clear particle excretion from the gastrointestinal tract into feces, while subchronic exposure led to tissue accumulation. This workflow enhances our ability to analyze and study NP uptake and biodistribution mechanisms down to the nanoscale in complex biological samples. A workflow to detect palladium-doped nanoplastic particles in biological samples combining three complimentary methods, namely inductively coupled plasma mass spectrometry, X-ray fluorescence imaging, and imaging mass cytometry.