A mathematical model for the dependence of keratin aggregate formation on the quantity of mutant keratin expressed in EGFP-K14 R125P keratinocytes

We examined keratin aggregate formation and the possible mechanisms involved. With this aim, we observed the effect that different ratios between mutant and wild-type keratins expressed in cultured keratinocytes may have on aggregate formation in vitro, as well as how keratin aggregate formation affects the mechanical properties of cells at the cell cortex. To this end we prepared clones with expression rates as close as possible to 25%, 50% and 100% of the EGFP-K14 proteins (either WT or R125P and V270M mutants). Our results showed that only in the case of the 25% EGFP-K14 R125P mutant significant differences could be seen. Namely, we observed in this case the largest accumulation of keratin aggregates and a significant reduction in cell stiffness. To gain insight into the possible mechanisms behind this observation, we extended our previous mathematical model of keratin dynamics by implementing a more complex reaction network that considers the coexistence of wild-type and mutant keratins in the cell. The new model, consisting of a set of coupled, non-linear, ordinary differential equations, allowed us to draw conclusions regarding the relative amounts of intermediate filaments and aggregates in cells, and suggested that aggregate formation by asymmetric binding between wild-type and mutant keratins could explain the data obtained on cells grown in culture.

Increased Platelet-CD4+ T Cell Aggregates Are Correlated With HIV-1 Permissiveness and CD4+ T Cell Loss

Chronic HIV-1 infection is associated with persistent inflammation, which contributes to disease progression. Platelet-T cell aggregates play a critical role in maintaining inflammation. However, the phenotypic characteristics and clinical significance of platelet-CD4+ T cell aggregates remain unclear in different HIV-infected populations. In this study, we quantified and characterized platelet-CD4+ T cell aggregates in the peripheral blood of treatment-naïve HIV-1-infected individuals (TNs), immunological responders to antiretroviral therapy (IRs), immunological non-responders to antiretroviral therapy (INRs), and healthy controls (HCs). Flow cytometry analysis and immunofluorescence microscopy showed increased platelet-CD4+ T cell aggregate formation in TNs compared to HCs during HIV-1 infection. However, the frequencies of platelet-CD4+ T cell aggregates decreased in IRs compared to TNs, but not in INRs, which have shown severe immunological dysfunction. Platelet-CD4+ T cell aggregate frequencies were positively correlated with HIV-1 viral load but negatively correlated with CD4+ T cell counts and CD4/CD8 ratios. Furthermore, we observed a higher expression of CD45RO, HIV co-receptors, HIV activation/exhaustion markers in platelet-CD4+ T cell aggregates, which was associated with HIV-1 permissiveness. High levels of caspase-1 and caspase-3, and low levels of Bcl-2 in platelet-CD4+ T cell aggregates imply the potential role in CD4+ T cell loss during HIV-1 infection. Furthermore, platelet-CD4+ T cell aggregates contained more HIV-1 gag viral protein and HIV-1 DNA than their platelet-free CD4+ T cell counterparts. The platelet-CD4+ T cell aggregate levels were positively correlated with plasma sCD163 and sCD14 levels. Our findings demonstrate that platelet-CD4+ T cell aggregate formation has typical characteristics of HIV-1 permissiveness and is related to immune activation during HIV-1 infection.

Adverse Outcome in COVID-19 Is Associated With an Aggravating Hypo-Responsive Platelet Phenotype

Thromboembolic complications are frequently observed in Coronavirus disease 2019 (COVID-19). While COVID-19 is linked to platelet dysregulation, the association between disease outcome and platelet function is less clear. We prospectively monitored platelet activation and reactivity in 97 patients during the first week of hospitalization and determined plasma markers of platelet degranulation and inflammation. Adverse outcome in COVID-19 was associated with increased basal platelet activation and diminished platelet responses, which aggravated over time. Especially GPIIb/IIIa responses were abrogated, pointing toward impeded platelet aggregation. Moreover, platelet-leukocyte aggregate formation was diminished, pointing toward abrogated platelet-mediated immune responses in COVID-19. No general increase in plasma levels of platelet-derived granule components could be detected, arguing against platelet exhaustion. However, studies on platelets from healthy donors showed that plasma components in COVID-19 patients with unfavorable outcome were at least partly responsible for diminished platelet responses.Taken together this study shows that unfavorable outcome in COVID-19 is associated with a hypo-responsive platelet phenotype that aggravates with disease progression and may impact platelet-mediated immunoregulation.

Primary Human Trabecular Meshwork Model for Pseudoexfoliation

The lack of an animal model or an in vitro model limits experimental options for studying temporal molecular events in pseudoexfoliation syndrome (PXF), an age related fibrillopathy causing trabecular meshwork damage and glaucoma. Our goal was to create a workable in vitro model of PXF using primary human TM (HTM) cell lines simulating human disease. Primary HTM cells harvested from healthy donors (n = 3), were exposed to various concentrations (5 ng/mL, 10 ng/mL, 15 ng/mL) of transforming growth factor-beta1 (TGF-β1) for different time points. Morphological change of epithelial–mesenchymal transition (EMT) was analyzed by direct microscopic visualization and immunoblotting for EMT markers. Expression of pro-fibrotic markers were analyzed by quantitative RT-PCR and immunoblotting. Cell viability and death in treated cells was analyzed using FACS and MTT assay. Protein complex and amyloid aggregate formation was analyzed by Immunofluorescence of oligomer11 and amyloid beta fibrils. Effect of these changes with pharmacological inhibitors of canonical and non-canonical TGF pathway was done to analyze the pathway involved. The expression of pro-fibrotic markers was markedly upregulated at 10 ng/mL of TGF-β1 exposure at 48–72 h of exposure with associated EMT changes at the same time point. Protein aggregates were seen maximally at these time points that were found to be localized around the nucleus and in the extracellular matrix (ECM). EMT and pro-fibrotic expression was differentially regulated by different canonical and non-canonical pathways suggesting complex regulatory mechanisms. This in vitro model using HTM cells simulated the main characteristics of human disease in PXF like pro-fibrotic gene expression, EMT, and aggregate formation.

Huntingtin Ubiquitination Mechanisms and Novel Possible Therapies to Decrease the Toxic Effects of Mutated Huntingtin

Huntington Disease (HD) is a dominant, lethal neurodegenerative disorder caused by the abnormal expansion (>35 copies) of a CAG triplet located in exon 1 of the HTT gene encoding the huntingtin protein (Htt). Mutated Htt (mHtt) easily aggregates, thereby inducing ER stress that in turn leads to neuronal injury and apoptosis. Therefore, both the inhibition of mHtt aggregate formation and the acceleration of mHtt degradation represent attractive strategies to delay HD progression, and even for HD treatment. Here, we describe the mechanism underlying mHtt degradation by the ubiquitin–proteasome system (UPS), which has been shown to play a more important role than the autophagy–lysosomal pathway. In particular, we focus on E3 ligase proteins involved in the UPS and detail their structure–function relationships. In this framework, we discuss the possible exploitation of PROteolysis TArgeting Chimeras (PROTACs) for HD therapy. PROTACs are heterobifunctional small molecules that comprise two different ligands joined by an appropriate linker; one of the ligands is specific for a selected E3 ubiquitin ligase, the other ligand is able to recruit a target protein of interest, in this case mHtt. As a consequence of PROTAC binding, mHtt and the E3 ubiquitin ligase can be brought to a relative position that allows mHtt to be ubiquitinated and, ultimately, allows a reduction in the amount of mHtt in the cell.