ABSTRACT
induced-pluripotent stem cell (iPSC)-derived neurospheroid (NSPH) models are an emerging in vitro toolkit to study the influence of inflammatory triggers on neurodegeneration and repair in a 3D neural environment. In contrast to their human counterpart, the absence of murine iPSC-derived NSPHs for profound characterisation and validation studies is a major experimental research gap, even though they offer the only possibility to truly compare or validate in vitro NSPH responses with in vivo brain responses. To contribute to these developments, we here describe the generation and characterisation of 5-week-old CX3CR1eGFP+/- CCR2RFP+/- murine (m)iPSC-derived bi-partite (neurons + astrocytes) and tri-partite (neurons + astrocytes + microglia) NSPH models that can be subjected to cellular activation following pro-inflammatory stimulation. First, cytokine analysis demonstrates that both bi-partite and tri-partite NSPHs can be triggered to release IL6 and CXCL10 following three days of stimulation with, respectively, TNFα + IL1ß + IFNγ and LPS + IFNγ. Additionally, immunocytochemical analysis for G3BP1 and PABPC1 revealed the development of stress granules in both bi-partite and tri-partite NSPHs after 3 days of stimulation. To further investigate the observed signs of inflammatory response and cellular stress, we performed an untargeted transcriptomic and proteomic analysis of bi- and tri-partite NSPHs under steady-state and inflammatory conditions. Here, using the combined differential gene and protein expression profiles between unstimulated and stimulated NSPHs, Ingenuity Pathway Analysis (IPA) confirms the activation of canonical pathways associated with inflammation and cellular stress in both bi-partite and tri-partite NSPHs. Moreover, our multi-omics analysis suggests a higher level of downstream inflammatory responses, impairment of homeostatic and developmental processes, as well as activation of cell death processes in stimulated tri-partite NSPHs compared to bi-partite NSPHs. Concluding, these results emphasise the advantages of including microglia in NSPH research to study inflammation-induced neurodegeneration in a 3D neural environment.
ABSTRACT
Human papillomavirus (HPV) vaccines, primarily relying on neutralizing antibodies, have proven highly effective. Recently, HPV-specific antibodies have been detected in the female genital tract secretions captured by first-void urine (FVU), offering a minimally invasive diagnostic approach. In this study, we investigated whether HPV16-specific antibodies present in FVU samples retain their neutralizing capacity by using pseudovirion-based neutralization assays. Paired FVU and serum samples (vaccinated n = 25, unvaccinated n = 25, aged 18-25) were analyzed using two orthogonal pseudovirion-based neutralization assays, one using fluorescence microscopy and the other using luminescence-based spectrophotometry. Results were compared with HPV16-specific IgG concentrations and correlations between neutralizing antibodies in FVU and serum were explored. The study demonstrated the presence of neutralizing antibodies in FVU using both pseudovirion-based neutralization assays, with the luminescence-based assay showing higher sensitivity for FVU samples, while the fluorescence microscopy-based assay exhibited better specificity for serum and overall higher reproducibility. High Spearman correlation values were calculated between HPV16-IgG and HPV16-neutralizing antibodies for both protocols (rs: 0.54-0.94, p < .001). Significant Spearman correlations between FVU and serum concentrations were also established for all assays (rs: 0.44-0.91, p < .01). This study demonstrates the continued neutralizing ability of antibodies captured with FVU, supporting the hypothesis that HPV vaccination may reduce autoinoculation and transmission risk to the sexual partner. Although further protocol optimizations are warranted, these findings provide a foundation for future research and larger cohort studies that could have implications for the optimal design, evaluation, and implementation of HPV vaccination programs.
Subject(s)
Papillomavirus Infections , Papillomavirus Vaccines , Humans , Female , Adolescent , Young Adult , Adult , Papillomavirus Infections/prevention & control , Reproducibility of Results , Antibodies, Viral , Antibodies, Neutralizing , Neutralization Tests/methods , Genitalia, Female , Human papillomavirus 16 , Immunoglobulin GABSTRACT
BACKGROUND: Huntington's disease (HD) is marked by a CAG-repeat expansion in the huntingtin gene that causes neuronal dysfunction and loss, affecting mainly the striatum and the cortex. Alterations in the neurovascular coupling system have been shown to lead to dysregulated energy supply to brain regions in several neurological diseases, including HD, which could potentially trigger the process of neurodegeneration. In particular, it has been observed in cross-sectional human HD studies that vascular alterations are associated to impaired cerebral blood flow (CBF). To assess whether whole-brain changes in CBF are present and follow a pattern of progression, we investigated both resting-state brain perfusion and vascular reactivity longitudinally in the zQ175DN mouse model of HD. METHODS: Using pseudo-continuous arterial spin labelling (pCASL) MRI in the zQ175DN model of HD and age-matched wild-type (WT) mice, we assessed whole-brain, resting-state perfusion at 3, 6 and 9 and 13 months of age, and assessed hypercapnia-induced cerebrovascular reactivity (CVR), at 4.5, 6, 9 and 15 months of age. RESULTS: We found increased perfusion in cortical regions of zQ175DN HET mice at 3 months of age, and a reduction of this anomaly at 6 and 9 months, ages at which behavioural deficits have been reported. On the other hand, under hypercapnia, CBF was reduced in zQ175DN HET mice as compared to the WT: for multiple brain regions at 6 months of age, for only somatosensory and retrosplenial cortices at 9 months of age, and brain-wide by 15 months. CVR impairments in cortical regions, the thalamus and globus pallidus were observed in zQ175DN HET mice at 9 months, with whole brain reactivity diminished at 15 months of age. Interestingly, blood vessel density was increased in the motor cortex at 3 months, while average vessel length was reduced in the lateral portion of the caudate putamen at 6 months of age. CONCLUSION: Our findings reveal early cortical resting-state hyperperfusion and impaired CVR at ages that present motor anomalies in this HD model, suggesting that further characterization of brain perfusion alterations in animal models is warranted as a potential therapeutic target in HD.
Subject(s)
Huntington Disease , Humans , Mice , Animals , Infant , Huntington Disease/genetics , Cross-Sectional Studies , Hypercapnia , Brain , Disease Models, Animal , PerfusionABSTRACT
BACKGROUND & AIMS: Mounting evidence suggests the gastrointestinal microbiome is a determinant of peripheral immunity and central neurodegeneration, but the local disease mechanisms remain unknown. Given its potential relevance for early diagnosis and therapeutic intervention, we set out to map the pathogenic changes induced by bacterial amyloids in the gastrointestinal tract and its enteric nervous system. METHODS: To examine the early response, we challenged primary murine myenteric networks with curli, the prototypical bacterial amyloid, and performed shotgun RNA sequencing and multiplex enzyme-linked immunosorbent assay. Using enteric neurosphere-derived glial and neuronal cell cultures, as well as in vivo curli injections into the colon wall, we further scrutinized curli-induced pathogenic pathways. RESULTS: Curli induced a proinflammatory response, with strong up-regulation of Saa3 and the secretion of several cytokines. This proinflammatory state was induced primarily in enteric glia, was accompanied by increased levels of DNA damage and replication, and triggered the influx of immune cells in vivo. The addition of recombinant Serum Amyloid A3 (SAA3) was sufficient to recapitulate this specific proinflammatory phenotype while Saa3 knock-out attenuated curli-induced DNA damage and replication. Similar to curli, recombinant SAA3 caused a strong up-regulation of Saa3 transcripts, illustrating its self-amplifying potential . Since colonization of curli-producing Salmonella and dextran sulfate sodium-induced colitis triggered a significant increase in Saa3 transcripts as well, we assume SAA3plays a central role in enteric dysfunction. Inhibition of dual leucine zipper kinase, an upstream regulator of the c-Jun N-terminal kinase pathway responsible for SAA3 production, attenuated curli- and recombinant SAA3-induced Saa3 up-regulation, DNA damage, and replication in enteric glia. CONCLUSIONS: Our results position SAA3 as an important mediator of gastrointestinal vulnerability to bacterial-derived amyloids and demonstrate the potential of dual leucine zipper kinase inhibition to dampen enteric pathology.
Subject(s)
Enteric Nervous System , Serum Amyloid A Protein , Animals , Enteric Nervous System/metabolism , Enteric Nervous System/pathology , Enteric Nervous System/immunology , Serum Amyloid A Protein/metabolism , Serum Amyloid A Protein/genetics , Mice , Bacterial Proteins/metabolism , Inflammation/immunology , Inflammation/pathology , Inflammation/metabolism , Neuroglia/metabolism , Neuroglia/immunology , Neuroglia/pathology , Mice, Inbred C57BL , Cytokines/metabolism , Gastrointestinal Microbiome/immunology , Mice, Knockout , Colitis/immunology , Colitis/microbiology , Colitis/pathology , Neurons/metabolism , Neurons/pathologyABSTRACT
Neurological disorders are defined by synaptic dysfunction. We present a workflow to quantify morphological and functional aspects of synaptic connectivity in neuronal cultures and obtain an integrated readout. We describe steps for measuring synchronous calcium bursting in GCaMP6f-transduced neurons and labeling mature synapses using a proximity ligation assay. The integration of functional and morphological information from the same cultures provides a rich fingerprint of synaptic connectivity, deployable in different experimental conditions. For complete details on the use and execution of this protocol, please refer to Verstraelen et al. and Verschuuren et al.1,2.
Subject(s)
Neurons , Synapses , Neurons/cytology , Neurons/physiology , Animals , Synapses/physiology , Cells, Cultured , Calcium/metabolism , Mice , Cell Culture Techniques/methodsABSTRACT
Human papillomavirus (HPV) infection is a primary cause of cervical and head-and-neck cancers. The HPV genome enters the nucleus during mitosis when the nuclear envelope disassembles. Given that lamins maintain nuclear integrity during interphase, we asked to what extent their loss would affect early HPV infection. To address this question, we infected human cervical cancer cells and keratinocytes lacking the major lamins with a HPV16 pseudovirus (HP-PsV) encoding an EGFP reporter. We found that a sustained reduction or complete loss of lamin B1 significantly increased HP-PsV infection rate. A corresponding greater nuclear HP-PsV load in LMNB1 knockout cells was directly related to their prolonged mitotic window and extensive nuclear rupture propensity. Despite the increased HP-PsV presence, EGFP transcript levels remained virtually unchanged, indicating an additional defect in protein turnover. Further investigation revealed that LMNB1 knockout led to a substantial decrease in autophagic capacity, possibly linked to the persistent activation of cGAS by cytoplasmic chromatin exposure. Thus, the attrition of lamin B1 increases nuclear perviousness and attenuates autophagic capacity, creating an environment conducive to unrestrained accumulation of HPV capsids. Our identification of lower lamin B1 levels and nuclear BAF foci in the basal epithelial layer of several human cervix samples suggests that this pathway may contribute to an increased individual susceptibility to HPV infection.
Subject(s)
Lamin Type B , Papillomavirus Infections , Female , Humans , Lamin Type B/genetics , Lamin Type B/metabolism , Papillomavirus Infections/genetics , Nuclear Envelope/metabolism , Mitosis , Chromosomes/metabolism , Lamin Type A/genetics , Lamin Type A/metabolismABSTRACT
Adoptive T cell transfer has shown great success in treating blood cancers, resulting in a growing number of FDA-approved therapies using chimeric antigen receptor (CAR)-engineered T cells. However, the effectiveness of this treatment for solid tumors is still not satisfactory, emphasizing the need for improved T cell engineering strategies and combination approaches. Currently, CAR T cells are mainly manufactured using gammaretroviral and lentiviral vectors due to their high transduction efficiency. However, there are concerns about their safety, the high cost of producing them in compliance with current Good Manufacturing Practices (cGMP), regulatory obstacles, and limited cargo capacity, which limit the broader use of engineered T cell therapies. To overcome these limitations, researchers have explored non-viral approaches, such as membrane permeabilization and carrier-mediated methods, as more versatile and sustainable alternatives for next-generation T cell engineering. Non-viral delivery methods can be designed to transport a wide range of molecules, including RNA, which allows for more controlled and safe modulation of T cell phenotype and function. In this review, we provide an overview of non-viral RNA delivery in adoptive T cell therapy. We first define the different types of RNA therapeutics, highlighting recent advancements in manufacturing for their therapeutic use. We then discuss the challenges associated with achieving effective RNA delivery in T cells. Next, we provide an overview of current and emerging technologies for delivering RNA into T cells. Finally, we discuss ongoing preclinical and clinical studies involving RNA-modified T cells.
Subject(s)
Neoplasms , T-Lymphocytes , Humans , RNA/metabolism , Immunotherapy, Adoptive/methods , Neoplasms/pathology , Cell Engineering/methodsABSTRACT
Long considered to fluctuate between pro- and anti-inflammatory states, it has now become evident that microglia occupy a variegated phenotypic landscape with relevance to aging and neurodegeneration. However, whether specific microglial subsets converge in or contribute to both processes that eventually affect brain function is less clear. To investigate this, we analyzed microglial heterogeneity in a tauopathy mouse model (K18-seeded P301L) and an accelerated aging model (Senescence-Accelerated Mouse-Prone 8, SAMP8) using cellular indexing of transcriptomes and epitopes by sequencing. We found that widespread tau pathology in K18-seeded P301L mice caused a significant change in the number and morphology of microglia, but only a mild overrepresentation of disease-associated microglia. At the cell population-level, we observed a marked upregulation of the calprotectin-encoding genes S100a8 and S100a9. In 9-month-old SAMP8 mice, we identified a unique microglial subpopulation that showed partial similarity with the disease-associated microglia phenotype and was additionally characterized by a high expression of the same calprotectin gene set. Immunostaining for S100A8 revealed that this population was enriched in the hippocampus, correlating with the cognitive impairment observed in this model. However, incomplete colocalization between their residence and markers of neuronal loss suggests regional specificity. Importantly, S100A8-positive microglia were also retrieved in brain biopsies of human AD and tauopathy patients as well as in a biopsy of an aged individual without reported pathology. Thus, the emergence of S100A8-positive microglia portrays a conspicuous commonality between accelerated aging and tauopathy progression, which may have relevance for ensuing brain dysfunction.
Subject(s)
Aging , Brain , Calgranulin A , Microglia , Animals , Microglia/metabolism , Mice , Brain/metabolism , Brain/pathology , Calgranulin A/metabolism , Calgranulin A/genetics , Aging/metabolism , tau Proteins/metabolism , tau Proteins/genetics , Humans , Disease Models, Animal , Tauopathies/metabolism , Tauopathies/pathology , Male , Mice, TransgenicABSTRACT
Neuromedin U (NMU) is an evolutionary conserved neuropeptide that has been implicated in multiple processes, such as circadian regulation, energy homeostasis, reward processing and stress coping. Although the central expression of NMU has been addressed previously, the lack of specific and sensitive tools has prevented a comprehensive characterization of NMU-expressing neurons in the brain. We have generated a knock-in mouse model constitutively expressing Cre recombinase under the Nmu promoter. We have validated the model using a multi-level approach based on quantitative reverse-transcription polymerase chain reactions, in situ hybridization, a reporter mouse line and an adenoviral vector driving Cre-dependent expression of a fluorescent protein. Using the Nmu-Cre mouse, we performed a complete characterization of NMU expression in adult mouse brain, unveiling a potential midline NMU modulatory circuit with the ventromedial hypothalamic nucleus (VMH) as a key node. Moreover, immunohistochemical analysis suggested that NMU neurons in the VMH mainly constitute a unique population of hypothalamic cells. Taken together, our results suggest that Cre expression in the Nmu-Cre mouse model largely reflects NMU expression in the adult mouse brain, without altering endogenous NMU expression. Thus, the Nmu-Cre mouse model is a powerful and sensitive tool to explore the role of NMU neurons in mice.
Subject(s)
Neuropeptides , Peptide Hormones , Animals , Mice , Neurons , Integrases/genetics , Neuropeptides/genetics , Disease Models, AnimalABSTRACT
Auranofin (AF) is a potent, off-patent thioredoxin reductase (TrxR) inhibitor that efficiently targets cancer via reactive oxygen species (ROS)- and DNA damage-mediated cell death. The goal of this study is to enhance the efficacy of AF as a cancer treatment by combining it with the poly(ADP-ribose) polymerase-1 (PARP) inhibitor olaparib (referred to as 'aurola'). Firstly, we investigated whether mutant p53 can sensitize non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) cancer cells to AF and olaparib treatment in p53 knock-in and knock-out models with varying p53 protein expression levels. Secondly, we determined the therapeutic range for synergistic cytotoxicity between AF and olaparib and elucidated the underlying molecular cell death mechanisms. Lastly, we evaluated the effectiveness of the combination strategy in a murine 344SQ 3D spheroid and syngeneic in vivo lung cancer model. We demonstrated that high concentrations of AF and olaparib synergistically induced cytotoxicity in NSCLC and PDAC cell lines with low levels of mutant p53 protein that were initially more resistant to AF. The aurola combination also led to the highest accumulation of ROS, which resulted in ROS-dependent cytotoxicity of mutant p53 NSCLC cells through distinct types of cell death, including caspase-3/7-dependent apoptosis, inhibited by Z-VAD-FMK, and lipid peroxidation-dependent ferroptosis, inhibited by ferrostatin-1 and alpha-tocopherol. High concentrations of both compounds were also needed to obtain a synergistic cytotoxic effect in 3D spheroids of the murine lung adenocarcinoma cell line 344SQ, which was interestingly absent in 2D. This cell line was used in a syngeneic mouse model in which the oral administration of aurola significantly delayed the growth of mutant p53 344SQ tumors in 129S2/SvPasCrl mice, while either agent alone had no effect. In addition, RNA sequencing results revealed that AF- and aurola-treated 344SQ tumors were negatively enriched for immune-related gene sets, which is in accordance with AF's anti-inflammatory function as an anti-rheumatic drug. Only 344SQ tumors treated with aurola showed the downregulation of genes related to the cell cycle, potentially explaining the growth inhibitory effect of aurola since no apoptosis-related gene sets were enriched. Overall, this novel combination strategy of oxidative stress induction (AF) with PARP inhibition (olaparib) could be a promising treatment for mutant p53 cancers, although high concentrations of both compounds need to be reached to obtain a substantial cytotoxic effect.
ABSTRACT
Photoporation is an up-and-coming technology for the gentle and efficient transfection of cells. Inherent to the application of photoporation is the optimization of several process parameters, such as laser fluence and sensitizing particle concentration, which is typically done one factor at a time (OFAT). However, this approach is tedious and runs the risk of missing a global optimum. Therefore, in this study, we explored whether response surface methodology (RSM) would allow for more efficient optimization of the photoporation procedure. As a case study, FITC-dextran molecules of 500 kDa were delivered to RAW264.7 mouse macrophage-like cells, making use of polydopamine nanoparticles (PDNPs) as photoporation sensitizers. Parameters that were varied to obtain an optimal delivery yield were PDNP size, PDNP concentration and laser fluence. Two established RSM designs were compared: the central composite design and the Box-Behnken design. Model fitting was followed by statistical assessment, validation, and response surface analysis. Both designs successfully identified a delivery yield optimum five- to eight-fold more efficiently than when using OFAT methodology while revealing a strong dependence on PDNP size within the design space. In conclusion, RSM proves to be a valuable approach to efficiently optimize photoporation conditions for a particular cell type.
Subject(s)
Nanoparticles , Animals , Mice , Transfection , LightABSTRACT
Immunohistochemical stains (IHC) reveal differences between liver lobule zones in health and disease, including nonalcoholic fatty liver disease (NAFLD). However, such differences are difficult to accurately quantify. In NAFLD, the presence of lipid vacuoles from macrovesicular steatosis further hampers interpretation by pathologists. To resolve this, we applied a zonal image analysis method to measure the distribution of hypoxia markers in the liver lobule of steatotic livers.The hypoxia marker pimonidazole was assessed with IHC in the livers of male C57BL/6 J mice on standard diet or choline-deficient L-amino acid-defined high-fat diet mimicking NAFLD. Another hypoxia marker, carbonic anhydrase IX, was evaluated by IHC in human liver tissue. Liver lobules were reconstructed in whole slide images, and staining positivity was quantified in different zones in hundreds of liver lobules. This method was able to quantify the physiological oxygen gradient along hepatic sinusoids in normal livers and panlobular spread of the hypoxia in NAFLD and to overcome the pronounced impact of macrovesicular steatosis on IHC. In a proof-of-concept study with an assessment of the parenchyma between centrilobular veins in human liver biopsies, carbonic anhydrase IX could be quantified correctly as well.The method of zonated quantification of IHC objectively quantifies the difference in zonal distribution of hypoxia markers (used as an example) between normal and NAFLD livers both in whole liver as well as in liver biopsy specimens. It constitutes a tool for liver pathologists to support visual interpretation and estimate the impact of steatosis on IHC results.
Subject(s)
Non-alcoholic Fatty Liver Disease , Animals , Mice , Male , Humans , Carbonic Anhydrase IX , Immunohistochemistry , Mice, Inbred C57BL , Liver/pathology , Hypoxia/pathologyABSTRACT
Ex vivo modification of T cells with exogenous cargo is a common prerequisite for the development of T cell therapies, such as chimeric antigen receptor therapy. Despite the clinical success and FDA approval of several such products, T cell manufacturing presents unique challenges related to therapeutic efficacy after adoptive cell transfer and several drawbacks of viral transduction-based manufacturing, such as high cost and safety concerns. To generate cellular products with optimal potency, engraftment potential and persistence in vivo, recent studies have shown that minimally differentiated T cell phenotypes are preferred. However, genetic engineering of quiescent T cells remains challenging. Photoporation is an upcoming alternative non-viral transfection method which makes use of photothermal nanoparticles, such as polydopamine nanoparticles (PDNPs), to induce transient membrane permeabilization by distinct photothermal effects upon laser irradiation, allowing exogenous molecules to enter cells. In this study, we analyzed the capability of PDNP-photoporation to deliver large model macromolecules (FITC-dextran 500 kDa, FD500) in unstimulated and expanded human T cells. We compared different sizes of PDNPs (150, 250 and 400 nm), concentrations of PDNPs and laser fluences and found an optimal condition that generated high delivery yields of FD500 in both T cell phenotypes. A multiparametric analysis of cell proliferation, surface activation markers and cytokine production, revealed that unstimulated T cells photoporated with 150 nm and 250 nm PDNPs retained their propensity to become activated, whereas those photoporated with 400 nm PDNPs did less. Our findings show that PDNP-photoporation is a promising strategy for transfection of quiescent T cells, but that PDNPs should be small enough to avoid excessive cell damage.
Subject(s)
Nanoparticles , T-Lymphocytes , Humans , Organophosphorus Compounds , Macromolecular SubstancesABSTRACT
BACKGROUND: Imbalanced synaptic transmission appears to be an early driver in Alzheimer's disease (AD) leading to brain network alterations. Early detection of altered synaptic transmission and insight into mechanisms causing early synaptic alterations would be valuable treatment strategies. This study aimed to investigate how whole-brain networks are influenced at pre- and early-plague stages of AD and if these manifestations are associated with concomitant cellular and synaptic deficits. METHODS: To this end, we used an established AD rat model (TgF344-AD) and employed resting state functional MRI and quasi-periodic pattern (QPP) analysis, a method to detect recurrent spatiotemporal motifs of brain activity, in parallel with state-of-the-art immunohistochemistry in selected brain regions. RESULTS: At the pre-plaque stage, QPPs in TgF344-AD rats showed decreased activity of the basal forebrain (BFB) and the default mode-like network. Histological analyses revealed increased astrocyte abundance restricted to the BFB, in the absence of amyloid plaques, tauopathy, and alterations in a number of cholinergic, gaba-ergic, and glutamatergic synapses. During the early-plaque stage, when mild amyloid-beta (Aß) accumulation was observed in the cortex and hippocampus, QPPs in the TgF344-AD rats normalized suggesting the activation of compensatory mechanisms during this early disease progression period. Interestingly, astrogliosis observed in the BFB at the pre-plaque stage was absent at the early-plaque stage. Moreover, altered excitatory/inhibitory balance was observed in cortical regions belonging to the default mode-like network. In wild-type rats, at both time points, peak activity in the BFB preceded peak activity in other brain regions-indicating its modulatory role during QPPs. However, this pattern was eliminated in TgF344-AD suggesting that alterations in BFB-directed neuromodulation have a pronounced impact in network function in AD. CONCLUSIONS: This study demonstrates the value of rsfMRI and advanced network analysis methods to detect early alterations in BFB function in AD, which could aid early diagnosis and intervention in AD. Restoring the global synaptic transmission, possibly by modulating astrogliosis in the BFB, might be a promising therapeutic strategy to restore brain network function and delay the onset of symptoms in AD.
Subject(s)
Alzheimer Disease , Basal Forebrain , Alzheimer Disease/pathology , Amyloid beta-Peptides , Animals , Basal Forebrain/diagnostic imaging , Cholinergic Agents , Disease Models, Animal , Gliosis , Plaque, Amyloid , Rats , Rats, Inbred F344 , Rats, Transgenic , gamma-Aminobutyric AcidABSTRACT
Human papillomaviruses (HPV) are small, non-enveloped DNA viruses, which upon chronic infection can provoke cervical and head-and-neck cancers. Although the infectious life cycle of HPV has been studied and a vaccine is available for the most prevalent cancer-causing HPV types, there are no antiviral agents to treat infected patients. Hence, there is a need for novel therapeutic entry points and a means to identify them. In this work, we have used high-content microscopy to quantitatively investigate the early phase of HPV infection. Human cervical cancer cells and immortalized keratinocytes were exposed to pseudoviruses (PsV) of the widespread HPV type 16, in which the viral genome was replaced by a pseudogenome encoding a fluorescent reporter protein. Using the fluorescent signal as readout, we measured differences in infection between cell lines, which directly correlated with host cell proliferation rate. Parallel multiparametric analysis of nuclear organization revealed that HPV PsV infection alters nuclear organization and inflates promyelocytic leukemia protein body content, positioning these events at the early stage of HPV infection, upstream of viral replication. Time-resolved analysis revealed a marked heterogeneity in infection kinetics even between two daughter cells, which we attribute to differences in viral load. Consistent with the requirement for mitotic nuclear envelope breakdown, pharmacological inhibition of the cell cycle dramatically blunted infection efficiency. Thus, by systematic image-based single cell analysis, we revealed phenotypic alterations that accompany HPV PsV infection in individual cells, and which may be relevant for therapeutic drug screens.
Subject(s)
Papillomavirus Infections , Humans , Papillomavirus Infections/genetics , Human papillomavirus 16/genetics , Human papillomavirus 16/metabolism , Keratinocytes , Cell Nucleus , Cell LineABSTRACT
To find drivers of healthy ageing, a genome-wide association study (GWAS) was performed in healthy and unhealthy older individuals. Healthy individuals were defined as free from cardiovascular disease, stroke, heart failure, major adverse cardiovascular event, diabetes, dementia, cancer, chronic obstructive pulmonary disease (COPD), asthma, rheumatism, Crohn's disease, malabsorption or kidney disease. Six single nucleotide polymorphisms (SNPs) with unknown function associated with ten human genes were identified as candidate healthspan markers. Thirteen homologous or closely related genes were selected in the model organism C. elegans for evaluating healthspan after targeted RNAi-mediated knockdown using pathogen resistance, muscle integrity, chemotaxis index and the activity of known longevity and stress response pathways as healthspan reporters. In addition, lifespan was monitored in the RNAi-treated nematodes. RNAi knockdown of yap-1, wwp-1, paxt-1 and several acdh genes resulted in heterogeneous phenotypes regarding muscle integrity, pathogen resistance, chemotactic behaviour, and lifespan. Based on these observations, we hypothesize that their human homologues WWC2, CDKN2AIP and ACADS may play a role in health maintenance in the elderly.
Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Aged , Animals , Caenorhabditis elegans/physiology , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Carrier Proteins , Genome-Wide Association Study , Humans , Longevity/genetics , Phenotype , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , YAP-Signaling ProteinsABSTRACT
Radiotherapy is the standard of care for breast cancer. However, surviving radioresistant cells can repopulate following treatment and provoke relapse. Better understanding of the molecular mechanisms of radiation resistance may help to improve treatment of radioresistant tumours. To emulate radiation therapy at the cellular level, we exposed MCF7 breast cancer cells to daily radiation doses of 2 Gy up to an accumulated dose of 20 Gy. Fractionally irradiated cells (FIR20) displayed increased clonogenic survival and population doubling time as compared with age-matched sham-irradiated cells and untreated parental MCF7 cells. RNA-sequencing revealed a core signature of 229 mRNAs and 7 circular RNAs of which the expression was significantly altered in FIR20 cells. Dysregulation of several top genes was mirrored at the protein level. The FIR20 cell transcriptome overlapped significantly with canonical radiation response signatures and demonstrated a remarkable commonality with radiation and endocrine therapy resistance expression profiles, suggesting crosstalk between both acquired resistance pathways, as indicated by reduced sensitivity to tamoxifen cytotoxicity of FIR20 cells. Using predictive analyses and functional enrichment, we identified a gene-regulatory network that promotes stemness and inflammatory signalling in FIR20 cells. We propose that these phenotypic traits render breast cancer cells more radioresistant but may at the same time serve as potential targets for combination therapies.
Subject(s)
Breast Neoplasms , Radiation Tolerance , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/radiotherapy , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic , Humans , MCF-7 Cells , Phenotype , RNA, Circular , Radiation Tolerance/genetics , Tamoxifen/pharmacologyABSTRACT
Despite the considerable impact of stroke on both the individual and on society, a neuroprotective therapy for stroke patients is missing. This is partially due to the current lack of a physiologically relevant human in vitro stroke model. To address this problem, we have developed a luminescent human iPSC-derived neurospheroid model that enables real-time read-out of neural viability after ischemia-like conditions. We subjected 1- and 4-week-old neurospheroids, generated from iPSC-derived neural stem cells, to 6 h of oxygen-glucose deprivation (OGD) and measured neurospheroid luminescence. For both, we detected a decrease in luminescent signal due to ensuing neurotoxicity, as confirmed by conventional LDH assay and flow cytometric viability analysis. Remarkably, 1-week-old, but not 4-week-old neurospheroids recovered from OGD-induced injury, as evidenced by their reduced but overall increasing luminescence over time. This underscores the need for more mature neurospheroids, more faithfully recapitulating the in vivo situation. Furthermore, treatment of oxygen- and glucose-deprived neurospheroids with the pan-caspase inhibitor Z-VAD-FMK did not increase overall neural survival, despite its successful attenuation of apoptosis, in a human-based 3D environment. Nevertheless, owing to its three-dimensional organization and real-time viability reporting potential, the luminescent neurospheroids may become readily adopted in high-throughput screens aimed at identification of new therapeutic agents to treat acute ischemic stroke patients.
Subject(s)
Induced Pluripotent Stem Cells , Ischemic Stroke , Stroke , Apoptosis , Cell Survival/physiology , Glucose , Humans , Luminescence , Oxygen/adverse effectsABSTRACT
Delivery of nanomaterials into cells is of interest for fundamental cell biological research as well as for therapeutic and diagnostic purposes. One way of doing so is by physically disrupting the plasma membrane (PM). Several methods that exploit electrical, mechanical or optical cues have been conceived to temporarily disrupt the PM for intracellular delivery, with variable effects on cell viability. However, apart from acute cytotoxicity, subtler effects on cell physiology may occur as well. Their nature and timing vary with the severity of the insult and the efficiency of repair, but some may provoke permanent phenotypic alterations. With the growing palette of nanoscale delivery methods and applications, comes a need for an in-depth understanding of this cellular response. In this review, we summarize current knowledge about the chronology of cellular events that take place upon PM injury inflicted by different delivery methods. We also elaborate on their significance for cell homeostasis and cell fate. Based on the crucial nodes that govern cell fitness and functionality, we give directions for fine-tuning nano-delivery conditions.
ABSTRACT
Neuroblastoma is a tumour that arises from the sympathoadrenal lineage occurring predominantly in children younger than five years. About half of the patients are diagnosed with high-risk tumours and undergo intensive multi-modal therapy. The success rate of current treatments for high-risk neuroblastoma is disappointingly low and survivors suffer from multiple therapy-related long-term side effects. Most chemotherapeutics drive cancer cells towards cell death or senescence. Senescence has long been considered to represent a terminal non-proliferative state and therefore an effective barrier against tumorigenesis. This dogma, however, has been challenged by recent observations that infer a much more dynamic and reversible nature for this process, which may have implications for the efficacy of therapy-induced senescence-oriented treatment strategies. Neuroblastoma cells in a dormant, senescent-like state may escape therapy, whilst their senescence-associated secretome may promote inflammation and invasiveness, potentially fostering relapse. Conversely, due to its distinct molecular identity, senescence may also represent an opportunity for the development of novel (combination) therapies. However, the limited knowledge on the molecular dynamics and diversity of senescence signatures demands appropriate models to study this process in detail. This review summarises the molecular knowledge about cellular senescence in neuroblastoma and investigates current and future options towards therapeutic exploration.