技术资料

There is increasing evidence that coronavirus disease 2019 (COVID-19) produces more severe symptoms and higher mortality among men than among women1-5. However,whether immune responses against severe acute respiratory syndrome coronavirus (SARS-CoV-2) differ between sexes,and whether such differences correlate with the sex difference in the disease course of COVID-19,is currently unknown. Here we examined sex differences in viral loads,SARS-CoV-2-specific antibody titres,plasma cytokines and blood-cell phenotyping in patients with moderate COVID-19 who had not received immunomodulatory medications. Male patients had higher plasma levels of innate immune cytokines such as IL-8 and IL-18 along with more robust induction of non-classical monocytes. By contrast,female patients had more robust T cell activation than male patients during SARS-CoV-2 infection. Notably,we found that a poor T cell response negatively correlated with patients' age and was associated with worse disease outcome in male patients,but not in female patients. By contrast,higher levels of innate immune cytokines were associated with worse disease progression in female patients,but not in male patients. These findings provide a possible explanation for the observed sex biases in COVID-19,and provide an important basis for the development of a sex-based approach to the treatment and care of male and female patients with COVID-19. View Publication

The pulmonary system is comprised of two main compartments,airways and alveolar space. Their tissue and cellular complexity ensure lung function and protection from external agents,for example,virus. Two-dimensional (2D) in vitro systems and animal models have been largely employed to elucidate the molecular mechanisms underlying human lung development,physiology,and pathogenesis. However,neither of these models accurately recapitulate the human lung environment and cellular crosstalk. More recently,human-derived three-dimensional (3D) models have been generated allowing for a deeper understanding of cell-to-cell communication. However,the availability and accessibility of primary human cell sources from which generate the 2D and 3D models may be limited. In the past few years,protocols have been developed to successfully employ human pluripotent stem cells (hPSCs) and differentiate them toward pulmonary fate in vitro. In the present review,we discuss the advantages and pitfalls of hPSC-derived lung 2D and 3D models,including the main characteristics and potentials for these models and their current and future applications for modeling development and diseases. Lung organoids currently represent the closest model to the human pulmonary system. We further focus on the applications of lung organoids for the study of human diseases such as pulmonary fibrosis,infectious diseases,and lung cancer. Finally,we discuss the present limitations and potential future applications of 3D lung organoids. This article is categorized under: Adult Stem Cells,Tissue Renewal,and Regeneration {\textgreater} Stem Cells and Disease Adult Stem Cells,Tissue Renewal,and Regeneration {\textgreater} Stem Cell Differentiation and Reversion. View Publication

The mechanism of inhibition of the Ca(2+)-ATPase from sarcoplasmic reticulum by the sesquiterpene lactones thapsigargin,trilobolide and thapsivillosin A (TvA) has been determined. A decrease in the affinity of the ATPase for Ca2+ is observed in the presence of the inhibitors (I),consistent with a shift in the E1/E2 equilibrium for the ATPase towards E2 forms. Amounts of inhibitor beyond a 1:1 molar ratio with ATPase produce no further decrease in affinity for Ca2+,inconsistent with the formation of a dead-end complex. Measurements of the rate of quenching of the tryptophan fluorescence of the ATPase by TvA are consistent with an association step to give E2I followed by an isomerization to a modified state E2AI. The kinetics of the reversal of the effects of TvA by Ca2+ at sub-stoichiometric amounts of TvA are bi-exponential,with a fast component whose rate is independent of TvA concentration and equal to the rate observed in the absence of TvA,and a slow component whose rate decreases with increasing TvA concentration. These observations are also consistent with the formation of a modified state E2AI following the initial binding of I to E2. The equilibrium constant E2AI/E2I increases in the order TvA {\textless} trilobolide {\textless} thapsigargin. The results suggest that the effects of the inhibitors on the overall ratio of E2 to E1 forms of the ATPase follow largely from the formation of E2AI from E2I,and that binding constants are very similar for E1Ca2,E1 and E2. View Publication

Expression of BAX,without another death stimulus,proved sufficient to induce a common pathway of apoptosis. This included the activation of interleukin 1 beta-converting enzyme (ICE)-like proteases with cleavage of the endogenous substrates poly(ADP ribose) polymerase and D4-GDI (GDP dissociation inhibitor for the rho family),as well as the fluorogenic peptide acetyl-Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC). The inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) successfully blocked this protease activity and prevented FAS-induced death but not BAX-induced death. Blocking ICE-like protease activity prevented the cleavage of nuclear and cytosolic substrates and the DNA degradation that followed BAX induction. However,the fall in mitochondrial membrane potential,production of reactive oxygen species,cytoplasmic vacuolation,and plasma membrane permeability that are downstream of BAX still occurred. Thus,BAX-induced alterations in mitochondrial function and subsequent cell death do not apparently require the known ICE-like proteases. View Publication

A novel fluorescent derivative of glucose was synthesized by reacting D-glucosamine and NBD-Cl. The TLC analysis of the reaction mixture showed the generation of a single spot with intense fluorescence (lambda Ex = 475 nm,lambda Em = 550 nm). The obtained novel fluorescent product,which was identified as 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) by 1H-NMR and FAB-MS spectrometries,was applied to the assessment of the glucose uptake activity of Escherichia coli B. 2-NBDG accumulated in living cells and not in dead cells. The uptake of 2-NBDG was competitively inhibited by D-glucose and not by L-glucose,which suggested the involvement of the glucose transporting system in the uptake of 2-NBDG. 2-NBDG taken into the cytoplasma of E. coli cells was supposedly converted into another derivative in the glucose metabolic pathway. View Publication

Respiratory syncytial virus (RSV) is increasingly recognized for causing severe morbidity and mortality in older adults,but there are few studies on the RSV-induced immune response in this population. Information on the immunological processes at play during RSV infection in specific risk groups is essential for the rational and targeted design of novel vaccines and therapeutics. Here,we assessed the antibody and local cytokine response to RSV infection in community-dwelling older adults (≥60 years of age). During three winters,serum and nasopharyngeal swab samples were collected from study participants during acute respiratory infection and recovery. RSV IgG enzyme-linked immunosorbent assays (ELISA) and virus neutralization assays were performed on serum samples from RSV-infected individuals (n = 41) and controls (n = 563 and n = 197,respectively). Nasal RSV IgA and cytokine concentrations were determined using multiplex immunoassays in a subset of participants. An in vitro model of differentiated primary bronchial epithelial cells was used to assess RSV-induced cytokine responses over time. A statistically significant increase in serum neutralization titers and IgG concentrations was observed in RSV-infected participants compared to controls. During acute RSV infection,a statistically significant local upregulation of beta interferon (IFN-$\beta$),IFN-$\lambda$1,IFN-$\gamma$,interleukin 1$\beta$ (IL-1$\beta$),tumor necrosis factor alpha (TNF-$\alpha$),IL-6,IL-10,CXCL8,and CXCL10 was found. IFN-$\beta$,IFN-$\lambda$1,CXCL8,and CXCL10 were also upregulated in the epithelial model upon RSV infection. In conclusion,this study provides novel insights into the basic immune response to RSV infection in an important and understudied risk population,providing leads for future studies that are essential for the prevention and treatment of severe RSV disease in older adults.IMPORTANCE Respiratory syncytial virus (RSV) can cause severe morbidity and mortality in certain risk groups,especially infants and older adults. Currently no (prophylactic) treatment is available,except for a partially effective yet highly expensive monoclonal antibody. RSV therefore remains a major public health concern. To allow targeted development of novel vaccines and therapeutics,it is of great importance to understand the immunological mechanisms that underlie (protection from) severe disease in specific risk populations. Since most RSV-related studies focus on infants,there are only very limited data available concerning the response to RSV in the elderly population. Therefore,in this study,RSV-induced antibody responses and local cytokine secretion were assessed in community-dwelling older adults. These data provide novel insights that will benefit ongoing efforts to design safe and effective prevention and treatment strategies for RSV in an understudied risk group. View Publication

Dendritic cells (DC) play an essential role in innate immunity and radiation-elicited immune responses. LGP2 is a RIG-I like receptor (RLR) involved in cytoplasmic RNA recognition and anti-viral responses. Although LGP2 has also been linked to cell survival of both tumor cells and T cells,the role of LGP2 in mediating DC function and anti-tumor immunity elicited by radiotherapy remains unclear. Here we report that tumor DC are linked to the clinical outcome of breast cancer patients who received radiotherapy (RT) and the presence of DC correlates with gene expression of LGP2 in the tumor microenvironment. In preclinical models,host LGP2 was essential for optimal anti-tumor control by ionizing radiation (IR). The absence of LGP2 in DC dampened type I interferon production and the priming capacity of DC. In the absence of LGP2,MDA5-mediated activation of type I IFN signaling was abrogated. The MDA5/LGP2 agonist high molecular weight poly I: C improved the anti-tumor effect of IR. This study reveals a previously undefined role of LGP2 in host immunity and provides a new strategy to improve the efficacy of radiotherapy. View Publication

SARS-CoV-2,a $\beta$-coronavirus,has rapidly spread across the world,highlighting its high transmissibility,but the underlying morphogenesis and pathogenesis remain poorly understood. Here,we characterize the replication dynamics,cell tropism and morphogenesis of SARS-CoV-2 in organotypic human airway epithelial (HAE) cultures. SARS-CoV-2 replicates efficiently and infects both ciliated and secretory cells in HAE cultures. In comparison,HCoV-NL63 replicates to lower titers and is only detected in ciliated cells. SARS-CoV-2 shows a similar morphogenetic process as other coronaviruses but causes plaque-like cytopathic effects in HAE cultures. Cell fusion,apoptosis,destruction of epithelium integrity,cilium shrinking and beaded changes are observed in the plaque regions. Taken together,our results provide important insights into SARS-CoV-2 cell tropism,replication and morphogenesis. View Publication

MEF2C encodes a transcription factor that is critical in nervous system development. Here,to examine disease-associated functions of MEF2C in human microglia,we profiled microglia differentiated from isogenic MEF2C-haploinsufficient and MEF2C-knockout induced pluripotent stem cell lines. Complementary transcriptomic and functional analyses revealed that loss of MEF2C led to a hyperinflammatory phenotype with broad phagocytic impairment,lipid accumulation,lysosomal dysfunction and elevated basal inflammatory cytokine secretion. Genome-wide profiling of MEF2C-bound sites coupled with the active regulatory landscape enabled inference of its transcriptional functions and potential mechanisms for MEF2C-associated cellular functions. Transcriptomic and epigenetic approaches identified substantial overlap with idiopathic autism datasets,suggesting a broader role of human microglial MEF2C dysregulation in idiopathic autism. In a mouse xenotransplantation model,loss of MEF2C led to morphological,lysosomal and lipid abnormalities in human microglia in vivo. Together,these studies reveal mechanisms by which reduced microglial MEF2C could contribute to the development of neurological diseases. Coufal and colleagues generated microglia from human iPS cells to examine mechanistic roles of the transcription factor MEF2C and how these roles might relate to the autism phenotype seen following the loss of MEF2C in human microglia. View Publication

Respiratory organoids have emerged as a powerful in vitro model for studying respiratory diseases and drug discovery. However,the high-throughput analysis of organoid images remains a challenge due to the lack of automated and accurate segmentation tools. This study presents a semi-automatic algorithm for image analysis of respiratory organoids (nasal and lung organoids),employing the U-Net architecture and CellProfiler for organoids segmentation. The algorithm processes bright-field images acquired through z-stack fusion and stitching. The model demonstrated a high level of accuracy,as evidenced by an intersection-over-union metric (IoU) of 0.8856,F1-score = 0.937 and an accuracy of 0.9953. Applied to forskolin-induced swelling assays of lung organoids,the algorithm successfully quantified functional differences in Cystic Fibrosis Transmembrane conductance Regulator (CFTR)-channel activity between healthy donor and cystic fibrosis patient-derived organoids,without fluorescent dyes. Additionally,an open-source dataset of 827 annotated respiratory organoid images was provided to facilitate further research. Our results demonstrate the potential of deep learning to enhance the efficiency and accuracy of high-throughput respiratory organoid analysis for future therapeutic screening applications. Author summaryIn this study,we developed a semi-automated tool to analyze images of respiratory organoids—3D cell structures that mimic the human respiratory system. These organoids are vital for studying diseases like cystic fibrosis and testing potential drugs,but manually analyzing their images is time-consuming and prone to errors. Our tool uses artificial intelligence (AI) to quickly and accurately measure organoid size and shape from bright-field microscope images,eliminating the need for fluorescent dyes that can harm cells. We trained our AI model on a publicly shared dataset of 827 annotated organoid images,achieving high accuracy in detecting and quantifying organoids. When applied to cystic fibrosis research,the tool successfully measured differences in organoid swelling (forskolin-induced swelling - a key test for drug response) between healthy and patient-derived samples. By making our dataset and method openly available,we hope to support further research into respiratory diseases. Our work bridges the gap between complex lab techniques and practical applications,offering a faster,more reliable way to study human health and disease. View Publication

A growing body of evidence implicates inflammation as a key hallmark in the pathophysiology of Parkinson’s disease (PD),with microglia playing a central role in mediating neuroinflammatory signaling in the brain. However,the molecular mechanisms linking microglial activation to dopaminergic neuron degeneration remain poorly understood. In this study,we investigated the contribution of the PD-associated LRRK2-G2019S mutation to microglial neurotoxicity using patient-derived induced pluripotent stem cell (iPSC) models. We found that LRRK2-G2019S mutant microglia exhibited elevated activation markers,enhanced phagocytic capacity,and increased secretion of pro-inflammatory cytokines such as TNF-α. These changes were associated with metabolic dysregulation,including upregulated glycolysis and impaired serine biosynthesis. In 3D midbrain organoids,these overactivated microglia resulted in dopaminergic neuron degeneration. Notably,treating LRRK2-G2019S microglia with oxamic acid,a glycolysis inhibitor,attenuated microglial inflammation and reduced neuronal loss. Our findings underscore the link between metabolic targeting in microglia and dopaminergic neuronal loss in LRRK2-G2019S mutation,and highlight a potential strategy that warrants further preclinical evaluation. View Publication

Chronic compressive cervical spinal cord injury (cCSCI),a debilitating condition,lacks effective treatment options. Addressing this gap,our study introduces a novel rat model of cCSCI developed through spinal cord compression via synthetic polyether sheet implantation,closely mimicking human pathology. We evaluated the model’s fidelity utilizing a comprehensive series of behavioral,electrophysiological,and histological assessments. Our research also explored the therapeutic potential of oligodendrogenic neural progenitor cells (oNPCs) derived from induced pluripotent stem cells. Transplanted oNPCs successfully integrated into the host spinal cord,differentiated into neurons,astrocytes,and oligodendrocytes,and demonstrated a remarkable capacity for enhancing neuroplasticity. Electrophysiological analyses revealed significant improvements in motor evoked potentials and a rectification of the excitability imbalance posttransplantation,indicating substantial recovery of motor circuits. Histological findings complemented these results,showing enhanced remyelination and a reduction in excitatory transmitter expression in the residual gray matter. Functionally,the transplantation of oNPCs led to marked improvements in grip strength,locomotor abilities,and sensory functions,surpassing those seen with standard treatments. This study not only provides a novel and reliable rat model of cCSCI for further research but also highlights the potential of oNPCs as a transformative approach for spinal cord injury therapy,suggesting their significant role in neural regeneration and repair. View Publication