Use of 2D minilungs from human embryonic stem cells to study the interaction of Cryptococcus neoformans with the respiratory tract.

Organoids can meet the needs between the use of cell culture and in vivo work, bringing together aspects of multicellular tissues, providing a more similar in vitro system for the study of various components, including host-interactions with pathogens and drug response. Organoids are structures that resemble organs in vivo, originating from pluripotent stem cells (PSCs) or adult stem cells (ASCs). There is great interest in deepening the understanding of the use of this technology to produce information about fungal infections and their treatments. This work aims the use 2D human lung organoid derived from human embryonic stem cells (hESCs), to investigate Cryptococcus neoformans-host interactions. C. neoformans is an opportunistic fungus acquired by inhalation that causes systemic mycosis mainly in immunocompromised individuals. Our work highlights the suitability of human minilungs for the study of C. neoformans infection (adhesion, invasion and replication), the interaction with the surfactant and induction of the host's alveolar pro-inflammatory response.

Modeling of Respiratory Diseases Evolving with Fibrosis from Organoids Derived from Human Pluripotent Stem Cells.

Respiratory disease is one of the leading causes of morbidity and mortality worldwide. There is no cure for most diseases, which are treated symptomatically. Hence, new strategies are required to deepen the understanding of the disease and development of therapeutic strategies. The advent of stem cell and organoid technology has enabled the development of human pluripotent stem cell lines and adequate differentiation protocols for developing both airways and lung organoids in different formats. These novel human-pluripotent-stem-cell-derived organoids have enabled relatively accurate disease modeling. Idiopathic pulmonary fibrosis is a fatal and debilitating disease that exhibits prototypical fibrotic features that may be, to some extent, extrapolated to other conditions. Thus, respiratory diseases such as cystic fibrosis, chronic obstructive pulmonary disease, or the one caused by SARS-CoV-2 may reflect some fibrotic aspects reminiscent of those present in idiopathic pulmonary fibrosis. Modeling of fibrosis of the airways and the lung is a real challenge due to the large number of epithelial cells involved and interaction with other cell types of mesenchymal origin. This review will focus on the status of respiratory disease modeling from human-pluripotent-stem-cell-derived organoids, which are being used to model several representative respiratory diseases, such as idiopathic pulmonary fibrosis, cystic fibrosis, chronic obstructive pulmonary disease, and COVID-19.

Spontaneous closure of a macular hole in an unusually long time after primary vitrectomy / Fechamento espontâneo de buraco macular em um tempo excepcionalmente longo após vitrectomia primária

ABSTRACT We report the case of a 68-year-old man who presented to our outpatient clinic for routine examination. Fifteen months before, he had undergone combined cataract and idiopathic full-thickness macular hole surgery in his right eye at another institution. In the present evaluation, the best-corrected visual acuity in his right eye was counting fingers. Fundus examination evidenced an idiopathic full-thickness macular hole in that eye, which was confirmed on spectral domain optical coherence tomography. A new surgery was offered, but the patient declined. Twenty-one months after his first consultation with us (36 months after the surgery), spectral domain optical coherence tomography revealed spontaneous closure of the idiopathic full-thickness macular hole, with a gap at the foveal ellipsoid zone. At the final visit, 22 months after the closure of the idiopathic full-thickness macular hole, the patient's best-corrected visual acuity was 20/25, and the gap at the ellipsoid zone had decreased.

RESUMO Este é o relato do caso de um homem de 68 anos que procurou nosso ambulatório para exames de rotina. Quinze meses antes, ele havia se submetido a uma cirurgia conjunta de catarata e buraco macular idiopático de espessura total em seu olho direito, em outra instituição. Durante a consulta em nosso ambulatório, a melhor acuidade visual corrigida no olho direito era de contagem de dedos. O exame do fundo evidenciou um buraco macular idiopático de espessura total naquele olho, o que foi confirmado por uma tomografia de coerência óptica de domínio espectral. Uma nova cirurgia foi oferecida, mas o paciente recusou. Vinte e um meses após sua primeira consulta (36 meses após a cirurgia), a tomografia de coerência óptica de domínio espectral revelou o fechamento espontâneo do buraco macular idiopático de espessura total, com uma lacuna na zona elipsoide foveal. Na última consulta, 22 meses após o fechamento do buraco macular idiopático de espessura total, a melhor acuidade visual corrigida foi de 20/25 e a lacuna na zona elipsoide havia diminuído.

Minilungs from Human Embryonic Stem Cells to Study the Interaction of Streptococcus pneumoniae with the Respiratory Tract.

The new generation of organoids derived from human pluripotent stem cells holds a promising strategy for modeling host-bacteria interaction studies. Organoids recapitulate the composition, diversity of cell types, and, to some extent, the functional features of the native organ. We generated lung bud organoids derived from human embryonic stem cells to study the interaction of Streptococcus pneumoniae (pneumococcus) with the alveolar epithelium. Invasive pneumococcal disease is an important health problem that may occur as a result of the spread of pneumococcus from the lower respiratory tract to sterile sites. We show here an efficient experimental approach to model the main events of the pneumococcal infection that occur in the human lung, exploring bacterial adherence to the epithelium and internalization and triggering an innate response that includes the interaction with surfactant and the expression of representative cytokines and chemokines. Thus, this model, based on human minilungs, can be used to study pneumococcal virulence factors and the pathogenesis of different serotypes, and it will allow therapeutic interventions in a reliable human context. IMPORTANCE Streptococcus pneumoniae is responsible for high morbidity and mortalities rates worldwide, affecting mainly children and adults older than 65 years. Pneumococcus is also the most common etiologic agent of bacterial pneumonia and nonepidemic meningitis, and it is a frequent cause of bacterial sepsis. Although the introduction of pneumococcal vaccines has decreased the burden of pneumococcal disease, the rise of antibiotic-resistant strains and nonvaccine types by serotype replacement is worrisome. To study the biology of pneumococcus and to establish a reliable human model for pneumococcal pathogenesis, we generated human minilungs from embryonic stem cells. The results show that these organoids can be used to model some events occurring during the interaction of pneumococcus with the lung, such as adherence, internalization, and the initial alveolar innate response. This model also represents a great alternative for studying virulence factors involved in pneumonia, drug screening, and other therapeutic interventions.

Effects of Hypocalcemic Vitamin D Analogs in the Expression of DNA Damage Induced in Minilungs from hESCs: Implications for Lung Fibrosis.

In our previous work, we evaluated the therapeutic effects of 1α,25-Dihydroxyvitamin D3, the biologically active form of vitamin D, in the context of bleomycin-induced lung fibrosis. Contrary to the expected, vitamin D supplementation increased the DNA damage expression and cellular senescence in alveolar epithelial type II cells and aggravated the overall lung pathology induced in mice by bleomycin. These effects were probably due to an alteration in the cellular DNA double-strand breaks' repair capability. In the present work, we have evaluated the effects of two hypocalcemic vitamin D analogs (calcipotriol and paricalcitol) in the expression of DNA damage in the context of minilungs derived from human embryonic stem cells and in the cell line A549.

Spontaneous closure of a macular hole in an unusually long time after primary vitrectomy.

We report the case of a 68-year-old man who presented to our outpatient clinic for routine examination. Fifteen months before, he had undergone combined cataract and idiopathic full-thickness macular hole surgery in his right eye at another institution. In the present evaluation, the best-corrected visual acuity in his right eye was counting fingers. Fundus examination evidenced an idiopathic full-thickness macular hole in that eye, which was confirmed on spectral domain optical coherence tomography. A new surgery was offered, but the patient declined. Twenty-one months after his first consultation with us (36 months after the surgery), spectral domain optical coherence tomography revealed spontaneous closure of the idiopathic full-thickness macular hole, with a gap at the foveal ellipsoid zone. At the final visit, 22 months after the closure of the idiopathic full-thickness macular hole, the patient's best-corrected visual acuity was 20/25, and the gap at the ellipsoid zone had decreased.

Identification of AAV serotypes for lung gene therapy in human embryonic stem cell-derived lung organoids.

Gene therapy is being investigated for a range of serious lung diseases, such as cystic fibrosis and emphysema. Recombinant adeno-associated virus (rAAV) is a well-established, safe, viral vector for gene delivery with multiple naturally occurring and artificial serotypes available displaying alternate cell, tissue, and species-specific tropisms. Efficient AAV serotypes for the transduction of the conducting airways have been identified for several species; however, efficient serotypes for human lung parenchyma have not yet been identified. Here, we screened the ability of multiple AAV serotypes to transduce lung bud organoids (LBOs)-a model of human lung parenchyma generated from human embryonic stem cells. Microinjection of LBOs allowed us to model transduction from the luminal surface, similar to dosing via vector inhalation. We identified the naturally occurring rAAV2 and rAAV6 serotypes, along with synthetic rAAV6 variants, as having tropism for the human lung parenchyma. Positive staining of LBOs for surfactant proteins B and C confirmed distal lung identity and suggested the suitability of these vectors for the transduction of alveolar type II cells. Our findings establish LBOs as a new model for pulmonary gene therapy and stress the relevance of LBOs as a viral infection model of the lung parenchyma as relevant in SARS-CoV-2 research.

Physiological and pathological effects of amyloid-ß species in neural stem cell biology.

Although amyloid-ß peptide is considered neurotoxic, it may mediate several physiological processes during embryonic development and in the adult brain. The pathological function of amyloid-ß peptide has been extensively studied due to its implication in Alzheimer's disease, but its physiological function remains poorly understood. Amyloid-ß peptide can be detected in non-aggregated (monomeric) and aggregated (oligomeric and fibrillary) forms. Each form has different cytotoxic and/or physiological properties, so amyloid-ß peptide and its role in Alzheimer's disease need to be studied further. Neural stem cells and neural precursor cells are good tools for the study on neurodegenerative diseases and can provide future therapeutic applications in diseases such as Alzheimer's disease. In this review, we provide an outline of the effects of amyloid-ß peptide, in monomeric and aggregated forms, on the biology of neural stem cells/neural precursor cells, and discuss the controversies. We also describe the possible molecular targets that could be implicated in these effects, especially GSK3ß. A better understanding of amyloid-ß peptide (both physiological and pathological), and the signaling pathways involved are essential to advance the field of Alzheimer's disease.

Physiological effects of amyloid precursor protein and its derivatives on neural stem cell biology and signaling pathways involved.

The pathological implication of amyloid precursor protein (APP) in Alzheimer's disease has been widely documented due to its involvement in the generation of amyloid-ß peptide. However, the physiological functions of APP are still poorly understood. APP is considered a multimodal protein due to its role in a wide variety of processes, both in the embryo and in the adult brain. Specifically, APP seems to play a key role in the proliferation, differentiation and maturation of neural stem cells. In addition, APP can be processed through two canonical processing pathways, generating different functionally active fragments: soluble APP-α, soluble APP-ß, amyloid-ß peptide and the APP intracellular C-terminal domain. These fragments also appear to modulate various functions in neural stem cells, including the processes of proliferation, neurogenesis, gliogenesis or cell death. However, the molecular mechanisms involved in these effects are still unclear. In this review, we summarize the physiological functions of APP and its main proteolytic derivatives in neural stem cells, as well as the possible signaling pathways that could be implicated in these effects. The knowledge of these functions and signaling pathways involved in the onset or during the development of Alzheimer's disease is essential to advance the understanding of the pathogenesis of Alzheimer's disease, and in the search for potential therapeutic targets.

Effects of chlorpyrifos on cell death and cellular phenotypic specification of human neural stem cells.

Chlorpyrifos (CPF) is an organophosphate pesticide widely used in agriculture, whose traditional and well-known mechanism of action is the inhibition of the enzyme Acetylcholinesterase (AChE). Subacute exposures to CPF have been associated with alterations different from the inhibition of AChE. Because of the vulnerability of the developing nervous system, prenatal and early postnatal exposures are of special concern. Human neural stem cells (hNSC) provide the opportunity to study early stages of neural development and may be a valuable tool for developmental neurotoxicology (DNT). In the current work, the cell line hNS1 was used as a model system with the aim of validating this cell line as a reliable testing method. To evaluate the effects of CPF on early developmental stages, hNS1 cells were exposed to different concentrations of the pesticide and cell death, proliferation and cell fate specification were analyzed under differentiation conditions. Since hNS1 cells responded to CPF in a similar way to other human cell lines, we consider it may be a valid model for DNT chemical assessment. CPF induced apoptotic cell death only at the highest doses tested, suggesting that it is not toxic for the specific developmental stage here addressed under short term exposure. In addition, the higher doses of CPF promoted the generation of astroglial cells, without affecting neurogenesis.

Early induction of senescence and immortalization in PGC-1α-deficient mouse embryonic fibroblasts.

AIMS: Oxidative stress is known to induce early replicative senescence. Senescence has been proposed to work as a barrier to immortalization and tumor development. Here, we aimed to evaluate the impact of the loss of peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α), a master regulator of oxidative metabolism and mitochondrial reactive oxygen species (ROS) generation, on replicative senescence and immortalization in mouse embryonic fibroblasts (MEFs). RESULTS: We found that primary MEFs lacking PGC-1α showed higher levels of ROS than wild-type MEFs at all cell passages tested. The elevated production of ROS was associated with higher levels of oxidative DNA damage and the increased formation of DNA double-strand breaks. Evaluation of the induction of DNA repair systems in response to γ-radiation indicated that the loss of PGC-1α also resulted in a small but significant reduction in their activity. DNA damage induced the early activation of senescence markers, including an increase in the number of ß-galactosidase-positive cells, the induction of p53 phosphorylation, and the increase in p16 and p19 protein. These changes were, however, not sufficient to reduce proliferation rates of PGC-1α-deficient MEFs at any cell passage tested. Moreover, PGC-1α-deficient cells escaped replicative senescence. INNOVATION & CONCLUSION: PGC-1α plays an important role in the control of cellular senescence and immortalization.

Encircling Scleral Buckling for Traumatic Severe Hypotony.

The authors report the use of an encircling scleral buckling procedure for the management of severe hypotony secondary to traumatic annular ciliochoroidal detachment (CCD) with cyclodialysis cleft. Medical records of patients with severe ocular hypotony were retrospectively reviewed. Four patients with traumatic annular CCD with cyclodialysis cleft were identified. Diagnosis of CCD was documented by ultrasound biomicroscopy and presence of cyclodialysis cleft was confirmed by gonioscopy or ultrasound biomicroscopy. All patients underwent scleral buckling surgery with an encircling band for annular CCD with cyclodialysis cleft. Intraocular pressure (IOP) and visual acuity (VA) significantly improved postoperatively. Mean IOP changed from 2.5 mm Hg ± 0.5 mm Hg to 10.75 mm Hg ± 1.1 mm Hg (P = .0129) and mean best-corrected VA changed from +0.50 ± 0.16 logMAR to +0.15 ± 0.17 logMAR (P = .0123). IOP normalization was achieved despite persistence of CCD. These results support the use of scleral buckling with an encircling band as an effective approach for severe hypotony in patients with annular CCD regardless the cyclodialysis cleft extension. [Ophthalmic Surg Lasers Imaging Retina. 2020;51:58-63.].

Aß42 Peptide Promotes Proliferation and Gliogenesis in Human Neural Stem Cells.

Amyloid-ß 42 [Aß1-42 (Aß42)] is one of the main Aß peptide isoforms found in amyloid plaques of brains with Alzheimer's disease (AD). Although Aß42 is associated with neurotoxicity, it might mediate several normal physiological processes during embryonic brain development and in the adult brain. However, due to the controversy that exists in the field, relatively little is known about its physiological function. In the present work, we have analyzed the effects of different concentrations of monomeric Aß42 on cell death, proliferation, and cell fate specification of human neural stem cells (hNSCs), specifically the hNS1 cell line, undergoing differentiation. Our results demonstrate that at higher concentrations (1 µM), Aß42 increases apoptotic cell death and DNA damage, indicating that prolonged exposure of hNS1 cells to higher concentrations of Aß42 is neurotoxic. However, at lower concentrations, Aß42 significantly promotes cell proliferation and glial cell specification of hNS1 cells by increasing the pool of proliferating glial precursors, without affecting neuronal differentiation, in a concentration-dependent manner. At the molecular level, these effects could be mediated, at least in part, by GSK3ß, whose expression is increased by treatment with Aß42 and whose inhibition prevents the glial specification induced by Aß42. Since the cellular and molecular effects are known to appear decades before the first clinical symptoms, these types of studies are important in discovering the underlying pathophysiological processes involved in the development of AD. This knowledge could then be used in diagnosing the disease at early stages and be applied to the development of new treatment options.

Detrimental pro-senescence effects of vitamin D on lung fibrosis.

BACKGROUND: The multiple biological effects of vitamin D and its novel activities on inflammation and redox homeostasis have raised high expectations on its use as a therapeutic agent for multiple fibrogenic conditions. We have assessed the therapeutic effects of 1α,25-Dihydroxyvitamin D3, the biologically active form of vitamin D, in the context of lung fibrosis. METHODS: We have used representative cellular models for alveolar type II cells and human myofibroblasts. The extension of DNA damage and cellular senescence have been assessed by immunofluorescence, western-blot and senescence-associated ß-galactosidase activity. We have also set up a murine model for lung fibrosis by intraperitoneal injections of bleomycin. RESULTS: Vitamin D induces cellular senescence in bleomycin-treated alveolar epithelial type II cells and aggravates the lung pathology induced by bleomycin. These effects are probably due to an alteration of the cellular DNA double-strand breaks repair in bleomycin-treated cells. CONCLUSIONS: The detrimental effects of vitamin D in the presence of a DNA damaging agent might preclude its use as an antifibrogenic agent for pulmonary fibrosis characterized by DNA damage occurrence and cellular senescence.

Effects of lung and airway epithelial maturation cocktail on the structure of lung bud organoids.

Organoids from human pluripotent stem cells are becoming suitable models for studies of organ development, drug screening, regenerative medicine, and disease modeling. Three-dimensional minilungs in Matrigel culture have recently been generated from human embryonic stem cells. These particular organoids, named lung bud organoids, showed branching airway and early alveolar structures resembling those present in lungs from the second trimester of human gestation. We show here that the treatment of such organoids with a lung and airway epithelial maturation cocktail containing dexamethasone drives lung bud organoids to the formation of paddle-racquet like structures. This strategy may help to increase the versatility of lung organoids and to generate structures more advanced than the original branching texture.

Role of Amyloid Precursor Protein (APP) and Its Derivatives in the Biology and Cell Fate Specification of Neural Stem Cells.

Amyloid precursor protein (APP) is a member of the APP family of proteins, and different enzymatic processing leads to the production of several derivatives that are shown to have distinct biological functions. APP is involved in the pathology of Alzheimer's disease (AD), the most common neurodegenerative disorder causing dementia. Furthermore, it is believed that individuals with Down syndrome (DS) have increased APP expression, due to an extra copy of chromosome 21 (Hsa21), that contains the gene for APP. Nevertheless, the physiological function of APP remains unclear. It is known that APP plays an important role in neural growth and maturation during brain development, possibly by influencing proliferation, cell fate specification and neurogenesis of neural stem cells (NSCs). Proteolytic cleavage of APP occurs mainly via two mutually exclusive pathways, the non-amyloidogenic pathway or the amyloidogenic pathway. Other alternative pathways (η-secretase, δ-secretase and meprin pathways) have also been described for the physiological processing of APP. The different metabolites generated from these pathways, including soluble APPα (sAPPα), soluble APPß (sAPPß), ß-amyloid (Aß) peptides and the APP intracellular domain (AICD), have different functions determined by their structural differences, equilibrium and concentration with respect to other fragments derived from APP. This review discusses recent observations regarding possible functions of APP and its proteolytic derivatives in the biology and phenotypic specification of NSCs. This can be important for a better understanding of the pathogenesis and the development of future therapeutic applications for AD and/or DS, diseases in which alterations in neurogenesis have been described.

A Two-Dimensional Human Minilung System (Model) for Respiratory Syncytial Virus Infections.

Human respiratory syncytial virus (HRSV) is a major cause of serious pediatric respiratory diseases that lacks effective vaccine or specific therapeutics. Although our understanding about HRSV biology has dramatically increased during the last decades, the need for adequate models of HRSV infection is compelling. We have generated a two-dimensional minilung from human embryonic stem cells (hESCs). The differentiation protocol yielded at least six types of lung and airway cells, although it is biased toward the generation of distal cells. We show evidence of HRSV replication in lung cells, and the induction of innate and proinflammatory responses, thus supporting its use as a model for the study of HRSV-host interactions.

Induction of DNA double-strand breaks and cellular senescence by human respiratory syncytial virus.

Human respiratory syncytial virus (HRSV) accounts for the majority of lower respiratory tract infections during infancy and childhood and is associated with significant morbidity and mortality. HRSV provokes a proliferation arrest and characteristic syncytia in cellular systems such as immortalized epithelial cells. We show here that HRSV induces the expression of DNA damage markers and proliferation arrest such as P-TP53, P-ATM, CDKN1A and γH2AFX in cultured cells secondary to the production of mitochondrial reactive oxygen species (ROS). The DNA damage foci contained γH2AFX and TP53BP1, indicative of double-strand breaks (DSBs) and could be reversed by antioxidant treatments such as N-Acetylcysteine (NAC) or reduced glutathione ethyl ester (GSHee). The damage observed is associated with the accumulation of senescent cells, displaying a canonical senescent phenotype in both mononuclear cells and syncytia. In addition, we show signs of DNA damage and aging such as γH2AFX and CDKN2A expression in the respiratory epithelia of infected mice long after viral clearance. Altogether, these results show that HRSV triggers a DNA damage-mediated cellular senescence program probably mediated by oxidative stress. The results also suggest that this program might contribute to the physiopathology of the infection, tissue remodeling and aging, and might be associated to long-term consequences of HRSV infections.

Thyroid hormone increases bulk histones expression by enhancing translational efficiency.

The expression of canonical histones is normally coupled to DNA synthesis during the S phase of the cell cycle. Replication-dependent histone mRNAs do not contain a poly(A) tail at their 3' terminus, but instead possess a stem-loop motif, the binding site for the stem-loop binding protein (SLBP), which regulates mRNA processing, stability, and relocation to polysomes. Here we show that the thyroid hormone can increase the levels of canonical histones independent of DNA replication. Incubation of mouse embryonic fibroblasts with T3 increases the total levels of histones, and expression of the thyroid hormone receptor ß induces a further increase. This is not restricted to mouse embryonic fibroblasts, because T3 also raises histone expression in other cell lines. T3 does not increase histone mRNA or SLBP levels, suggesting that T3 regulates histone expression by a posttranscriptional mechanism. Indeed, T3 enhanced translational efficiency, inducing relocation of histone mRNA to heavy polysomes. Increased translation was associated with augmented transcription of the eukaryotic translation initiation factor 4 γ2 (EIF4G2). T3 induced EIF4G2 protein and mRNA levels and the thyroid hormone receptor bound to the promoter region of the Eif4g2 gene. Induction of EIF4G2 was essential for T3-dependent histone induction, because depletion of this factor abolished histone increase. These results point out the importance of the thyroid hormones on the posttranscriptional regulation of histone biosynthesis in a cell cycle-independent manner and also suggest the potential regulation of eukaryotic translation by the modulation of the initiation factor EIF4G2, which also operates in the translation of canonical mRNAs.

The thyroid hormone receptor ß induces DNA damage and premature senescence.

There is increasing evidence that the thyroid hormone (TH) receptors (THRs) can play a role in aging, cancer and degenerative diseases. In this paper, we demonstrate that binding of TH T3 (triiodothyronine) to THRB induces senescence and deoxyribonucleic acid (DNA) damage in cultured cells and in tissues of young hyperthyroid mice. T3 induces a rapid activation of ATM (ataxia telangiectasia mutated)/PRKAA (adenosine monophosphate-activated protein kinase) signal transduction and recruitment of the NRF1 (nuclear respiratory factor 1) and THRB to the promoters of genes with a key role on mitochondrial respiration. Increased respiration leads to production of mitochondrial reactive oxygen species, which in turn causes oxidative stress and DNA double-strand breaks and triggers a DNA damage response that ultimately leads to premature senescence of susceptible cells. Our findings provide a mechanism for integrating metabolic effects of THs with the tumor suppressor activity of THRB, the effect of thyroidal status on longevity, and the occurrence of tissue damage in hyperthyroidism.