SARS-CoV-2 spike fails to induce inflammation in paediatric airway cells

Introduction/Aim: The spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enables it to recognise and bind host receptors. These dynamics have been modelled in various cell types and immortalised lines, but rarely in primary airway epithelial cells (AEC), and especially not in children. Therefore, this study on AEC recapitulated earlier work testing the hypothesis that exposure to the spike protein would induce airway immune responses in airway cells of young children. Method(s): Primary AEC monolayer cultures from healthy children (n = 5, <10 years old, males = 5) were exposed to the spike protein S1 subunit (0.01, 1, and 10 mug/mL) over 48 h. Induced inflammatory cytokines, interleukin (IL) 6 and IL8, and viral-associated chemokines, CCL5 and CXCL10 were measured via ELISA. Basal receptor gene expression (ACE2 and TMPRSS2) was measured in monolayer (n = 5) and terminally differentiated (air-liquid interface [ALI];n = 5) cell models as well as in ex-vivo cells obtained directly from nasal brushings (n = 71). Generalised linear modelling, accounting for individual variability, identified any statistical difference (p < 0.05). Result(s): Exposure to the spike protein resulted in no increase in IL6 and IL8 production, however a significant (p < 0.05) decrease was observed at the highest dose tested (10 mug/mL). CXCL10 was only significantly induced at the highest dose (10 mug/mL) whereas CCL5 was not induced. When compared to ex-vivo samples, baseline expression of ACE2 and TMPRSS2 was significantly lower in monolayer cultures (~57- and ~4- fold respectively, p < 0.05), whereas ALI cultures had similar expression levels. Conclusion(s): The use of recombinant spike protein and monolayer cultures appears to not accurately model SARS-CoV-2 spike protein-host interactions. The lack of inflammatory responses may be attributed to the lower receptor gene expression in monolayer cultures. Future studies should utilise live virus and ALI cultures as a more biologically relevant model to study virus-host interactions.

Electrophysiological approaches to study cystic fibrosis transmembrane conductance regulator function from human induced pluripotent stem cell-derived airway basal cells

Background: Induced pluripotent stem cells (iPSCs), self-renewable and reprogrammed from somatic cells using different transcription factors, are considered an ideal resource for regenerative medicine to replace diseased or damaged tissues. Airway basal cells not only serve as precursors for secretory and multiciliated cells, but also contribute to maintenance and regeneration of the airway epithelial barrier. Recently, it was reported that induced basal cells (iBCs) from human iPSCs recapitulate molecular and functional features from human iPSCs of airway basal cells, including selfrenewal and multilineage differentiation [1]. iBCs as in vitro model can be used in research on diseases affecting the airway, including COVID-19, influenza, asthma, and cystic fibrosis, although despite these advantages in generating iBCs, this is insufficient to support electrophysiological evidence. Our goal in this study is to define CFTR function in iPSCderived iBCs using electrophysiological methods. Method(s): An iPSC line containing dual reporter NKX2.1GFP and TP63tdTOMATO were used to generate iBCs according to a previously published protocol [1]). iBCs were differentiated into ciliated cells using air-liquid interface (ALI) culture. Short-circuit current measurements were taken on the cells cultured in ALI culture using an Ussing chamber using a previously described protocol [2]). To measure CFTR current using electrophysiological studies, fully differentiated monolayers on filters were dissociated into single cells, which were fixed onto a collagencoated cover glass using cytospin. Whole-cell patch-clamp recordingswere performed according to a previous published protocol [3]. Result(s): We generated proximal airway iBCs from iPSCs with the dualfluorescent reporter system of green fluorescent protein (marks lung progenitors) and tdTomato (marks subsequent airway progenitor) (Figure 1a). These cells on ALI culture demonstrated CFTR function using short-circuit current measurements (Figure 1b). We also measured CFTRdependent currents in iPSC-airway basal cells using whole-cell patchclamp recording (Figure 2). Conclusion(s): We identified CFTR function in electrophysiological experiments using airway iBCs in vitro from iPSCs. Therefore, our study helps advance the field of regenerative medicine, benefiting airway and lung diseases. This may ultimately allowfor development of individual, diseasespecific airway basal stem cells, leading to drug development and a platform on which targeted drug approaches can be tested. Copyright © 2022, European Cystic Fibrosis Society. All rights reserved

Anatomic Images-based 3d Bioprinting And Computational Simulation Of Human Lung Tissue

Purpose/Objectives: Bioprinted models of lung tissue are in high demand but in short supply, particularly for addressing the research needs in response to COVID-19 pandemic. The lung is arguably one of the most complex organs in the body, with a multiscale cytoarchitectural organization serving its multiple functions. In particular, the cellular structure of the alveolar sacs poses a big challenge to extrusion bioprinting, which is more adept at capturing the external shape of biological objects than their cell-level details.Methodology: Recently, we proposed a constructive compromise, attainable by bioprinting of equivalent 3D constructs derived from individual (such as 'precision cut lung slices') or stackable (serial histological sections) anatomic images. The advantage of this approach is that in these images, which can be obtained either from regular histology, or confocal fluorescence or electron microscopy (EM), is already incorporated a wealth of structural information. This can be first transferred to '2.5 dimensional' models (by giving them a finite thickness), and then these can be printed layer-by-layer and stacked as tissue-equivalent 3D volumes. Here we illustrate this proposed workflow with 3D printed human lung sections, and with a lung fragment reconstituted from serial sections, while also simulating the infection with SARS-CoV-2 virus in the same constructs by an agent-based modeling approach.Results: As proof of concept, we processed a human lung histological section in CAD, converted it as .stl file and then 3D printed it using as materials both polycaprolactone (by fused filament fabrication), and by the FRESH method using alginate as hydrogel bioink. Similarly, we extracted from a serial EM stack an image selection which was imported in CAD as well and printed as a self-standing object by photolithography. Here we also report the re-purposing of a simulation program of SARS-CoV-2 infection created on the CompuCell 3D (CC3D) platform, to analyze the propagation of infection in cellular patterns derived from the same histological and ultrastructural sections of human lungs. Using it, we explored the spatial distribution and kinetics of several cell classes (infected, virus shedding, apoptotic), the associated viral and cytokine fields, as well as the impact of the presence of generic inflammatory cells, in comparison with the comparable situations when the cell distribution was a uniform epithelial monolayer. We noted a good reproducibility of these simulations, in spite of the section-characteristic cell distribution patterns, and of the initial locations ('seeding') of the viral infection. In addition, we reconstructed thicker virtual tissue slices from multiple single-cell layers for the study of their viral infection as well.Conclusion/Significance: In conclusion, while more sophisticated methods to capture the tissue structure in 3D constructs certainly exist, the extrusion bioprinting is shown here to be capable to offer a simpler, more practical, and more affordable alternative. We also demonstrated how computational simulations on the same images as used in bioprinting, can be used as a useful heuristic instrument to anticipate the results of the interaction of viruses with bioprinted structures that are more complex than cellular monolayers.

Persistence of Transitional Cell State Without Senescence or Fibrosis During Alveolar Epithelial Regeneration in Human Acute Respiratory Distress Syndrome

RATIONALE: Acute respiratory distress syndrome (ARDS) results from injury to the alveolar epithelial cell (AEC) barrier leading to pulmonary edema;recovery requires epithelial regeneration. However, the specific defect resulting in ongoing barrier permeability in fatal early ARDS is unknown. In mouse models of ARDS, we found that AEC2s assume a transitional state characterized by transient cell cycle arrest during differentiation towards AEC1s. Transitional cells persist and are senescent in human idiopathic pulmonary fibrosis (IPF), leading some to speculate that persistence of transitional cells is pathognomonic of fibrosis in humans. We hypothesized that transitional cells also arise early in human ARDS and that incomplete AEC1 differentiation from the transitional state underlies barrier permeability and death from respiratory failure in early ARDS. We speculated that in contrast to IPF, transitional cells in early ARDS are in transient cell cycle arrest but not senescent and maintain capacity for an AEC1 fate. METHODS AND RESULTS: Lung tissue was obtained from patients who died within two weeks of hospitalization of ARDS due to COVID-19 or other etiologies, and from patients with IPF. Histology revealed diffuse alveolar damage without fibrosis in patients with ARDS. Immunostaining demonstrated AEC damage and abundant transitional cells in both ARDS and IPF. In ARDS, transitional cells existed in a monolayer on alveolar septa, filling gaps denuded of AEC1s and displaying spread morphologies without AEC1 marker expression, suggesting ongoing but incomplete differentiation. In fibrosis, transitional cells existed on a background of architectural distortion and fibrosis. Meta-analysis of single cell RNA sequencing (scRNAseq) datasets demonstrated that transitional cells were transcriptionally highly similar. However, the senescence marker p16 was expressed in transitional cells in human IPF but not in mouse models. Immunostaining confirmed that transitional cells in IPF but not human ARDS expressed p16. CONCLUSION: We conclude that transitional cells arise in early human ARDS without fibrosis. We propose that incomplete AEC1 differentiation from the transitional state is the specific defect in epithelial regeneration underlying barrier permeability and respiratory failure. We speculate that in early human ARDS, as in mouse models, transitional cells retain the capacity to differentiate into AEC1s, restoring alveolar architecture without fibrosis. However, in IPF and fibroproliferative ARDS, transitional cells become senescent, lose capacity for AEC1 differentiation, and fibrosis ensues. Evolution of transitional cells from a transient cell cycle arrest to a permanent cell cycle arrest (senescence) may be the key defect driving the pathogenesis of fibrosis after injury.

ANÁLISE DO TESTE DE MMA (MONOCYTE MONOLAYER ASSAY), SUBCLASSE DE ANTICORPOS E DESFECHO TRANSFUSIONAL EM PACIENTES COMPLEXOS ATENDIDOS NA FUNDAÇÃO HEMOCENTRO DE BRASÍLIA

Introdução: A técnica de monocamada de monócitos (Monocyte Monolayer Assay – MMA) é um ensaio celular funcional in vitro, capaz de prever a sobrevivência in vivo das hemácias transfundidas. É um teste utilizado para diferenciação entre anticorpos clinicamente significantes e não significantes, principalmente em pacientes complexos, para os quais não são encontrados hemocomponentes com provas de compatibilidade negativas. Objetivos: Analisar a relação entre os resultados dos testes de MMA, títulos e subclasses dos anticorpos envolvidos e o desfecho transfusional em pacientes complexos atendidos no Laboratório de Imuno-hematologia de Pacientes da Fundação Hemocentro de Brasília (LIHP-FHB). Materiais e métodos: As amostras dos pacientes foram coletadas nas agências transfusionais do DF e encaminhadas para os testes no LIHP-FHB. A monocamada dos monócitos foi obtida a partir de doadores saudáveis do sexo masculino (O RhD positivo), seguindo as etapas de isolamento (buffy-coat), separação (Ficoll) e cultivo (meio RPMI). A preparação das lâminas incluiu as fases de: aderência monocítica, adsorção do soro do paciente com as hemácias dos doadores, incubações, lavagens e coloração com Leishman. Os controles positivos (Control Cell, Immucor) e negativos (hemácias do doador) foram realizados nas mesmas condições. O Índice Monocitário (MI) foi calculado a partir da análise por microscopia óptica das hemácias fagocitadas ou aderidas, sendo considerados os resultados de MI ≤5%, 5,1%–20% e >20%, como baixo, moderado e alto risco de hemólise pós-transfusional, respectivamente. Foram realizados testes sorológicos em cartão gel-teste (Bio-Rad) para determinação da classe (IgG, IgM, IgA), subclasse (IgG1 e IgG3) e título dos anticorpos. Resultados: Foram avaliados 6 pacientes (13 a 51 anos), sendo 3 (50%) com múltiplos aloanticorpos e autoanticorpos, 2 (33,3%) com autoanticorpos e 1 (16,7%) com múltiplos aloanticorpos, autoanticorpos e anticorpos raros (anti-Hr, -hrs). Desses, 5 (83,3%) apresentavam anemia falciforme e 1 (16,7%) apresentava IRC e infecção por COVID-19. Metade dos pacientes apresentavam anticorpos IgG1 ou IgG3 com baixo risco hemolítico e a outra metade IgG1 ou IgG3 com alto risco. Foram testados 33 concentrados de hemácias (CHs) por meio do teste de MMA, sendo 8 (24,2%) com MI ≤5%, 20 (60,7%) com MI entre 5,1%–20% e 5 (15,1%) com MI >20%. Quatro pacientes receberam transfusões de CHs fenótipo-compatíveis para os sistemas Rh, Kell, Kidd, Duffy e MNS (MIs: 0%, 2%, 6% e 8%). Apenas o CH com MI de 8% resultou em redução dos índices de Hb (8,3 para 7,7 g/dL) e Ht (25 para 23,9%), após 1 hora da transfusão. Os demais CHs transfundidos promoveram incremento nos níveis de Hb e Ht, tanto após 1 hora (0,73 a 2,0 g/dL e 2,4 a 5,8%, respectivamente), quanto após 14 dias (0,8 a 1,2 g/dL e 2,9 a 4,2%, respectivamente). Discussão e conclusão: Nossos resultados revelaram que mais de 75% dos CHs testados apresentaram moderado ou alto risco de hemólise pós-transfusional. Das quatro unidades de CHs transfundidos, três (MIs: 0%, 2%, 6%) promoverem incremento e uma (MI: 8%) declínio, nos níveis de hemoglobina e hematócrito, indicando uma possível associação entre os índices de fagocitose e o risco de hemólise. Não foi evidenciado associação entre os resultados do MMA e as subclasses e títulos dos anticorpos. Apesar do pequeno número de amostras, nossos resultados revelam que o MMA pode ser uma importante ferramenta nas decisões transfusionais em pacientes complexos.

DEVELOPMENT of COVID-19 MONOCLONAL ANTIBODIES and RECOMBINANT PROTEINS AS REAGENTS for BIOMEDICAL RESEARCH and DIAGNOSTIC TESTS

Since SARS-COV-2 virus spread worldwide and COVID-19 turned rapidly into a pandemic illness, the necessity for vaccines and diagnostic tests became crucial. The viral surface is decorated with Spike, the major antigenic determinant and main target for vaccine development. Within Spike, the receptor binding domain (RBD), constitutes the main target of highly neutralizing antibodies found in COVID-19 convalescent plasma. Besides vaccination, another important aspect of Spike (and RBD) is their use as immunogen for the development of poli- and monoclonal antibodies (mAbs) for therapeutic and diagnostic purposes. Here we report the development and preliminary biochemical characterization of a set of monoclonal antibodies against the Spike RBD domain along with the recombinant expression of two mayor COVID-19 protein reagents: the viral Spike RBD domain and the extracellular domain of the human receptor ACE2. RBD and the extracellular domain of ACE2 (aa 1-740) were obtained through transient gene transfection (TGE) in two different mammalian cell culture systems: HEK293T adherent monolayers and Expi293F™ suspension cultures. Due to its low cost and ease scale-up, all transfections were carried with polyethyleneimine (PEI). Expressed proteins were purified from culture supernatants by immobilized metal affinity chromatography. Anti-RBD mAbs were developed from two different immunization schemes: one aimed to elicit antibodies with viral neutralizing potential, and the other with the ability to recognize denatured RBD for routine lab immunoassays. To achieve this, the first group of mice was immunized with RBD in aluminum salts (RBD/Al) and the other with RBD emulsified in Freunds adjuvant (RBD/FA). Polyclonal and monoclonal antibody reactivities against native or denatured RBD forms were then assessed by ELISA. Complete RBD denaturation was followed by intrinsic fluorescence spectral changes upon different physicochemical stress treatments. As expected, RBD/Al immunized mice developed an antibody response shifted to native RBD while those immunized with RBD/FA showed a high response against both forms of the protein. In accordance with the observed polyclonal response, RBD/FA derived mAbs recognize both, native and denatured RBD. On the contrary, hybridomas generated from the RBD/Al protocol mostly recognize RBD in its native state. Further ELISA binding assays revealed that all RBD/FA derived mAbs can form a trimeric complex with ACE2 and RBD, denoting they would not have viral neutralizing activity. ELISA competition assays with the RBD/ACE2 complex aimed to determine the neutralization potential of the RBD/Al derived mAbs are under way. Overall, the anti-Spike RBD mAbs and the recombinant RBD and ACE2 proteins presented here constitute valuable tools for diverse COVID-19 academic research projects and local immunity surveillance testing.

Circulating Levels of Ang/Tie2 and VEGF-a Pathway Mediators Are Associated with Clinical Severity, Endothelial Barrier Disruption and Coagulation Activation in COVID-19

Background: the pathogenesis of severe COVID-19 involves the deregulated activation of different compartments of immunothrombosis, which are otherwise important for pathogen eradication and tissue repair. Coagulation activation, angiogenesis and alterations of endothelial barrier (EB) are elements of immunothrombosis that have been shown to be involved in the pathogenesis of COVID-19. Angiopoietins (Ang) 1 and 2 and their receptor Tie2 and VEGF-A are well-known pro-angiogenic mediators that, during inflammation also mediate EB disruption. Recently, it has also been demonstrated that the Ang/Tie2 pathway is involved in coagulation activation. Here we explored whether increased levels of angiogenesis/EB regulators (which have been previously associated with disease severity in COVID-19) are also associated with both EB disruption and coagulation activation in this condition. Methods: the study population consisted of 30 patients with COVID-19 confirmed by RT-PCR and presenting typical CT findings admitted due to hypoxemia. Thirty sex- and age-matched healthy individuals were recruited at the same time, from the same geographic region. Patients were part of a clinical trial (REBEC: U1111-1250-1843) but samples were obtained before any study intervention, within 24 hours from diagnosis confirmation. Circulating levels of angiogenesis/EB regulation mediators and coagulation biomarkers were measured by commercial assays (immunological or functional). Monolayers of endothelial cells from umbilical veins (HUVECs) or lung (HULECs) were used for measurement of EB integrity using an impedance sensor system (ECIS, Electric Cell-substrate Impedance Sensing System). Cells were stimulated with serum from patients or healthy individuals and EB integrity was continuously monitored for 36 hours. Clinical outcomes were obtained from the digital medical records. Results: mean length of hospital stay (LOS) was 12.9 ± 9.8 days. Twelve patients (40%) required intensive care (ICU) and 28/30 patients survived. Mean D-dimer was 3,609 ± 14,440 ng/mL. Circulating levels of Ang1, Ang2, sTie2 and VEGF-A were all significantly increased in patients compared to healthy individuals (Ang1: 463.2 ± 194.6 vs 237.4 ± 104.9 pg/mL, p<0.0001;Ang2: 1,926 (1,275 - 3,134) vs 1,215 (9 - 1,440 pg/mL), p<0.0001;Tie2: 10,753 ± 2,377 vs 8,603 ± 1,851 pg/mL, p<0.0001 and VEGF-A: 94.7 (73.4 - 116.0) vs 45.9 (39.7 - 57.0 pg/mL), p<0.0001.). In contrast, soluble VE-cadherin levels were decreased in patients compared to healthy individuals (1,234 ± 318 vs 1,539 ± 363 ng/mL, P=0.001). Serum from COVID-19 patients induced decreases of EB integrity in monolayers of both HUVECs and HULECs as early as 15 minutes, lasting up to 5 hours after stimulation (figure 1). The magnitude of EB disruption was correlated with clinically relevant outcomes such as time of ICU stay and LOS (figure 1). Interestingly, levels of Ang1, Ang2 and soluble VE-cadherin levels were also significantly correlated with the magnitude of EB disruption, as well as with biomarkers of coagulation activation such as fibrinogen, Von Willebrand Factor antigen levels, PAI-1, P-selectin and urokinase receptor (uPAR). Conclusions: Ang-1/Ang-2 mediated Tie2 signaling has been shown to be important for the fine regulation of barrier integrity and coagulation activation at the endothelial level, which are two critical elements of immunothrombosis. Our results provide evidence supporting that the interplay between these processes can play a role in the mechanisms driving COVID-19 severity, and suggest that targeting the Ang/Tie2 and VEGF-A pathways could be attractive strategies to modulate not only changes of the alveolar-capillary barrier, but also of coagulation activation in COVID-19. Figure 1. In (a), endothelial barrier (EB) integrity of HUVEC monolayers upon stimulation by serum from COVID-19 patients and healthy individuals (n=27-30 per group). The lower the normalized resistance, the higher the magnitude of EB disruption. Significant differences (* to ****) are evident from 15 min to 5 hours (An va corrected for multiple comparisons). In the lower panels, the correlation of EB disruption with clinically relevant outcomes such as length of hospital stay (b) and days of intensive care (c) are shown. Negative correlations (Spearman test) indicate that the magnitude of EB disruption is associated with worse outcomes. [Formula presented] Disclosures: No relevant conflicts of interest to declare.