Quercetin inhibits SARS-CoV-2 infection and prevents syncytium formation by cells co-expressing the viral spike protein and human ACE2.

BACKGROUND: Several in silico studies have determined that quercetin, a plant flavonol, could bind with strong affinity and low free energy to SARS-CoV-2 proteins involved in viral entry and replication, suggesting it could block infection of human cells by the virus. In the present study, we examined the ex vivo ability of quercetin to inhibit of SARS-CoV-2 replication and explored the mechanisms of this inhibition. METHODS: Green monkey kidney Vero E6 cells and in human colon carcinoma Caco-2 cells were infected with SARS-CoV-2 and incubated in presence of quercetin; the amount of replicated viral RNA was measured in spent media by RT-qPCR. Since the formation of syncytia is a mechanism of SARS-CoV-2 propagation, a syncytialization model was set up using human embryonic kidney HEK293 co-expressing SARS-CoV-2 Spike (S) protein and human angiotensin converting enzyme 2 (ACE2), [HEK293(S + ACE2) cells], to assess the effect of quercetin on this cytopathic event by microscopic imaging and protein immunoblotting. RESULTS: Quercetin inhibited SARS-CoV-2 replication in Vero E6 cells and Caco-2 cells in a concentration-dependent manner with a half inhibitory concentration (IC50) of 166.6 and 145.2 µM, respectively. It also inhibited syncytialization of HEK293(S + ACE2) cells with an IC50 of 156.7 µM. Spike and ACE2 co-expression was associated with decreased expression, increased proteolytic processing of the S protein, and diminished production of the fusogenic S2' fragment of S. Furin, a proposed protease for this processing, was inhibited by quercetin in vitro with an IC50 of 116 µM. CONCLUSION: These findings suggest that at low 3-digit micromolar concentrations of quercetin could impair SARS-CoV-2 infection of human cells partly by blocking the fusion process that promotes its propagation.

Dental malocclusion among children with attention deficit hyperactivity disorder.

INTRODUCTION: Children with attention deficit hyperactivity disorder (ADHD) have more sleep breathing problems and parafunctional oral habits than individuals without ADHD. However, there is scarce information on the correlation between their dental malocclusion and these functional disorders. The objective of this study was to assess the severity of malocclusion in patients with and without ADHD and to evaluate the correlation between their functional disorders and dental malocclusion. METHODS: Eighty-eight patients aged 6-17 years were divided into 2 groups: ADHD (n = 44) and control (n = 44). A medical questionnaire to assess functional disorders and an orthodontic examination to evaluate malocclusion were completed for each patient. Distribution of the data was evaluated using Shapiro-Wilk test, whereas the 2 groups were compared with a t test, Mann-Whitney U test, Fisher exact test, and Spearman correlation. The association between parafunctional oral habits, ADHD drug intake, and malocclusion severity were assessed with a t test and Mann-Whitney U test. RESULTS: Patients with ADHD had significantly higher severity of malocclusion (P = 0.042), more dental rotation (P = 0.021) and more parafunctional oral habits (P = 0.001), specifically bruxism (P = 0.005), and a history of pacifier use (P = 0.009), than the control group. CONCLUSIONS: It is important to be aware of the increased risk of parafunctional oral habits and dental malocclusion among ADHD patients to develop preventive programs, as well as therapeutic strategies for them.

The Protein Coded by a Short Open Reading Frame, Not by the Annotated Coding Sequence, Is the Main Gene Product of the Dual-Coding Gene MIEF1.

Proteogenomics and ribosome profiling concurrently show that genes may code for both a large and one or more small proteins translated from annotated coding sequences (CDSs) and unannotated alternative open reading frames (named alternative ORFs or altORFs), respectively, but the stoichiometry between large and small proteins translated from a same gene is unknown. MIEF1, a gene recently identified as a dual-coding gene, harbors a CDS and a newly annotated and actively translated altORF located in the 5'UTR. Here, we use absolute quantification with stable isotope-labeled peptides and parallel reaction monitoring to determine levels of both proteins in two human cells lines and in human colon. We report that the main MIEF1 translational product is not the canonical 463 amino acid MiD51 protein but the small 70 amino acid alternative MiD51 protein (altMiD51). These results demonstrate the inadequacy of the single CDS concept and provide a strong argument for incorporating altORFs and small proteins in functional annotations.

Deep transcriptome annotation enables the discovery and functional characterization of cryptic small proteins.

Recent functional, proteomic and ribosome profiling studies in eukaryotes have concurrently demonstrated the translation of alternative open-reading frames (altORFs) in addition to annotated protein coding sequences (CDSs). We show that a large number of small proteins could in fact be coded by these altORFs. The putative alternative proteins translated from altORFs have orthologs in many species and contain functional domains. Evolutionary analyses indicate that altORFs often show more extreme conservation patterns than their CDSs. Thousands of alternative proteins are detected in proteomic datasets by reanalysis using a database containing predicted alternative proteins. This is illustrated with specific examples, including altMiD51, a 70 amino acid mitochondrial fission-promoting protein encoded in MiD51/Mief1/SMCR7L, a gene encoding an annotated protein promoting mitochondrial fission. Our results suggest that many genes are multicoding genes and code for a large protein and one or several small proteins.

Prevalence of malocclusion in Canadian children with autism spectrum disorder.

INTRODUCTION: The purposes of this study were to determine the prevalence of malocclusion among children with autism spectrum disorder (ASD) and to describe the most common malocclusion traits in this population. METHODS: This cross-sectional study included patients diagnosed with ASD aged between 5 and 18 years. Randomly selected healthy children with the same demographic characteristics comprised the control group. Dental charts were reviewed to obtain the children's sociodemographic characteristics and type of occlusion. Information on each child's molar occlusion classification (Angle classification), midline deviation, crossbite, open bite, overbite, overjet, and crowding were recorded. The statistical analysis used descriptive analysis, the Pearson chi-square test, and multivariate logistic regression. RESULTS: Ninety-nine children comprised the ASD group, and 101 children were in the control group. Our results demonstrated a significantly higher prevalence of malocclusion in children with ASD compared with the control group (P <0.001). Patients with ASD were significantly more likely to have posterior crossbite (P = 0.03), increased overjet (P <0.0001), and severe maxillary crowding (P <0.01). Furthermore, children with ASD were more likely to have malocclusion than non-ASD children, independently of their demographic characteristics (odds ratio, 2.6; 95% confidence interval, 1.46, -4.65). CONCLUSIONS: The prevalence of malocclusion was higher among children with ASD. Posterior crossbite, increased overjet, and severe maxillary crowding were the most common malocclusion traits in these children.

Flow cytometry assessment of in vitro generated CD138+ human plasma cells.

The in vitro CD40-CD154 interaction promotes human B lymphocytes differentiation into plasma cells. Currently, CD138 is the hallmark marker enabling the detection of human plasma cells, both in vitro and in vivo; its presence can be monitored by flow cytometry using a specific antibody. We have developed a culture system allowing for the differentiation of memory B lymphocytes. In order to detect the newly formed plasma cells, we have compared their staining using five anti-CD138 monoclonal antibodies (mAbs). As a reference, we also tested human cell lines, peripheral blood mononuclear cells, and bone marrow samples. The five anti-CD138 mAbs stained RPMI-8226 cells (>98%) with variable stain index (SI). The highest SI was obtained with B-A38 mAb while the lowest SI was obtained with DL-101 and 1D4 mAbs. However, the anti-CD138 mAbs were not showing equivalent CD138(+) cells frequencies within the generated plasma cells. B-A38, B-B4, and MI-15 were similar (15-25%) while DL-101 mAb stained a higher proportion of CD138-positive cells (38-42%). DL-101 and B-A38 mAbs stained similar populations in bone marrow samples but differed in their capacity to bind to CD138(high) and CD138(lo) cell lines. In conclusion, such cellular fluctuations suggest heterogeneity in human plasma cell populations and/or in CD138 molecules.

Bench-test comparison of 26 emergency and transport ventilators.

INTRODUCTION: Numerous emergency and transport ventilators are commercialized and new generations arise constantly. The aim of this study was to evaluate a large panel of ventilators to allow clinicians to choose a device, taking into account their specificities of use. METHODS: This experimental bench-test took into account general characteristics and technical performances. Performances were assessed under different levels of FIO2 (100%, 50% or Air-Mix), respiratory mechanics (compliance 30,70,120 mL/cmH2O; resistance 5,10,20 cmH2O/mL/s), and levels of leaks (3.5 to 12.5 L/min), using a test lung. RESULTS: In total 26 emergency and transport ventilators were analyzed and classified into four categories (ICU-like, n = 5; Sophisticated, n = 10; Simple, n = 9; Mass-casualty and military, n = 2). Oxygen consumption (7.1 to 15.8 L/min at FIO2 100%) and the Air-Mix mode (FIO2 45 to 86%) differed from one device to the other. Triggering performance was heterogeneous, but several sophisticated ventilators depicted triggering capabilities as efficient as ICU-like ventilators. Pressurization was not adequate for all devices. At baseline, all the ventilators were able to synchronize, but with variations among respiratory conditions. Leak compensation in most ICU-like and 4/10 sophisticated devices was able to correct at least partially for system leaks, but with variations among ventilators. CONCLUSION: Major differences were observed between devices and categories, either in terms of general characteristics or technical reliability, across the spectrum of operation. Huge variability of tidal volume delivery with some devices in response to modifications in respiratory mechanics and FIO2 should make clinicians question their use in the clinical setting.

Persistent Polyclonal B Cell Lymphocytosis B Cells Can Be Activated through CD40-CD154 Interaction.

Persistent polyclonal B cell lymphocytosis (PPBL) is a rare disorder, diagnosed primarily in adult female smokers and characterized by an expansion of CD19(+)CD27(+)IgM(+) memory B cells, by the presence of binucleated lymphocytes, and by a moderate elevation of serum IgM. The clinical course is usually benign, but it is not known whether or not PPBL might be part of a process leading to the emergence of a malignant proliferative disorder. In this study we sought to investigate the functional response of B cells from patients with PPBL by use of an optimal memory B cell culture model based on the CD40-CD154 interaction. We found that the proliferation of PPBL B cells was almost as important as that of B cells from normal controls, resulting in high immunoglobulin secretion with in vitro isotypic switching. We conclude that the CD40-CD154 activation pathway is functional in the memory B cell population of PPBL patients, suggesting that the disorder may be due to either a dysfunction of other cells in the microenvironment or a possible defect in another B cell activation pathway.

Modulation of CD40-activated B lymphocytes by N-acetylcysteine involves decreased phosphorylation of STAT3.

B lymphocyte activation, maturation and reshaping require the interaction of its receptor CD40 with its ligand CD154, which is expressed on activated T lymphocytes. Metabolism in activated B lymphocytes is also characterized with several REDOX changes including fluctuation of Reactive Oxygen Species (ROS). Herein, we first confirm that stimulation of human peripheral blood B lymphocyte with CD154 increases intracellular ROS level. Then, by treatments with two well-known antioxidants, N-acetylcysteine (NAC) and Trolox, we further investigate the influence of REDOX fluctuation in CD40-activated B lymphocyte homeostasis in long term culture (13 days). Treatments with NAC increase viability, decrease proliferation and Ig secretion and enhance homoaggregation of B lymphocytes while Trolox only induces a marginal increase of their Ig secretion. The NAC-induced homoaggregation phenotype is paralleled with increased expressions of CD54, CD11a, CD27 and CD38. Mechanistically, a 24h exposure of B lymphocytes with NAC is sufficient to show strong inhibition of STAT3 phosphorylation. Besides, the treatment of B lymphocytes with the STAT3 inhibitor VI increases viability and decreases proliferation and secretion as in NAC-treated cells thus showing a role for STAT3 in these NAC-induced phenotypes. This study done in a human-based model provides new findings on how REDOX fluctuations may modulate CD40-activated B lymphocytes during immune response and provide additional hints on NAC its immunomodulatory functions.

Large-scale in vitro expansion of polyclonal human switched-memory B lymphocytes.

Polyclonal preparations of therapeutic immunoglobulins, namely intravenous immunoglobulins (IVIg), are essential in the treatment of immunodeficiency and are increasingly used for the treatment of autoimmune and inflammatory diseases. Currently, patients' accessibility to IVIg depends exclusively upon volunteer blood donations followed by the fractionation of pooled human plasma obtained from thousands of individuals. Presently, there are no in vitro cell culture procedures allowing the preparation of polyclonal human antibodies. All in vitro human therapeutic antibodies that are currently generated are based on monoclonal antibodies, which are mostly issued from genetic engineering or single cell antibody technologies. Here, we describe an in vitro cell culture system, using CD40-CD154 interactions, that leads to a 1×10(6)-fold expansion of switched memory B lymphocytes in approximately 50 days. These expanded cells secrete polyclonal IgG, which distribution into IgG(1), IgG(2), IgG(3) and IgG(4) is similar to that of normal human serum. Such in vitro generated IgG showed relatively low self-reactivity since they interacted moderately with only 24 human antigens among a total of 9484 targets. Furthermore, up to one liter of IgG secreting cells can be produced in about 40 days. This experimental model, providing large-scale expansion of human B lymphocytes, represents a critical step toward the in vitro production of polyclonal human IgG and a new method for the ex vivo expansion of B cells for therapeutic purposes.

Effective in vitro expansion of CD40-activated human B lymphocytes in a defined bovine protein-free medium.

CD40-CD154 interaction is used to culture human B lymphocytes, which are now viewed as effectors to potentially promote T lymphocyte response against malignant cells in cell-based therapy. Currently, the media used, based on bovine serum, are raising concerns for patient safety in such therapy. In this study, we have investigated whether human B lymphocytes could be cultured in the absence of bovine serum. Blood CD19(+) B lymphocytes were activated using interaction through CD40 in medium containing defined animals or human proteins and lipids, and were monitored during short-term periods (≤15 days). Conventional stem-cell medium and a medium containing human albumin instead of bovine albumin were tested. We observed that the response of B lymphocytes appeared influenced by lot-to-lot variability in low density lipoproteins (LDL). Nevertheless, B lymphocyte proliferation and secretion in serum-free and bovine protein-free media were quite similar to that of cells cultured in medium containing FBS. Our results show that CD40-activated B lymphocytes can be cultured for up to 15 days in a serum-free medium containing human albumin, LDL, α-tocopherol and chemically-defined lipids.

Bench tests of simple, handy ventilators for pandemics: performance, autonomy, and ergonomy.

BACKGROUND: It has been pointed out that in the wake of a virulent flu strain, patients with survivable illness will die from lack of resources unless more ventilators are made available. Numerous disaster-type ventilators are available, but few evaluations have been performed. OBJECTIVE: To compare simple, lightweight, and handy ventilators that could be used in the initial care of patients with respiratory distress. METHODS: We bench-tested 4 volume-cycled ventilators (Carevent ALS, EPV100, Pneupac VR1, and Medumat Easy) and 2 pressure-cycled ventilators (Oxylator EMX and VAR-Plus). We studied their general physical characteristics, sonometry, gas consumption, technical performance, ergonomy, and user-friendliness. With a test lung we assessed performance at F(IO(2)) of 0.50 and 1.0, set compliance of 30, 70, and 120 mL/cm H(2)O, and set resistance of 5, 10, and 20 cm H(2)O/L/s. To study user-friendliness and ergonomy we conducted, in randomized order, 7 or 8 objective, quantitative tests and 2 subjective tests. RESULTS: Compliance and resistance strongly affected tidal volume with the pressure-cycled ventilators (from 418 ± 49 mL to 1,377 ± 444 mL with the VAR-Plus, at the lowest pressure level), whereas the volume-cycled ventilators provided a consistent tidal volume in the face of changing test lung characteristics. CONCLUSIONS: We are concerned that the pressure-cycled ventilators did not provide a consistent tidal volume, and under certain conditions the volume delivered would be unsafe (too large or too small). Most of the volume-cycled ventilators proved to be technically efficient and reliable. Their reliability, portability, and ease of use could make them valuable in natural disasters and mass-casualty events.

Estimation of the number of CD154 molecules in membrane extracts used as a source of CD40 stimulation of human B lymphocytes.

The CD40-CD154 interaction is better exemplified by a rheostat than by an on-off switch, and variations in its intensity can play a role in the regulation of B lymphocyte activation following primary and/or secondary humoral immune response. The CD40-CD154 interaction is often studied in co-culture models using CD154+ adherent cells, which can be problematic when performing protein or gene analyses. The use of membrane extracts prepared from CD154+-transfected cells can eliminate possible interferences caused by the presence of contaminating feeder cells. Given the dose-response effect of CD154 on target B cells, it is important to measure the amount of CD154 when using soluble membranes. We hereby report a simple method, based on cytometry analysis, to estimate the relative number of CD154 molecules in membrane extracts, allowing reproducibility in human B-cell activation level.

Peripheral blood CD27+ IgG+ B cells rapidly proliferate and differentiate into immunoglobulin-secreting cells after exposure to low CD154 interaction.

In vitro CD40 stimulation of human B cells isolated from lymphoid organs is dominated by memory B cells undergoing faster proliferation and higher differentiation than naive B cells. In contrast, we previously reported that blood memory B cells mainly differentiate into immunoglobulin-secreting cells in response to CD40 stimulation. However, variations in CD40-CD154 interaction are now recognized to influence B-cell fate. In this study, we have compared the in vitro response of blood CD27(-) and CD27(-) IgG(-) to CD27(+) and CD27(+) IgG(+) B cells following low-density exposure to CD154 in the presence of a mixture of interleukin-2 (IL-2), IL-4 and IL-10. The evolution of these cell populations was monitored during initiation and following long-term stimulation. Over a 5-day period, CD27(+) B cells underwent differentiation into immunoglobulin-secreting cells more readily than CD27(-) cells, and CD27(+) IgG(+) B cells gave rise to a near homogeneous population of CD19(+) CD27(++) CD38(+) IgG(lo) cells capable of high immunoglobulin G (IgG) secretion. During the same period, CD27(-) IgG(-) B cells partially became CD19(++) CD27(-) CD38(-) IgG(++) cells but showed no IgG secretion. Long-term stimulation revealed that CD27(+) IgG(+) B cells retained a high expansion capacity and could maintain their momentum towards differentiation over naive B cells. In addition, long-term stimulation was driving CD27(-) IgG(-) and total CD19(+) B cells to evolve into similar CD27(+) and CD27(-) subsets, suggesting naive homeostatic proliferation. Overall, these results tend to reconcile memory B cells from blood and lymphoid organs regarding their preferential differentiation capacity compared to naive cells, and further suggest that circulating memory IgG(+) cells may be intrinsically prone to rapid activation upon appropriate stimulation.

Characterization of mononuclear cells remaining in the leukoreduction system chambers of apheresis instruments after routine platelet collection: a new source of viable human blood cells.

BACKGROUND: The yield of white blood cells (WBCs) extracted from whole-blood leukoreduction filters can be affected by the storage conditions and delay before filtration. Platelets (PLTs) collected with apheresis instruments (Trima Accel, Gambro BCT) are leukoreduced during the procedure on a fluidized particle bed in a leukoreduction chamber (LRS chamber). In this report, the residual cell content of these LRS chambers was characterized to determine whether it would be a valuable source of viable human blood cells. STUDY DESIGN AND METHODS: The content of LRS chambers was eluted by gravity, and peripheral blood mononuclear cells (PBMNCs) were purified on a Ficoll-Paque gradient. Analyses were performed before and after freezing. Proportions of CD3+, CD14+, CD16+, CD19+, CD34+, and CD45+ cells were determined by flow cytometry. The frequency of T cells expressing CD4, CD8, and CD27 and of B cells expressing immunoglobulin G (IgG), IgM, and CD27 was also determined. RESULTS: LRS chambers held approximately 10(9) CD45+ cells representing the normal proportions of CD3+, CD14+, CD16+, and CD19+ cell populations of PBMNCs. A small fraction of these CD45+ cells were CD34+CD38+ cells (0.3 +/- 0.2%). The viability of these cells, measured before and after freezing, was more than 95 percent. CONCLUSION: The residual cell content of Trima Accel LRS chambers recovered after PLT collection is a good source of viable monocytes and lymphocytes. These PBMNCs, containing CD3+, CD14+, CD16+, CD19+, and CD34+ cells can be frozen to prepare cell banks, which opens new avenues for utilization in several physiologic studies or even in cellular therapy applications.

B cell proliferation following CD40 stimulation results in the expression and activation of Src protein tyrosine kinase.

Resting normal human B cells express negligible c-src mRNA or Src protein tyrosine kinase; however, upon induction of proliferation, these cells express high levels of both mRNA and protein and show a concomitant increase in tyrosine kinase activity of immunoprecipitated Src. Src expression was most pronounced upon stimulation with CD154, and to a lesser extent CD70, Staphylococcus aureus, Cowan strain I and phorbol ester, and correlated with the activation of the cells. Transfection of cDNA for human wild-type or kinase-dead Src into Raji B cells resulted in an increase and decrease, respectively, of the cell numbers in culture, showing a direct correlation of proliferation to the expression of Src that was corroborated using anti-sense oligodeoxynucleotides and chemical inhibitors. Furthermore, the human B cell lines, Namalwa, Daudi and Raji express low levels of Src but express very high levels of Src after stimulation with CD154 that showed a correlation with increased activation. This is the first report of Src detectable in normal B cells. The finding that Src expression is inducible and correlates with stimulation by CD154 and the proliferation of the B cells suggests that Src may play a specific role in normal and transformed B cell activation/proliferation pathways mediated primarily through CD40 stimulation.

Differential responses of human B-lymphocyte subpopulations to graded levels of CD40-CD154 interaction.

Naïve and memory B-lymphocyte populations are activated by CD154 interaction through cell-surface CD40. This interaction plays an important role in the regulation of the humoral immune response, and increasing evidence indicates that fine variation in CD40 binding influences B lymphocytes, macrophages and dendritic cells in murine models. Here we have investigated whether and how variations in the intensity of the CD40-CD154 interaction could contribute to differential regulation of human B-lymphocyte populations. Proliferation and differentiation of B lymphocytes were monitored in response to graded levels of CD40 stimulation in the presence of interleukin (IL)-2, IL-4 and IL-10. Our results show that the level of CD154 binding to CD40 on B lymphocytes can directly influence the evolution of CD19(+) CD27(-) and CD19(+) CD27(+) cell populations. Furthermore, proliferation, global expansion of CD19(+) cells and emergence of CD38(++) CD138(+) cells, as well as immunoglobulin G (IgG) and IgM secretion, were affected by the level of exposure of B lymphocytes to CD154. These results suggest that the CD40-CD154 interaction is more like a rheostat than an on/off switch, and its variation of intensity may play a role in the regulation of B-lymphocyte activation following the primary and/or secondary humoral immune response.

Intravenous immunoglobulins induce the in vitro differentiation of human B lymphocytes and the secretion of IgG.

The therapeutic effects of intravenous immunoglobulins (IVIGs) in several autoimmune diseases are characterized by a decrease in pathologic autoantibody levels. Although little direct evidence has been reported in humans, the large repertoire of natural immunoglobulin G (IgG) antibodies in IVIGs is expected to be involved in the regulation of autoreactive B lymphocytes. In normal adult mice, IVIGs have been reported to modulate immature B cells as well as peripheral B lymphocytes through V-region connections. Studies with human serum also indicated that anti-idiotypic antibodies, present in IVIG preparations, could recognize both natural and pathologic autoantibodies. We have used an in vitro culture system to characterize the direct effect of IVIGs on human B lymphocytes. This in vitro culture system involves CD40 activation of B lymphocytes by its ligand CD154 in the presence of cytokines. In this system, addition of IVIGs decreased by 50% to 80% the expansion of B lymphocytes. This reduced expansion was due to a decrease in the proliferation rate. In addition, a portion of B lymphocytes was differentiated into IgG-secreting cells in the presence of IVIGs and the secreted IgGs were reactive with antigens such as nucleoprotamine, dsDNA, tetanus toxin, and human IgG F(ab')(2) fragments. These observations indicate that IVIGs can have direct effects on B lymphocytes and suggest that such IVIG regulation of B lymphocytes could be involved in the therapeutic effects of IVIGs in autoimmune diseases.