Leveraging metabolic modeling to identify functional metabolic alterations associated with COVID-19 disease severity.

OBJECTIVE: Since the COVID-19 pandemic began in early 2020, SARS-CoV2 has claimed more than six million lives world-wide, with over 510 million cases to date. To reduce healthcare burden, we must investigate how to prevent non-acute disease from progressing to severe infection requiring hospitalization. METHODS: To achieve this goal, we investigated metabolic signatures of both non-acute (out-patient) and severe (requiring hospitalization) COVID-19 samples by profiling the associated plasma metabolomes of 84 COVID-19 positive University of Virginia hospital patients. We utilized supervised and unsupervised machine learning and metabolic modeling approaches to identify key metabolic drivers that are predictive of COVID-19 disease severity. Using metabolic pathway enrichment analysis, we explored potential metabolic mechanisms that link these markers to disease progression. RESULTS: Enriched metabolites associated with tryptophan in non-acute COVID-19 samples suggest mitigated innate immune system inflammatory response and immunopathology related lung damage prevention. Increased prevalence of histidine- and ketone-related metabolism in severe COVID-19 samples offers potential mechanistic insight to musculoskeletal degeneration-induced muscular weakness and host metabolism that has been hijacked by SARS-CoV2 infection to increase viral replication and invasion. CONCLUSIONS: Our findings highlight the metabolic transition from an innate immune response coupled with inflammatory pathway inhibition in non-acute infection to rampant inflammation and associated metabolic systemic dysfunction in severe COVID-19.

Dysregulation of the CD4+ T cells lineage differentiation in dyslipidemic patients and impact of lipoprotein-apheresis treatment: A case study.

BACKGROUND AND AIM: Lipoprotein-apheresis (LA) is a therapeutic approach used against severe forms of dyslipidemia in patients who are non-responders or intolerant to pharmacological treatments. However, little is known about the potential pleiotropic effects of LA, particularly regarding the immune system and its regulation. Thus, in an attempt to analyse the potential effects of dyslipidemia and LA on the regulation of CD4+ T cells activation and lineage differentiation, we compared the CD4+ T cells cytokines secretion profiles of dyslipidemic patients before and after LA with the profiles observed in healthy donors. METHODS: CD4+ T cells were isolated from 5 LA patients and 5 healthy donors and activated with anti-CD3 or anti-CD3 + anti-CD46 antibodies. The supernatants were collected after 36 h incubation and levels of secreted cytokines analysed by flow cytometry. RESULTS: Our results revealed a deep remodelling of CD4+ T cells cytokines secretion patterns in dyslipidemic patients compared to healthy donors, as reflected by a 15 times higher IFN-γ secretion rate after CD3 + CD46 co-activation in dyslipidemic patients after LA compared to healthy subjects and 8 times higher after CD3 activation alone (p = 0.0187 and p = 0.0118 respectively). Moreover, we demonstrated that LA itself also modifies the phenotype and activation pattern of CD4+ T-cells in dyslipidemic patients. CONCLUSION: These observations could be of fundamental importance in the improvement of LA columns/systems engineering and in developing new therapeutic approaches regarding dyslipidemia and associated pathologies such as atherosclerosis and type 2 diabetes.

Adenylyl cyclase 8 is central to glucagon-like peptide 1 signalling and effects of chronically elevated glucose in rat and human pancreatic beta cells.

AIMS/HYPOTHESIS: Glucose and incretins regulate beta cell function, gene expression and insulin exocytosis via calcium and cAMP. Prolonged exposure to elevated glucose (also termed glucotoxicity) disturbs calcium homeostasis, but little is known about cAMP signalling. We therefore investigated long-term effects of glucose on this pathway with special regard to the incretin glucagon-like peptide 1 (GLP-1). METHODS: We exposed INS-1E cells and rat or human islets to different levels of glucose for 3 days and determined functional responses in terms of second messengers (cAMP, Ca(2+)), transcription profiles, activation of cAMP-responsive element (CRE) and secretion by measuring membrane capacitance. Moreover, we modulated directly the abundance of a calcium-sensitive adenylyl cyclase (ADCY8) and GLP-1 receptor (GLP1R). RESULTS: GLP-1- or forskolin-mediated increases in cytosolic calcium, cAMP-levels or insulin secretion were largely reduced in INS-1E cells cultured at elevated glucose (>5.5 mmol/l). Statistical analysis of transcription profiles identified cAMP pathways as major targets regulated by glucose. Quantitative PCR confirmed these findings and unravelled marked downregulation of the calcium-sensitive adenylyl cyclase ADCY8 also in rat and in human islets. Re-expression of ADCY8, but not of the GLP1R, recovered GLP-1 signalling in glucotoxicity in INS-1E cells and in rat islets. Moreover, knockdown of this adenylyl cyclase showed that GLP-1-induced cAMP generation, calcium signalling, activation of the downstream target CRE and direct amplification of exocytosis by cAMP-raising agents (evaluated by capacitance measurement) proceeds via ADCY8. CONCLUSIONS/INTERPRETATION: cAMP-mediated pathways are modelled by glucose, and downregulation of the calcium-sensitive ADCY8 plays a central role herein, including signalling via the GLP1R.

Human eosinophils and human high affinity IgE receptor transgenic mouse eosinophils express low levels of high affinity IgE receptor, but release IL-10 upon receptor activation.

FcepsilonRI expressed by human eosinophils is involved in IgE-mediated cytotoxicity reactions toward the parasite Schistosoma mansoni in vitro. However, because receptor expression is low on these cells, its functional role is still controversial. In this study, we have measured surface and intracellular expression of FcepsilonRI by blood eosinophils from hypereosinophilic patients and normal donors. The number of unoccupied receptors corresponded to approximately 4,500 Ab binding sites per cell, whereas 50,000 Ab binding sites per cell were detected intracellularly. Eosinophils from patients displayed significantly more unoccupied receptors than cells from normal donors. This number correlated to both serum IgE concentrations and to membrane-bound IgE. The lack of FcepsilonRI expression by mouse eosinophils has hampered further studies. To overcome this fact and experimentally confirm our findings on human eosinophils, we engineered IL-5 x hFcepsilonRIalpha double-transgenic mice, whose bone marrow, blood, spleen, and peritoneal eosinophils expressed FcepsilonRI levels similar to levels of human eosinophils, after 4 days culture with IgE in the presence of IL-5. Both human and mouse eosinophils were able to secrete IL-10 upon FcepsilonRI engagement. Thus, comparative analysis of cells from patients and from a relevant animal model allowed us to clearly demonstrate that FcepsilonRI-mediated eosinophil activation leads to IL-10 secretion. Through FcepsilonRI expression, these cells are able to contribute to both the regulation of the immune response and to its effector mechanisms.

Differential regulation of the overlapping Kaposi's sarcoma-associated herpesvirus vGCR (orf74) and LANA (orf73) promoters.

Similar to that of other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) lytic replication destroys the host cell, while the virus can persist in a latent state in synchrony with the host. During latency only a few genes are transcribed, and the question becomes one of what determines latent versus lytic gene expression. Here we undertake a detailed analysis of the latency-associated nuclear antigen (LANA [orf73]) promoter (LANAp). We characterized a minimal region that is necessary and sufficient to maintain high-level transcription in all tissues tested, including primary endothelial cells and B cells, which are the suspected natural host for KSHV. We show that in transient-transfection assays LANAp mimics the expression pattern observed for the authentic promoter in the context of the KSHV episome. Unlike other KSHV promoters tested thus far, LANAp is not affected by tetradecanoyl phorbol acetate or viral lytic cycle functions. It is, however, subject to control by LANA itself and cellular regulatory factors, such as p53. This is in contrast to the K14/vGCR (orf74) promoter, which overlaps LANAp and directs transcription on the opposite strand. We isolated a minimal cis-regulatory region sufficient for K14/vGCR promoter activity and show that it, too, mimics the regulation observed for the authentic viral promoter. In particular, we demonstrate that its activity is absolutely dependent on the immediate-early transactivator orf50, the KSHV homolog of the Epstein-Barr virus Rta transactivator.

Expression of a functional Fc epsilon RI on rat eosinophils and macrophages.

Besides its crucial role in type I hypersensitivity reactions, IgE is involved in anti-parasite immunity. This role has been clearly demonstrated in both human and rat schistosomiasis, but remains controversial in the mouse. Since the cellular distribution of the high affinity IgE receptor, Fc epsilon RI, differs in humans and mice, it might explain the differences in effector function of IgE between the two species. In humans, eosinophils and macrophages induce IgE-dependent cytotoxicity toward Schistosoma mansoni larvae, which involves Fc epsilon RI in the case of eosinophils. In the present study, we have investigated the expression and function of Fc epsilon RI in rat eosinophils and macrophages. We demonstrate, by flow cytometry, fluorescence microscopy, and western blot analysis, that in rats, as in humans, a functional alpha gamma 2 trimeric Fc epsilon RI is expressed on eosinophils and macrophages. We also show that these two cell types can induce IgE-mediated, Fc epsilon RI-dependent cellular cytotoxicity toward schistosomula. These results thus provide a molecular basis for the differences observed between rat and mouse regarding IgE-mediated anti-parasite immunity.

Selectin and Lewis(x) are required as co-receptors in antibody-dependent cell-mediated cytotoxicity of human eosinophils to Schistosoma mansoni schistosomula.

Killing of Schistosoma mansoni larvae by human eosinophils via antibody-dependent cell-mediated cytotoxicity (ADCC) mechanisms requires adherence between effector cells and parasite targets. The role of adhesion molecules in this mechanism was investigated using blocking monoclonal antibodies (mAb) and soluble ligands. We show that, along with the Mac-1 alpha chain, interactions between selectins and LewisX-related structures, both expressed by eosinophils and parasite targets, play a critical part in the antibody-dependent cytotoxic function of eosinophils. To further elucidate the interactions between adhesion molecules and eosinophil Fc receptors, ADCC was performed with IgG1 or IgA mAb. We found that mAb directed against Mac-1 alpha chain or against LewisX could significantly inhibit the IgG1-, but not IgA cytotoxicity. This result might be explained, at least in part, by the inhibitory effect of these mAb on the release by eosinophils of eosinophil cationic protein, one of the major mediators involved in target killing. Taken together, these results suggest novel interactions between Fc receptors and selectins and LewisX-related structures which might act as co-receptors for eosinophil-mediated cytotoxicity.

Inhibitory effects of lodoxamide on eosinophil activation.

Recent reports describe the beneficial use of lodoxamide, an anti-allergic compound, for the treatment of asthma and allergic conjunctivitis. Lodoxamide is known as a mast cell stabilizer, however, the association of a significant clinical improvement with a specific decrease in eosinophil infiltrate suggested possible direct effects of lodoxamide on eosinophils. The chemotactic response of eosinophils to fMLP as well as to IL-5, in vitro, was very significantly and dose-dependently inhibited by Lodoxamide. Lodoxamide was also able to strongly inhibit the release of eosinophil peroxidase after IgA-dependent activation and, to a lesser extent, the release of eosinophil cationic protein and eosinophil-derived neurotoxin. Moreover, the release of cytotoxic mediators evaluated in an antibody-dependent cytotoxicity assay against parasitic targets was also significantly reduced, not only in the case of human eosinophils but also in a rat eosinophil-mast cell model of cytotoxicity. Taken together, these results indicate that lodoxamide can exert potent inhibitory effects on eosinophil activation in vitro combined with a strong inhibition of eosinophil attraction, leading therefore to a reduction in their pathological potential in vivo.

Role of adhesion molecules of the selectin-carbohydrate families in antibody-dependent cell-mediated cytoxicity to schistosome targets.

The role of adhesion molecules in antibody-dependent cell-mediated cytotoxicity (ADCC) of macrophages toward the extracellular parasite Schistosoma mansoni was investigated by using 1) a panel of blocking mAbs directed against adhesion molecules and 2) different soluble ligands as candidate inhibitors of ADCC. The results show that the beta2-integrin Mac-1 (CD11b/CD18), L-selectin (CD62-L), and the carbohydrate determinant sialyl Lewis(x) (sLe(x); sCD15) are required for macrophage effector function toward schistosomula targets. On the other hand, the parasite counter-receptors involved in ADCC were found to share common motifs with the mammalian selectin-carbohydrate families. One family of parasite receptor(s) involved in ADCC carries the Lewis(x) (Le(x); CD15) carbohydrate structure, whereas a second family of receptor(s) appears to display selectin-like properties with affinity for the sLe(x) tetrasaccharide. Immunostaining experiments confirmed that schistosomula express on their surface hostlike molecules recognized by anti-Le(x) (CD15) and by anti-human E-selectin (CD62-E) mAbs. The double receptor-ligand interaction between macrophages and parasite targets provides new insights into the biologic roles of selectins and Le(x)-related structures in immunity against helminthic parasites.

From allergy to schistosomes: role of Fc receptors and adhesion molecules in eosinophil effector function.

The dual function of eosinophils has been evidenced in protective immunity against parasites as well as in pathological manifestations during allergic disorders. We have demonstrated that a new class of IgE receptors, Fc epsilon RII/CD23, was involved in the functional duality of eosinophils and other proinflammatory cells. More recently, we have shown that Fc epsilon RI, the high affinity IgE receptor thought to be only expressed by basophils and most cells, was involved in eosinophil-mediated cytotoxicity against schistosomes as well as in mediator release. These results favour the view that both IgE and its receptors have been primarily associated to a protective immune response, rather than to pathology. Not only IgE receptors but also members belonging to the family of adhesion molecules can participate as co-receptors in eosinophil effector function. The inhibitory role of monoclonal antibodies to Lewis(X) (Le(X) CD15) or to selectins in eosinophil-mediated cytotoxicity towards schistosomes and the detection of Le(X) and selectin-like molecules on schistosomula surface indicate a double interaction mediated by selectins and their carbohydrate ligands between eosinophils and schistosomula. These results suggest new functions for these adhesion molecules, previously known to be involved mainly in cell infiltration.

Human neutrophils express immunoglobulin E (IgE)-binding proteins (Mac-2/epsilon BP) of the S-type lectin family: role in IgE-dependent activation.

It has been suggested that neutrophils may be involved in the late-phase reaction of immunoglobulin E (IgE)-dependent hypersensitivity states. However, the identity of neutrophil-associated molecules inducing the release of mediators remains unclear. In this report, we demonstrate that human neutrophils from normal donors or from patients with inflammatory disorders could bind myeloma IgE proteins, especially after desialylation. Northern blot, immunoprecipitation, and flow cytometry analyses revealed that neutrophils did not express Fc epsilon RII/CD23, but rather Mac-2/epsilon binding protein (BP), belonging to the S-type lectin family. Similarly to IgA used as positive control, myeloma IgE proteins, as well as polyclonal IgE antibodies with or without antibody specificity, were both capable of inducing a neutrophil respiratory burst. Anti-Mac-2 but not anti-CD23 mAb strongly decreased the IgE-dependent activation of neutrophils, induced either by the specific antigen or by anti-IgE antibodies. These findings open new perspectives on the functional role of neutrophils in IgE-associated diseases including allergic states or parasitic infections.

[Presence and functional role of an epitope of the second receptor for IgE (FcE RII) in mast cells and basophils in the rat]. / Présence et rôle fonctionnel d'un épitope du second récepteur pour l'IgE (FcE RII) sur les mastocytes et les basophiles de rat.

Mast cells and basophils express the high affinity IgE receptor (FcERI) whereas the low affinity receptor for monomeric IgE (FcE RII) is present on macrophages, lymphocytes, eosinophils, platelets and Langerhans cells. Recent studies confirmed that the two receptors were totally distinct. The present work shows that a monoclonal antibody (BB10), able to bind to FcE RII on different cell populations, interacts with FcE RI expressing cells: rat peritoneal mast cells and a rat basophilic leukemia cell line (RBL 2 H 3). The structure recognized by BB10 is distinct from FcE RI and modulates the IgE-dependent histamine release. In conclusion, it appears that a common epitope with FcE RII is present on mast cells and basophils and that a functional relation might exist between this structure and FcE RI.

[Existence and function of a receptor for immunoglobulin A on human eosinophils]. / Existence et fonctions d'un récepteur pour l'immunoglobuline A sur les éosinophiles humains.

The existence of receptors for immunoglobulin A on human eosinophils is demonstrated by flow cytofluorometry. Between 5 and 60% of eosinophils purified from peripheral blood of hypereosinophilic patients are able to bind monomeric serum IgA. The addition of antihuman IgA antibodies to surface IgA-bearing eosinophils induces the exocytosis of peroxidase contained in the granules suggesting a cell activation due to IgA receptors. The inhibition of antiparasitic cytotoxicity by eosinophils preincubated with IgA under a polymeric form only, indicates the low affinity of IgA receptors as well as their participation in the effector function of eosinophils.

Functional role of the alpha-chain of complement receptor type 3 in human eosinophil-dependent antibody-mediated cytotoxicity against schistosomes.

The participation of complement receptor type 3 (CR3) in antibody-dependent effector function of human eosinophils against parasites was studied by using monoclonal antibodies directed against various surface molecules. Both adherence and cytotoxicity of hypodense eosinophils to IgE-coated schistosomula of Schistosoma mansoni were strongly inhibited by anti-CR3 antibodies (OKM1 or Mo1). The specificity of the inhibitory effect for the alpha-chain of CR3 was shown by the lack of inhibition of anti-beta-chain or anti-LFA1 alpha-chain monoclonal antibodies, although these antigens were expressed on human eosinophils. These results associated to previous works on IgE receptors demonstrate that both receptor for Fc fragments of IgE and CR3 are essential in IgE-dependent cytotoxicity of human eosinophils. Flow microfluorometry analysis revealed that hypodense eosinophils were more intensively stained by OKM1 antibodies than the normodense populations. In the case of IgG-mediated cytotoxicity by normodense eosinophils, only the enhancement of cytotoxicity due to monokine activation was inhibited by anti-CR3 alpha-chain antibodies. These findings suggest an increased expression of CR3 on eosinophils after activation either in vivo or in vitro. The participation of CR3 in IgE-mediated cytotoxicity against schistosomes was also required in the case of blood monocytes but not for platelet-mediated killing, which does not require prior adherence. The biologic role of CR3 is therefore extended to effector mechanisms involving eosinophils and two different isotypes of antibodies and possibly implied in immunity against schistosomes.

Functional study of a monoclonal antibody to IgE Fc receptor (Fc epsilon R2) of eosinophils, platelets, and macrophages.

An IgM mAb (BB10) was produced by immunization of mice with human eosinophils purified according to their abnormal low density ("hypodense" cells), and previously shown to exhibit increased IgE-dependent antiparasite cytotoxicity. This BB10 antibody, selected for positive fluorescence staining of hypodense blood or lung eosinophils and low or negative staining of normodense eosinophils or neutrophils, could strongly inhibit IgE-dependent cytotoxicity of human eosinophils and platelets. The specificity for the IgE Fc receptor was suggested by the high levels of inhibition of IgE rosettes formed by eosinophils after incubation with the purified IgM fraction of BB10, whereas other receptors (Fc gamma R, CR1) were not affected. On the other hand, BB10, able to inhibit rat eosinophil Fc epsilon R, did not react with the IgE Fc receptor on mast cells or basophils. A technique using radioiodinated BB10 allowed us to quantify the specific binding of BB10 to human eosinophils and platelets. Competition experiments revealed a crossinhibition between the binding of BB10 and IgE, suggesting the specificity of BB10 for the IgE binding site of eosinophil, platelet, and monocyte Fc epsilon R. Three proteins having extrapolated Mr of 32,000, 43,000-45,000, and 97,000 were found in the platelet extract eluted from a BB10 or from an IgE immunosorbent column. These findings confirm the similarities between IgE Fc receptors on human eosinophils, platelets, and macrophages, already observed with polyclonal antibodies directed against the B lymphocyte Fc epsilon receptor. They suggest, moreover, that the mAb BB10 can represent a good reagent for further investigations on the structure and the functions of this IgE Fc receptor (Fc epsilon R2).

Analysis of visual scanning patterns of pistol shooters.

Visual behaviour of pistol shooters of various levels of experience was studied using a video-oculometric technique to record the direction of gaze. A spatio-temporal analysis of the fixation patterns of subjects (five international pistol shooters and five international pentathletes) found that patterns were related to the level of skill. The pentathletes, whose level was lower, positioned their gaze on the weapon sight and visually followed the movement of the weapon towards the target. The pistol shooters appeared to use negative feedback in bringing up their weapons, so reducing the angle formed by the weapon, target and gaze. They fixed their gaze directly on the target or between the target and the weapon. Observations showed a prolonged time of attack (means = 1.107 s vs 0.558 s; P less than 0.001) and a shorter stabilization time (means = 0.714 s vs 1.216 s; P less than 0.001) for the most expert shooters. The psycho-physiological mechanisms which were the basis of these patterns and the reasons why shooters chose them are discussed.

Interactions between eosinophils and antibodies: in vivo protective role against rat schistosomiasis.

An original protocol of cell transfer from Schistosoma mansoni-infected rats to normal recipient rats is used to investigate the protective role of phagocytic cell populations, described as effector cells in vitro, against a challenge infection with S. mansoni. Nonadherent, eosinophil-enriched and -adherent, macrophage-rich cell preparations, injected via intradermal and subcutaneous routes at the precise site of exposure to cercariae, were able to significantly protect the recipient rats. The time-course study of this protective effect according to the time after infection of donor rats revealed that eosinophils were the major cell population involved in the early phase of infection (4 to 5 weeks), whereas macrophages could also be incriminated thereafter. A rosette assay using anti-immunoglobulin-coated erythrocytes indicated a sequence of the various antibody isotypes under study (IgG1, IgG2a, IgE) on the eosinophil surface, during the course of infection. As previously shown in vitro, cytophilic antibodies seemed to participate in the protective effect of eosinophils, since eosinophil-enriched cells from normal rats, sensitized in vitro with immune complexes present in infected rat serum, could also confer significant protection. These observations establish therefore the relevance between our previous in vitro studies and rat resistance to a challenge infection with S. mansoni, underlining the major role played by the interaction between antibodies and phagocytic cells (eosinophils and macrophages).

[Complement markers Bf and C4 in multiple sclerosis]. / Les marqueurs Bf et C4 du complément dans la sclérose en plaques.

Some immunogenetic HLA markers are significantly correlated with multiple sclerosis, e.g.: the antigens B7, DR2, and the associations B7-DR2, A3-B7-DR2. In addition, the polymorphism of the allotypes Bf and C4 is also controlled by chromosome 6; a study of these markers is therefore of interest. The study of Bf and C4 in multiple sclerosis included a population of genotyped unrelated patients: 50 patients for Bf markers and 41 for C4A and C4B markers. This study revealed an over-representation of allotype S and homozygous BfSS in multiple sclerosis. BfSS homozygote was significantly more frequent in the B7 negative and/or DR2 negative patients, i.e. when the risk markers per se were absent. No correlation could be evidenced with the remittent or progressive character of the disease. These data, obtained from the study of C4, are still preliminary ones. The results found with the Bf markers confirm the existence of a genetic factor in multiple sclerosis and suggest that the susceptibility gene of the disease could be closer to locus Bf than to locus DR.

Pickup of visual information by the pilot during a ground control approach in a fighter aircraft simulator.

Before providing the new single-seat fighter aircraft with selective visual information display systems, it is necessary to conduct new studies of the visual behavior of pilots flying these aircraft in order to determine the nature of information to be displayed. The authors describe a modified NAC Eye Mark recorder which can be used in tight spaces without any interfering light source and given an example of its use in an experiment conducted in a Mirage III R training simulator. The reported experiment was designed to analyse the visual behavior of 12 pilots of four different qualification levels who flew a ground control approach (GCA) test each day for five consecutive days. The results show that the pilot's visual behavior is stable, both on an intra- and inter-individual basis. In addition, it is possible to classify the control panel instruments as a function of the number of times and length of time they are checked.