The spectrum of biochemical alterations associated with organ dysfunction and inflammatory status and their association with disease outcomes in severe COVID-19: A longitudinal cohort and time-series design study.

BACKGROUND: In patients with severe COVID-19, no data are available on the longitudinal evolution of biochemical abnormalities and their ability to predict disease outcomes. METHODS: Using a retrospective, longitudinal cohort study design on consecutive patients with severe COVID-19, we used an extensive biochemical dataset of serial data and time-series design to estimate the occurrence of organ dysfunction and the severity of the inflammatory reaction and their association with acute respiratory failure (ARF) and death. FINDINGS: On the 162 studied patients, 1151 biochemical explorations were carried out for up to 59 biochemical markers, totaling 15,260 biochemical values. The spectrum of biochemical abnormalities and their kinetics were consistent with a multi-organ involvement, including lung, kidney, heart, liver, muscle, and pancreas, along with a severe inflammatory syndrome. The proportion of patients who developed an acute kidney injury (AKI) stage 3, increased significantly during follow-up (0·9%, day 0; 21·4%, day 14; P<0·001). On the 20 more representative biochemical markers (>250 iterations), only CRP >90 mg/L (odds ratio [OR] 6·87, 95% CI, 2·36-20·01) and urea nitrogen >0·36 g/L (OR 3·91, 95% CI, 1·15-13·29) were independently associated with the risk of ARF. Urea nitrogen >0·42 g/L was the only marker associated with the risk of COVID-19 related death. INTERPRETATION: Our results point out the lack of the association between the inflammatory markers and the risk of death but rather highlight a significant association between renal dysfunction and the risk of COVID-19 related acute respiratory failure and death.

Long-term ACE Inhibitor/ARB Use Is Associated With Severe Renal Dysfunction and Acute Kidney Injury in Patients With Severe COVID-19: Results From a Referral Center Cohort in the Northeast of France.

BACKGROUND: In patients with severe coronavirus disease 2019 (COVID-19), data are scarce and conflicting regarding whether chronic use of angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) influences disease outcomes. In patients with severe COVID-19, we assessed the association between chronic ACEI/ARB use and the occurrence of kidney, lung, heart, and liver dysfunctions and the severity of the inflammatory reaction as evaluated by biomarkers kinetics, and their association with disease outcomes. METHODS: We performed a retrospective longitudinal cohort study on consecutive patients with newly diagnosed severe COVID-19. Independent predictors were assessed through receiver operating characteristic analysis, time-series analysis, logistic regression analysis, and multilevel modeling for repeated measures. RESULTS: On the 149 patients included in the study 30% (44/149) were treated with ACEI/ARB. ACEI/ARB use was independently associated with the following biochemical variations: phosphorus >40 mg/L (odds ratio [OR], 3.35, 95% confidence interval [CI], 1.83-6.14), creatinine >10.1 mg/L (OR, 3.22, 2.28-4.54), and urea nitrogen (UN) >0.52 g/L (OR, 2.65, 95% CI, 1.89-3.73). ACEI/ARB use was independently associated with acute kidney injury stage ≥1 (OR, 3.28, 95% CI, 2.17-4.94). The daily dose of ACEI/ARB was independently associated with altered kidney markers with an increased risk of +25 to +31% per each 10 mg increment of lisinopril-dose equivalent. In multivariable multilevel modeling, UN >0.52 g/L was independently associated with the risk of acute respiratory failure (OR, 3.54, 95% CI, 1.05-11.96). CONCLUSIONS: Patients chronically treated with ACEI/ARB who have severe COVID-19 are at increased risk of acute kidney injury. In these patients, the increase in UN associated with ACEI/ARB use could predict the development of acute respiratory failure.

Efficacy of low dose nitisinone in the management of alkaptonuria.

AIM: To study the efficacy of low dosage of nitisinone in alkaptonuria. BACKGROUND: Alkaptonuria (AKU) is a rare genetic disease which induces deposition of homogentisic acid (HGA) in connective inducing premature arthritis, lithiasis, cardiac valve disease, fractures, muscle and tendon ruptures and osteopenia. Recent studies showed that nitisinone decreases HGA and is a beneficial therapy in AKU. This treatment induces an increase in tyrosine levels which can induces adverse effects as keratopathy. METHODS: We described the evolution HGA excretion and tyrosine evolution in 3 AKU patients treated by very low dosage of nitisinone with regards to their daily protein intakes. We also described the first pregnancy in an AKU patient treated by nitisinone. RESULTS: We found mild clinical signs of alkaptonuria on vertebra MRI in two young adults and homogentisate deposition in teeth of a 5 years old girl. Very low dose of nitisinone (10% of present recommended dose: 0.2 mg/day) allowed to decrease homogentisic acid by >90% without increasing tyrosine levels above 500 µmol/ in these three patients. INTERPRETATIONS: The analysis of the follow-up data shows that, in our three patients, a low-dosage of nitisinone is sufficient to decrease urinary HGA without increasing plasma tyrosine levels above the threshold of 500 µmol/L.

GC-MS-based urine metabolic profiling of autism spectrum disorders.

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders resulting from multiple factors. Diagnosis is based on behavioural and developmental signs detected before 3 years of age, and there is no reliable biological marker. The purpose of this study was to evaluate the value of gas chromatography combined with mass spectroscopy (GC-MS) associated with multivariate statistical modeling to capture the global biochemical signature of autistic individuals. GC-MS urinary metabolic profiles of 26 autistic and 24 healthy children were obtained by liq/liq extraction, and were or were not subjected to an oximation step, and then were subjected to a persilylation step. These metabolic profiles were then processed by multivariate analysis, in particular orthogonal partial least-squares discriminant analysis (OPLS-DA, R(2)Y(cum) = 0.97, Q(2)(cum) = 0.88). Discriminating metabolites were identified. The relative concentrations of the succinate and glycolate were higher for autistic than healthy children, whereas those of hippurate, 3-hydroxyphenylacetate, vanillylhydracrylate, 3-hydroxyhippurate, 4-hydroxyphenyl-2-hydroxyacetate, 1H-indole-3-acetate, phosphate, palmitate, stearate, and 3-methyladipate were lower. Eight other metabolites, which were not identified but characterized by a retention time plus a quantifier and its qualifier ion masses, were found to differ between the two groups. Comparison of statistical models leads to the conclusion that the combination of data obtained from both derivatization techniques leads to the model best discriminating between autistic and healthy groups of children.

Phospholipase D activation mediates cobalamin-induced downregulation of Multidrug Resistance-1 gene and increase in sensitivity to vinblastine in HepG2 cells.

Failure of cancer chemotherapy due to multidrug resistance is often associated with altered Multidrug Resistance-1 gene expression. Cobalamin is the cofactor of methionine synthase, a key enzyme of the methionine cycle which synthesizes methionine, the precursor of cell S-adenosyl-methionine synthesis. We previously showed that cobalamin was able to down-regulate Multidrug Resistance-1 gene expression. Herein we report that this effect occurs through cobalamin-activation of phospholipase D activity in HepG2 cells. Cobalamin-induced down-regulation of Multidrug Resistance-1 gene expression was similar to that induced by the phospholipase D activator oleic acid and was negatively modulated by the phospholipase D inhibitor n-butanol. Cobalamin increased cell S-adenosyl-methionine content, which is the substrate for phosphatidylethanolamine-methyltransferase-dependent phosphatidylcholine production. We showed that cobalamin-induced increase in cell phosphatidylcholine production was phosphatidylethanolamine-methyltransferase-dependent. Oleic acid-dependent activation of phospholipase D was accompanied by an increased sensitivity to vinblastine of HepG2 cells while n-butanol enhanced the resistance of the cells to vinblastine. These data indicate that cobalamin mediates down-regulation of Multidrug Resistance-1 gene expression through increased S-adenosyl-methionine and phosphatidylcholine productions and phospholipase D activation. This points out phospholipase D as a potential target to down-regulate Multidrug Resistance-1 gene expression for improving chemotherapy efficacy.

Impaired interleukin-8 chemokine secretion by staphylococcus aureus-activated epithelium and T-cell chemotaxis in cystic fibrosis.

Staphylococcus aureus is frequently isolated from lungs of patients with cystic fibrosis (CF). Upon lung infection with S. aureus, airway epithelial cells (AEC) produce high levels of chemokines that enhance T-cell chemotaxis. Although the number of lymphocytes is increased in the airways and bronchoalveolar lavage fluid of patients with CF, the mechanisms responsible for their accumulation and the role of S. aureus in this process are largely unknown. This study investigated early S. aureus impact on chemokine secretion by CF epithelial cells and chemotaxis of CF T cells. CF and non-CF AEC were grown in a cell culture model and apically stimulated with S. aureus. Supernatants were quantified for chemokine secretions and assayed for T-cell chemotaxis. CF AEC secreted constitutively larger amounts of IL-8, GROalpha, MIG, MIP-3beta, and MCP-1 than non-CF epithelial cells. S. aureus interaction with epithelial cells increased chemokine production by non-CF cells but had no effect on CF cells. Chemotaxis of T cells derived from patients with CF was greater than that of T cells from subjects without CF. Moreover, there were more CF T cells expressing CXCR1 as compared with non-CF T cells. Under our experimental conditions, inhibition of IL-8 or its receptor CXCR1 resulted in a considerable decrease in T-cell chemotaxis (up to 80%). These data suggest that IL-8 and its receptor CXCR1 are key players in the chemotaxis of CF T cells and could be used as targets to develop therapies for CF.

Evidence of cataplerosis in a patient with neonatal classical galactosemia presenting as citrin deficiency.

Classical galactosemia is an autosomal recessive disorder caused by a deficiency of the enzyme galactose-1-phosphate uridyltransferase. Undoubtedly, some of the short term complications are linked to the toxic effects of the accumulated abnormal metabolites (galactose-1-phosphate and galactitol). However, the physiopathology of neonatal liver failure remains unclear. We report the case of a 7-week-old girl who was first diagnosed with liver failure, hypoprotidaemia, ascites and generalized edemas. High citrulline (293 micromol/L), on initial plasma amino acid, suggested the diagnosis of citrin deficiency. As the citric acid cycle intermediates were non-detectable (oxoglutarate, succinate and citrate), a cataplerotic state was suspected. As a result, citrate (as an anaplerotic treatment) induced a clear improvement in her liver function. Four weeks later, this patient was switched to a galactose-free formula (as recommended in citrin deficiency with galactosemia) and her pathological status returned to normal. Citrin deficiency was later ruled out by molecular biology studies; then we reintroduced a galactose-containing formula which re-evoked rapidly vomiting, galactose aversion and hepatic cytolysis and the diagnosis of classical galactosemia was established. Our case clearly shows that cataplerosis could play a role in the pathophysiology of the neonatal liver disease observed in classical galactosemia.

Cobalamin potentiates vinblastine cytotoxicity through downregulation of mdr-1 gene expression in HepG2 cells.

BACKGROUND: P-glycoprotein (Pgp), produced by multidrug resistance-1 gene (mdr-1), is a main mechanism developed by cancer cells to guard against anti-cancer drugs. Alterations of DNA methylation of the mdr-1 gene promoter are known to be linked to mdr-1 gene expression and are probably related to intracellular S-adenosyl-methionine. We here used HepG2 cells to determine the role of the methionine cycle (through the use of the Methionine-Synthase (MS) cofactor, cobalamin) on mdr-1 gene expression. METHODS: Semiquantitative RT-PCR of mdr-1 gene, cellular retention of rhodamine-123, and vinblastine cytotoxicity were carried out on cells cultivated with and without cobalamin. Methylation status of the mdr-1 gene promoter was determined by methylation-specific PCR. RESULTS: Addition of cobalamin to the cells led to an increase in MS activity, to a significant decrease in mdr-1 gene expression which is correlated to an increase in retention of the Pgp substrate Rhodamine 123. Furthermore, cobalamin potentiated cell sensitivity to vinblastine to the same range as that of the Pgp blocker verapamil and prevented methotrexate-induced up-regulation of mdr-1 gene expression. However, no modification in methylation of the mdr-1 gene promoter was observed. CONCLUSION: Cobalamin downregulates mdr-1 gene expression, as well as Pgp expression and function, and significantly increases cytotoxicity of vinblastine. The identification of this novel way of diminishing cellular resistance to the chemotherapeutic agent vinblastine holds promises of leading to better treatments for cancer patients.

Inhibition by TNF-alpha and IL-4 of cationic lipid mediated gene transfer in cystic fibrosis tracheal gland cells.

BACKGROUND: In vivo, tracheal gland serous cells highly express the cystic fibrosis transmembrane conductance regulator (cftr) gene. This gene is mutated in the lethal monogenic disease cystic fibrosis (CF). Clinical trials in which the human CFTR cDNA was delivered to the respiratory epithelia of CF patients have resulted in weak and transient gene expression. METHODS AND RESULTS: As CF is characterized by mucus inspissation, airway infection, and severe inflammation, we tested the hypothesis that inflammation and especially two cytokines involved in the Th1/Th2 inflammatory response, interleukin 4 (IL-4) and TNFalpha, could inhibit gene transfer efficiency using a model of human CF tracheal gland cells (CF-KM4) and Lipofectamine reagent as a transfection reagent. The specific secretory defects of CF-KM4 cells were corrected by Lipofectamine-mediated human CFTR gene transfer. However, this was altered when cells were pre-treated with IL-4 and TNFalpha. Inhibition of luciferase reporter gene expression by IL-4 and TNFalpha pre-treated CF-KM4 cells was measured by activity and real-time RT-PCR. Both cytokines induced similar and synergistic inhibition of transgene expression and activity. This cytokine-mediated inhibition could be prevented by anti-inflammatory agents such as glucocorticoids but not by non-steroidal (NSAI) agents. CONCLUSIONS: This data suggests that an inflammatory context generated by IL-4 and TNFalpha can inhibit human CFTR gene transfer in CF tracheal gland cells and that glucocorticoids may have a protecting action.

Live Staphylococcus aureus and bacterial soluble factors induce different transcriptional responses in human airway cells.

To characterize the response of respiratory epithelium to infection by Staphylococcus aureus (S. aureus), human airway cells were incubated for 1 to 24 h with a supernatant of a S. aureus culture (bacterial supernatant), then profiled with a pangenomic DNA microarray. Because an upregulation of many genes was noticed around 3 h, three independent approaches were then used to characterize the host response to a 3-h contact either with bacterial supernatant or with live bacteria: 1) a DNA microarray containing 4,200 sequence-verified probes, 2) a semiquantitative RT-PCR with a set of 537 pairs of validated primers, or 3) ELISA assay of IL-8, IL-6, TNFalpha, and PGE(2). Among others, Fos, Jun, and EGR-1 were upregulated by the bacterial supernatant and by live bacteria. Increased expression of bhlhb2 and Mig-6, promoter regions which harbor HIF responding elements, was explained by an increased expression of the HIF-1alpha protein. Activation of the inducible form of cyclooxygenase, COX-2, and of the interleukins IL-1, IL-6, and IL-8, as well as of the NF-kappaB pathway, was observed preferentially in cells in contact with bacterial supernatant. Early infection was characterized by an upregulation of anti-apoptotic genes and a downregulation of pro-apoptotic genes. This correlated with a necrotic, rather than apoptotic cell death. Overall, this first global description of an airway epithelial infection by S. aureus demonstrates a larger global response to bacterial supernatant (in term of altered genes and variation factors) than to exponentially growing live bacteria.

Homocysteine increases methionine synthase mRNA level in Caco-2 cells.

BACKGROUND: Methionine synthase (MTR) synthesizes methionine from homocysteine, using cobalamin as a cofactor and 5-methyltetrahydrofolate as a cosubstrate. AIM: To determine the influence of homocystine (Hcy, dimer of homocysteine) in the presence of either cobalamin or methionine on the transcription and the activity of methionine synthase in Caco-2, a human adenocarcinoma cell line. METHODS: Methionine synthase activity and quantification of its mRNA by real-time RT-PCR were determined in cells cultivated under four differents conditions: Hcy with cobalamin (Hcy+ Cbl+), Hcy with methionine (Hcy+Met+), methionine with Cbl (Met+ Cbl+) and methionine only (Met+). RESULTS: Activity (nmol/h/mg protein) was maximal in cells cultivated in Hcy+Cbl+ (2.45 +/- 0.35), compared to cells cultivated in Hcy+Met+ (0.18 +/- 0.01, p<0.001), in Met+ Cbl+ (1.60 +/- 0.06, p<0.05), and in Met+ (0.40 +/- 0.05, p<0.001), suggesting an adaptation of the cells to requirement in synthesized methionine. The mRNA level of MTR in Hcy+ Cbl+ and Hcy+Met+ (2.82 +/-0.49 and 3.33 +/- 0.48 AU, respectively ) was about 2.5 / 3.0-fold higher than that in Met+ Cbl+ and in Met+ (1.00 +/-0.13 and 1.20 +/-0.20 AU, respectively, p<0.001). CONCLUSION: Methionine synthase expression of Caco-2 cell is under a transcriptional control influenced by Hcy.

Gene transduction and cell entry pathway of fiber-modified adenovirus type 5 vectors carrying novel endocytic peptide ligands selected on human tracheal glandular cells.

Monolayers of cystic fibrosis transmembrane conductance regulator (CFTR)-deficient human tracheal glandular cells (CF-KM4) were subjected to phage biopanning, and cell-internalized phages were isolated and sequenced, in order to identify CF-KM4-specific peptide ligands that would confer upon adenovirus type 5 (Ad5) vector a novel cell target specificity and/or higher efficiency of gene delivery into airway cells of patients with cystic fibrosis (CF). Three different ligands, corresponding to prototypes of the most represented families of phagotopes recovered from intracellular phages, were designed and individually inserted into Ad5-green fluorescent protein (GFP) (AdGFP) vectors at the extremities of short fiber shafts (seven repeats [R7]) terminated by scissile knobs. Only one vector, carrying the decapeptide GHPRQMSHVY (abbreviated as QM10), showed an enhanced gene transduction of CF-KM4 cells compared to control nonliganded vector with fibers of the same length (AdGFP-R7-knob). The enhancement in gene transfer efficiency was not specific to CF-KM4 cells but was observed in other mammalian cell lines tested. The QM10-liganded vector was referred to as AdGFP-QM10-knob in its knobbed version and as AdGFP-QM10 in its proteolytically deknobbed version. AdGFP-QM10 was found to transduce cells with a higher efficiency than its knob-bearing version, AdGFP-QM10-knob. Consistent with this, competition experiments indicated that the presence of knob domains was not an absolute requirement for cell attachment of the QM10-liganded vector and that the knobless AdGFP-QM10 used alternative cell-binding domains on its capsid, including penton base capsomer, via a site(s) different from its RGD motifs. The QM10-mediated effect on gene transduction seemed to take place at the step of endocytosis in both quantitative and qualitative manners. Virions of AdGFP-QM10 were endocytosed in higher numbers than virions of the control vector and were directed to a compartment different from the early endosomes targeted by members of species C Ad. AdGFP-QM10 was found to accumulate in late endosomal and low-pH compartments, suggesting that QM10 acted as an endocytic ligand of the lysosomal pathway. These results validated the concept of detargeting and retargeting Ad vectors via our deknobbing system and redirecting Ad vectors to an alternative endocytic pathway via a peptide ligand inserted in the fiber shaft domain.

Dynamic interaction between airway epithelial cells and Staphylococcus aureus.

Staphylococcus aureus is a major cause of pulmonary infection, particularly in cystic fibrosis (CF) patients. However, few aspects of the interplay between S. aureus and host airway epithelial cells have been investigated thus far. We investigated by videomicroscopy the time- and bacterial concentration-dependent (10(4), 10(6), and 10(8) CFU/ml) effect of S. aureus on adherence, internalization, and the associated damage of the airway epithelial cells. The balance between the secretion by S. aureus of the alpha-toxin virulence factor and by the airway cells of the antibacterial secretory leukoproteinase inhibitor (SLPI) was also analyzed. After 1 h of interaction, whatever the initial bacterial concentration, a low percentage of S. aureus (<8%) adhered to airway cells, and no airway epithelial cell damage was observed. In contrast, after 24 h of incubation, more bacteria adhered to airway epithelial cells, internalized bacteria were observed, and a bacterial concentration-dependent effect on airway cell damage was observed. At 24 h, most airway cells incubated with bacteria at 10(8) CFU/ml exhibited a necrotic phenotype. The necrosis was preceded by a transient apoptotic process. In parallel, we observed a time- and bacterial concentration-dependent decrease in SLPI and increase in alpha-toxin expression. These results suggest that airway cells can defend against S. aureus in the early stages of infection. However, in later phases, there is a marked imbalance between the bactericidal capacity of host cells and bacterial virulence. These findings reinforce the potential importance of S. aureus in the pathogenicity of airway infections, including those observed early in CF patients.

Uptake of plasmid/glycosylated polymer complexes and gene transfer efficiency in differentiated airway epithelial cells.

BACKGROUND: We have studied gene transfer efficiency of glycosylated polylysines and glycosylated polyethylenimines as vectors in immortalized differentiated airway gland serous cells and primary cultures of human airway surface epithelial cells. METHODS AND RESULTS: In both cell types, lactosylated PEI was more efficient for gene transfer than unsubstituted PEI and lactosylated polylysine which requires the presence of endosomolytic agents. However, for all the vectors tested, gene transfer efficiency was lower in differentiated cells as compared with poorly differentiated cells. The presence of membrane lectins, i.e. cell surface sugar-specific receptors, was evaluated using fluorescein-conjugated neoglycoproteins and microscopy or flow cytometry. In differentiated airway surface epithelial cells, membrane lectins were not expressed and plasmid DNA/fluorescein-conjugated glycosylated polymer complexes were not incorporated. This accounted in part for the lack of gene transfer efficiency in these cells. In contrast, in differentiated airway gland serous cells, expression of lectins and their endocytotic properties appeared to be similar to that observed in undifferentiated cells, and plasmid DNA/fluorescein-conjugated glycosylated polymer complexes were incorporated in similar amounts by cells in both differentiated states CONCLUSIONS: Glycosylated PEI appears to be a promising gene delivery system since it is more efficient than the sugar-free polymer and does not require endosomolytic agents. However, in differentiated airway gland serous cells, a low gene transfer efficiency was observed that could not be attributed to low expression of membrane lectins or low uptake of glycosylated complexes. An impaired intracellular trafficking of glycosylated complexes in differentiated airway gland serous cells is suggested.