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Objectives Sars-CoV-2 acute infection is clinically heterogeneous, ranging from asymptomatic cases to patients with a severe, systemic clinical course. Among the involved factors age and preexisting morbidities play a major role; genetic host susceptibility contributes to modulating the clinical expression and outcome of the disease. Mannose-binding lectin is an acute-phase protein that activates the lectin-complement pathway, promotes opsonophagocytosis and modulates inflammation, and is involved in several bacterial and viral infections in humans. Understanding its role in Sars-CoV-2 infection could help select a better therapy.Methods We studied MBL2 haplotypes in 419 patients with acute COVID-19 in comparison to the general population and related the haplotypes to clinical and laboratory markers of severity.Results We recorded an enhanced frequency of MBL2 null alleles in patients with severe acute COVID-19. The homozygous null genotypes were significantly more frequent in patients with advanced WHO score 4-7 (OR of about 4) and related to more severe inflammation, neutrophilia, and lymphopenia.Conclusions Subjects with a defective MBL2 genotype (i.e., 0/0) are predisposed to a more severe acute Sars-CoV-2 infection; they may benefit from early replacement therapy with recombinant MBL. Furthermore, a subset of subjects with the A/A MBL genotype develop a relevant increase of serum MBL during the early phases of the disease and develop a more severe pulmonary disease; in these patients, the targeting of the complement may help. Therefore, COVID-19 patients should be tested at hospitalization with serum MBL analysis and MBL2 genotype, to define the optimal therapy.

dynamics of a rotating colony and trajectory of cells

Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in Cftr(F508del) homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation.

We have previously reported that TLR4 signaling is increased in lipopolysaccharide (LPS) -stimulated Cystic Fibrosis (CF) macrophages (MΦs), contributing to the robust production of pro-inflammatory cytokines. The heme oxygenase (HO-1)/carbon monoxide (CO) pathway modulates cellular redox status, inflammatory responses, and cell survival. The HO-1 enzyme, together with the scaffold protein caveolin 1 (CAV-1), also acts as a negative regulator of TLR4 signaling in MΦs. Here, we demonstrate that in LPS-challenged CF MΦs, HO-1 does not compartmentalize normally to the cell surface and instead accumulates intracellularly. The abnormal HO-1 localization in CF MΦs in response to LPS is due to decreased CAV-1 expression, which is controlled by the cellular oxidative state, and is required for HO-1 delivery to the cell surface. Overexpression of HO-1 or stimulating the pathway with CO-releasing molecules (CORM2)enhancesCAV-1 expression in CF MΦs, suggesting a positive-feed forward loop between HO-1/CO induction and CAV-1 expression. These manipulations reestablished HO-1 and CAV-1 cell surface localization in CF MΦ's. Consistent with restoration of HO-1/CAV-1 negative regulation of TLR4 signaling, genetic or pharmacological (CORM2)-induced enhancement of this pathway decreased the inflammatory response of CF MΦs and CF mice treated with LPS. In conclusion, our results demonstrate that the counter-regulatory HO-1/CO pathway, which is critical in balancing and limiting the inflammatory response, is defective in CF MΦs through a CAV-1-dependent mechanism, exacerbating the CF MΦ's response to LPS. This pathway could be a potential target for therapeutic intervention for CF lung disease.

In Italy, from January 2021, the Ministry of Health indicated a vaccination plan against COVID for frail patients and physicians based on a three-dose scheme. However, conflicting results have been reported on which biomarkers permit immunization assessment. We used several laboratory approaches (i.e., antibodies serum levels, flow cytometry analysis, and cytokines release by stimulated cells) to investigate the immune response in a cohort of 53 family pediatricians (FPs) at different times after the vaccine. We observed that the BNT162b2-mRNA vaccine induced a significant increase of specific antibodies after the third (booster) dose; however, the antibody titer was not predictive of the risk of developing the infection in the six months following the booster dose. The antigen stimulation of PBMC cells from subjects vaccinated with the third booster jab induced the increase of the activated T cells (i.e., CD4+ CD154+); the frequency of CD4+ CD154+ TNF-α+ cells, as well as the TNF-α secretion, was not modified, while we observed a trend of increase of IFN-γ secretion. Interestingly, the level of CD8+ IFN-γ+ (independently from antibody titer) was significantly increased after the third dose and predicts the risk of developing the infection in the six months following the booster jab. Such results may impact also other virus vaccinations.

Transglutaminase 2 (TG2) is a ubiquitously expressed member of the transglutaminase family with Ca2+-dependent protein crosslinking activity. Its subcellular localization is crucial in determining its function, and indeed, TG2 is found in the extracellular matrix, mitochondria, recycling endosomes, plasma membrane, cytosol, and nucleus because it is associated with cell growth, differentiation, and apoptosis. It is involved in several pathologies, such as celiac disease, cardiovascular, hepatic, renal, and fibrosis diseases, carrying out opposite functions of up and down regulation in the progression of the same pathology. Therefore, this fine regulation requires a very sensitive and specific method of identification of TG2, which is to be detected in very small quantities in a deregulated condition. Here, we demonstrate the possibility of detecting TG2 down to attomolar concentration by using organic electrochemical transistors driven by gold electrodes functionalized with anti-TG2 antibodies. In particular, a direct correlation between the TG2 concentration and the transistor transconductance values, as extracted from typical transfer curves, was found. Overall, our findings highlight the potentialities of this new biosensing approach for the detection of TG2 in the context of pathological diseases, offering a rapid and cost-effective alternative to traditional methods.

Cystic fibrosis (CF), the most common lethal monogenic disease in Caucasians, is characterized by recurrent bacterial infections and colonization, mainly by Pseudomonas aeruginosa, resulting in unresolved airway inflammation. CF is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which functions as a chloride channel in epithelial cells, macrophages, and other cell types. Impaired bacterial handling by macrophages is a feature of CF airways, although it is still debated how defective CFTR impairs bacterial killing. Recent evidence indicates that a defective autophagy in CF macrophages leads to alterations of bacterial clearance upon infection. Here we use bone marrow-derived macrophages from transgenic mice to provide the genetic proof that defective CFTR compromises both uptake and clearance of internalized Pseudomonas aeruginosa. We demonstrate that the proteostasis regulator cysteamine, which rescues the function of the most common F508del-CFTR mutant and hence reduces lung inflammation in CF patients, can also repair the defects of CF macrophages, thus restoring both bacterial internalization and clearance through a process that involves upregulation of the pro-autophagic protein Beclin 1 and re-establishment of the autophagic pathway. Altogether these results indicate that cysteamine restores the function of several distinct cell types, including that of macrophages, which might contribute to its beneficial effects on CF.

Phase II clinical trials indicate that the combination of cysteamine plus epigallocatechin gallate (EGCG) is effective against cystic fibrosis in patients bearing the most frequent etiological mutation (CFTRΔF508). Here, we investigated the interaction between both agents on cultured respiratory epithelia cells from normal and CFTRΔF508-mutated donors. We observed that the combination of both agents affected metabolic circuits (and in particular the tricarboxylic acid cycle) in a unique way and that cysteamine plus EGCG reduced cytoplasmic protein acetylation more than each of the 2 components alone. In a cell-free system, protein cross-linking activity of EGCG was suppressed by cysteamine. Finally, EGCG was able to enhance the conversion of cysteamine into taurine in metabolic flux experiments. Altogether, these results indicate that multiple pharmacological interactions occur between cysteamine and EGCG, suggesting that they contribute to the unique synergy of both agents in restoring the function of mutated CFTRΔF508.

International audience; Cystic fibrosis (CF) is a lethal monogenic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that entails the (diagnostic) increase in sweat electrolyte concentrations, progressive lung disease with chronic inflammation and recurrent bacterial infections, pancreatic insufficiency, and male infertility. Therapies aimed at restoring the CFTR defect have emerged. Thus, a small molecule which facilitates chloride channel opening, the potentiator Ivacaftor, has been approved for the treatment of CF patients bearing a particular class of rare CFTR mutations. However, small molecules that directly target the most common misfolded CFTR mutant, F508del, and improve its intracellular trafficking in vitro, have been less effective than expected when tested in CF patients, even in combination with Ivacaftor. Thus, new strategies are required to circumvent the F508del-CFTR defect. Airway and intestinal epithelial cells from CF patients bearing the F508del-CFTR mutation exhibit an impressive derangement of cellular proteostasis, with oxidative stress, overactivation of the tissue transglutaminase (TG2), and disabled autophagy. Proteostasis regulators such as cysteamine can rescue and stabilize a functional F508del-CFTR protein through suppressing TG2 activation and restoring autophagy in vivo in F508del-CFTR homozygous mice, in vitro in CF patient-derived cell lines, ex vivo in freshly collected primary patient's nasal cells, as well as in a pilot clinical trial involving homozygous F508del-CFTR patients. Here, we discuss how the therapeutic normalization of defective proteostasis can be harnessed for the treatment of CF patients with the F508del-CFTR mutation.

Cystic fibrosis (CF) is most frequently due to homozygous ΔF508-CFTR mutation. The ΔF508-CFTR protein is unstable in the plasma membrane (PM), even if it is rescued by pharmacological agents that prevent its intracellular retention and degradation. Restoring defective autophagy in CF airways by proteostasis regulators (such as cystamine and its reduced form, cysteamine) can rescue and stabilize ΔF508-CFTR at the PM, thus enabling the action of CFTR potentiators, which are pharmacological agents that stimulate the function of CFTR as an ion channel. The effects of cystamine extend for days (in vitro) and weeks (in vivo) beyond washout, suggesting that once peripheral proteostasis has been re-established, PM-resident ΔF508-CFTR sustains its own stability. We demonstrated that the pharmacological inhibition of wild-type CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette subfamily C, member 7)], in bronchial epithelial cells decreases the stability of the CFTR protein by inhibiting autophagy, elevating the abundance of SQSTM1/p62 and its interaction with CFTR at the PM, increasing the ubiqutination of CFTR, stimulating the lysosomal degradation of CFTR and avoiding its recycling. All these effects could be inhibited by cystamine. Moreover, CFTR-sufficient epithelia generate permissive conditions for incorporating ΔF508-CFTR into the PM and stabilizing it at this location. These results provide the rationale for a combination therapy of CF in which pretreatment with cystamine or cysteamine enables the later action of CFTR potentiators.