BNT162b2 mRNA SARS-CoV-2 Vaccine Elicits High Avidity and Neutralizing Antibodies in Healthcare Workers.

The BNT162b2 vaccine, containing lipid nanoparticles-formulated mRNA encoding the full-length spike protein of SARS-CoV-2, has been employed to immunize health care workers in Italy, administered in two doses 21 days apart. In this study, we characterized the antibody response induced by the BNT162b2 vaccine in a group of health care workers, tested at baseline, after the first dose and after the booster. Thirty-nine subjects without previous exposure to SARS-CoV-2 were vaccinated with the BNT162b2 vaccine. IgM, IgG, and IgA anti-receptor binding domain (RBD) were tested by ELISA. Neutralizing antibodies were evaluated testing the inhibition of RBD binding to ACE2. Antibody avidity was measured by urea avidity ELISA. IgM anti-RBD are produced after the first dose of vaccine and persist after the booster. IgG and IgA anti-RBD antibodies are detected in high amounts in all the subjects after the first dose and further increase after the booster. A few subjects, already after the first dose, produce antibodies inhibiting RBD interaction with ACE2. After the booster, high levels of inhibitory antibodies are detected in all the subjects. Affinity maturation takes place with boosting and IgG anti-RBD avidity increases with the number of immunizations. A less pronounced increase is observed with IgA. These data indicate that the BNT162b2 vaccine can induce high levels of protective antibodies of high avidity in vaccinated subjects; both IgG and IgA anti-RBD antibodies are produced. Further studies are needed to evaluate antibody persistence over time.

Enteric glial cells counteract Clostridium difficile Toxin B through a NADPH oxidase/ROS/JNK/caspase-3 axis, without involving mitochondrial pathways.

Enteric glial cells (EGCs) are components of the intestinal epithelial barrier essential for regulating the enteric nervous system. Clostridium difficile is the most common cause of antibiotic-associated colitis, toxin B (TcdB) being the major virulence factor, due to its ability to breach the intestinal epithelial barrier and to act on other cell types. Here we investigated TcdB effects on EGCs and the activated molecular mechanisms. Already at 2 hours, TcdB triggered ROS formation originating from NADPH-oxidase, as demonstrated by their reduction in the presence of the NADPH-oxidase inhibitor ML171. Although EGCs mitochondria support almost completely the cellular ATP need, TcdB exerted weak effects on EGCs in terms of ATP and mitochondrial functionality, mitochondrial ROS production occurring as a late event. ROS activated the JNK signalling and overexpression of the proapoptotic Bim not followed by cytochrome c or AIF release to activate the downstream apoptotic cascade. EGCs underwent DNA fragmentation through activation of the ROS/JNK/caspase-3 axis, evidenced by the ability of ML171, N-acetylcysteine, and the JNK inhibitor SP600125 to inhibit caspase-3 or to contrast apoptosis. Therefore, TcdB aggressiveness towards EGCs is mainly restricted to the cytosolic compartment, which represents a peculiar feature, since TcdB primarily influences mitochondria in other cellular types.

Enteric glial cells are susceptible to Clostridium difficile toxin B.

Clostridium difficile causes nosocomial/antibiotic-associated diarrhoea and pseudomembranous colitis. The major virulence factors are toxin A and toxin B (TcdB), which inactivate GTPases by monoglucosylation, leading to cytopathic (cytoskeleton alteration, cell rounding) and cytotoxic effects (cell-cycle arrest, apoptosis). C. difficile toxins breaching the intestinal epithelial barrier can act on underlying cells, enterocytes, colonocytes, and enteric neurons, as described in vitro and in vivo, but until now no data have been available on enteric glial cell (EGC) susceptibility. EGCs are crucial for regulating the enteric nervous system, gut homeostasis, the immune and inflammatory responses, and digestive and extradigestive diseases. Therefore, we evaluated the effects of C. difficile TcdB in EGCs. Rat-transformed EGCs were treated with TcdB at 0.1-10 ng/ml for 1.5-48 h, and several parameters were analysed. TcdB induces the following in EGCs: (1) early cell rounding with Rac1 glucosylation; (2) early G2/M cell-cycle arrest by cyclin B1/Cdc2 complex inactivation caused by p27 upregulation, the downregulation of cyclin B1 and Cdc2 phosphorylated at Thr161 and Tyr15; and (3) apoptosis by a caspase-dependent but mitochondria-independent pathway. Most importantly, the stimulation of EGCs with TNF-α plus IFN-γ before, concomitantly or after TcdB treatment strongly increased TcdB-induced apoptosis. Furthermore, EGCs that survived the cytotoxic effect of TcdB did not recover completely and showed not only persistent Rac1 glucosylation, cell-cycle arrest and low apoptosis but also increased production of glial cell-derived neurotrophic factor, suggesting self-rescuing mechanisms. In conclusion, the high susceptibility of EGCs to TcdB in vitro, the increased sensitivity to inflammatory cytokines related to apoptosis and the persistence of altered functions in surviving cells suggest an important in vivo role of EGCs in the pathogenesis of C. difficile infection.