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The dysregulated immune response and inflammation resulting in severe COVID-19 are still incompletely understood. Having recently determined that aberrant death-ligand-induced cell death can cause lethal inflammation, we hypothesized that this process might also cause or contribute to inflammatory disease and lung failure following SARS-CoV-2 infection. To test this hypothesis, we developed a novel mouse-adapted SARS-CoV-2 model (MA20) that recapitulates key pathological features of COVID-19. Concomitantly with occurrence of cell death and inflammation, FasL expression was significantly increased on inflammatory monocytic macrophages and NK cells in the lungs of MA20-infected mice. Importantly, therapeutic FasL inhibition markedly increased survival of both, young and old MA20-infected mice coincident with substantially reduced cell death and inflammation in their lungs. Intriguingly, FasL was also increased in the bronchoalveolar lavage fluid of critically-ill COVID-19 patients. Together, these results identify FasL as a crucial host factor driving the immuno-pathology that underlies COVID-19 severity and lethality, and imply that patients with severe COVID-19 may significantly benefit from therapeutic inhibition of FasL.

Abstract: Background: Staphylococcal implant infections’ response to treatment may be correlated with cytokine production. We investigated the effect of certain antibiotics on the cytokine response in experimental osteomyelitis. Methods: A stainless steel needle with an adherent slime-producing Staphylococcus aureus was implanted intramedullarly in the left tibia of 40 adult male Wistar rats. At 42 days after implantation, cefuroxime, vancomycin, tobramycin, and ciprofloxacin were administered intramuscularly every 12 h for 21 days. The control group was given no antibiotic. At the end of the treatment, implants and tibias were retrieved, and the bacterial numbers were estimated. Cytokines [interleukin-1α (IL-1α), IL-6, and IL-10] were determined (ELISA) in the tibial extract. Results: Vancomycin and cefuroxime inhibited bone colonization in all tibias, and tobramycin and ciprofloxacin inhibited it only partially. Cefuroxime reduced the number of bacteria that adhered to the implants more than the other antibiotics. IL-1α and IL-6 showed higher levels in the ciprofloxacin-treated group than in the cefuroxime-treated and control groups. IL-6 levels in rats treated with cefuroxime were lower than in rats treated with tobramycin or vancomycin and the control group. Cefuroxime decreased IL-10 levels more than ciprofloxacin or vancomycin or those seen in the control group. Conclusions: The cefuroxime group showed the greatest decrease of pro-inflammatory cytokines. Different antibiotics produce different cytokine reactions that should be studied to choose the best treatment.

Staphylococcal implant infections’ response to treatment may be correlated with cytokine production. We investigated the effect of certain antibiotics on the cytokine response in experimental osteomyelitis.

Staphylococcus aureus is a common cause of intramammary infections, which frequently become chronic, associated with the ability of the bacteria to produce biofilm. Here, we report a relationship between the ability to produce chronic bovine mastitis and biofilm formation. We have classified bovine mastitis S. aureus isolates into three groups based on the presence of particular genetic elements required for biofilm formation: group 1 (ica(+) bap(+)), group 2 (ica(+), bap negative), and group 3 (ica negative, bap negative). Overall, animals naturally infected with group 1 and 2 isolates had a lower milk somatic cell count than those infected with isolates of group 3. In addition, Bap-positive isolates were significantly more able to colonize and persist in the bovine mammary gland in vivo and were less susceptible to antibiotic treatments when forming biofilms in vitro. Analysis of the structural bap gene revealed the existence of alternate forms of expression of the Bap protein in S. aureus isolates obtained under field conditions throughout the animal's life. The presence of anti-Bap antibodies in serum samples taken from animals with confirmed S. aureus infections indicated the production of Bap during infection. Furthermore, disruption of the ica operon in a bap-positive strain had no effect on in vitro biofilm formation, a finding which strongly suggested that Bap could compensate for the deficiency of the PIA/PNAG product (a biofilm matrix polysaccharide). Altogether, these results demonstrate that, in the bovine intramammary gland, the presence of Bap may facilitate a biofilm formation connected with the persistence of S. aureus.

ABSTRACTStaphylococcus aureusis a common cause of intramammary infections, which frequently become chronic, associated with the ability of the bacteria to produce biofilm. Here, we report a relationship between the ability to produce chronic bovine mastitis and biofilm formation. We have classified bovine mastitis S. aureusisolates into three groups based on the presence of particular genetic elements required for biofilm formation: group 1 (ica+bap+), group 2 (ica+, bapnegative), and group 3 (icanegative, bapnegative). Overall, animals naturally infected with group 1 and 2 isolates had a lower milk somatic cell count than those infected with isolates of group 3. In addition, Bap-positive isolates were significantly more able to colonize and persist in the bovine mammary gland in vivo and were less susceptible to antibiotic treatments when forming biofilms in vitro. Analysis of the structural bapgene revealed the existence of alternate forms of expression of the Bap protein in S. aureusisolates obtained under field conditions throughout the animal's life. The presence of anti-Bap antibodies in serum samples taken from animals with confirmed S. aureusinfections indicated the production of Bap during infection. Furthermore, disruption of the icaoperon in a bap-positive strain had no effect on in vitro biofilm formation, a finding which strongly suggested that Bap could compensate for the deficiency of the PIA/PNAG product (a biofilm matrix polysaccharide). Altogether, these results demonstrate that, in the bovine intramammary gland, the presence of Bap may facilitate a biofilm formation connected with the persistence of S. aureus.

One of the “gold standard” techniques for postmortem confirmation of scrapie diagnosis in sheep and goats is immunohistochemical examination of brain tissue. Active surveillance for scrapie is mainly performed by rapid diagnostic tests on the basis of postmortem immunochemical detection of prion protein (PrP) in the obex tissue. The aim of this study was to determine the performance of 2 rapid tests, Prionics®-Check LIA (a chemiluminescence sandwich enzyme-linked immunosorbent assay) and Prionics®-Check Western blot for scrapie diagnosis when applied to brain areas other than the obex, in comparison with the recognized immunohistochemistry. Prion protein was detected in the obex, cervical spinal cord, and thalamus from all the scrapie-positive sheep by the 3 tests. Western blot and LIA were negative in other areas of the brain, although weak immunohistochemical staining was detected. The results show that the 2 rapid tests studied may detect PrP in brain areas other than the obex, although with a lower sensitivity than immunohistochemistry when there is minimal PrP deposition.

The constitutive and inducible isoforms of nitric oxide synthase (NOS) and the end-product of nitration, nitrotyrosine, were analyzed by immunohistochemistry, Western blotting, and enzymatic activity in sheep at different stages of the prion disease, scrapie. Four groups were studied: 1) nonaffected (control), 2) preclinical, 3) clinical, and 4) terminal. Constitutive neuronal NOS (nNOS) was the most abundant isoform present in cerebellar neurons of the sheep. Expression of nNOS increased in preclinical animals but diminished in the late stages of the disease. The Purkinje cells that usually are not immunoreactive for this protein became immunopositive in the clinical phase. In unaffected sheep, the inducible isoform (iNOS) was slightly positive in the Purkinje cells. As the disease progressed, the immunoreactivity of Purkinje neurons for iNOS increased. At the final stages, numerous iNOS-positive microglial cells were found in the molecular layer. There was a basal level of protein nitration in the cerebellum of unaffected sheep, especially in the molecular layer. As the disease progressed, the distal prolongations of the Purkinje cells and the astroglia became immunoreactive for nitrotyrosine. Our results suggest that the nitrergic system reacts to the progression of spongiform diseases and may be part of their pathogenesis mechanism.