Lactoferrin Against SARS-CoV-2: In Vitro and In Silico Evidences.

Lactoferrin (Lf) is a cationic glycoprotein synthetized by exocrine glands and is present in all human secretions. It is also secreted by neutrophils in infection and inflammation sites. This glycoprotein possesses antimicrobial activity due to its capability to chelate two ferric ions per molecule, as well as to interact with bacterial and viral anionic surface components. The cationic features of Lf bind to cells, protecting the host from bacterial and viral injuries. Its anti-inflammatory activity is mediated by the ability to enter inside the nucleus of host cells, thus inhibiting the synthesis of proinflammatory cytokine genes. In particular, Lf down-regulates the synthesis of IL-6, which is involved in iron homeostasis disorders and leads to intracellular iron overload, favoring viral replication and infection. The well-known antiviral activity of Lf has been demonstrated against DNA, RNA, and enveloped and naked viruses and, therefore, Lf could be efficient in counteracting also SARS-CoV-2 infection. For this purpose, we performed in vitro assays, proving that Lf exerts an antiviral activity against SARS-COV-2 through direct attachment to both SARS-CoV-2 and cell surface components. This activity varied according to concentration (100/500 µg/ml), multiplicity of infection (0.1/0.01), and cell type (Vero E6/Caco-2 cells). Interestingly, the in silico results strongly supported the hypothesis of a direct recognition between Lf and the spike S glycoprotein, which can thus hinder viral entry into the cells. These in vitro observations led us to speculate a potential supplementary role of Lf in the management of COVID-19 patients.

Bromelain Degrades Aß1-42 Monomers and Soluble Aggregates: An In Vitro Study in Cerebrospinal Fluid of Alzheimer's Disease Patients.

BACKGROUND: Therapeutic approaches targeting amyloid ß42 (Aß42) oligomers may represent a promising neuroprotective strategy for the prevention and treatment of Alzheimer's disease (AD). OBJECTIVE: In this study we evaluated the ability of bromelain, a plant cysteine protease derived from pineapple stems, to interact with synthetic Aß42 monomers and oligomers. We also examined the ability of bromelain to interfere in vitro with synthetic Aß42 aggregates in the cerebrospinal fluid (CSF) of Alzheimer's disease as well as of control patients affected by other neurological diseases. METHOD: Both synthetic monomers and aggregates of Aß42 were incubated in CSF with varying concentrations of bromelain. The effects of digestion were evaluated by Western Blot analysis using the specific monoclonal antibody 4G8 to identify the patterns of residual content of Aß42. We further used rat primary cortical culture neurons (CN) to examine the cytotoxic action of this natural compound. RESULTS: We found that bromelain successfully degraded Aß42 monomers and low and high molecular weight oligomers. Indeed, when bromelain preparations of 3 and 6 mU were added to the CSF, the residual amount of Aß42 monomers and oligomers were significantly reduced when compared to the same standard Aß42 preparations incubated in CSF without bromelain. Moreover, bromelain incubations of 0.1, 0.5, and 1 mU/ml were not toxic to CN, as compared to vehicle treated cells. CONCLUSION: Overall, these results represent an important insight into the action of bromelain on Aß42 oligomers, suggesting its potential use in the therapy of AD.

Hypothalamic dysfunction is related to sleep impairment and CSF biomarkers in Alzheimer's disease.

Hypothalamus is a key brain region regulating several essential homeostatic functions, including the sleep-wake cycle. Alzheimer's disease (AD) pathology affects nuclei controlling sleep-wake rhythm sited in this brain area. Since only post-mortem studies documented the relationship between hypothalamic atrophy and sleep-wake cycle impairment, we investigated in AD patients the possible hypothalamic in vivo alteration using 2-deoxy-2-(18F) fluoro-D-glucose ([18F]FDG) positron emission tomography ([18F]FDG PET), and its correlations with sleep impairment and cerebrospinal-fluid (CSF) AD biomarkers (tau proteins and ß-amyloid42). We measured sleep by polysomnography, CSF AD biomarkers and orexin levels, and hypothalamic [18F]FDG PET uptake in a population of AD patients compared to age- and sex-matched controls. We documented the significant reduction of hypothalamic [18F]FDG PET uptake in AD patients (n = 18) compared to controls (n = 18) (p < 0.01). Moreover, we found the increase of CSF orexin levels coupled with the marked alteration of nocturnal sleep in AD patients than controls. We observed the significant association linking the reduction of both sleep efficiency and REM sleep to the reduction of hypothalamic [18F]FDG PET uptake in the AD group, which in turn negatively correlated with the total-tau/beta-amyloid42 ratio (index of more marked neurodegeneration). Moreover, controls but not AD patients showed [18F]FDG PET interconnections between hypothalamus and limbic system. We documented the in vivo dysfunction of hypothalamus in AD patients, which lost the physiological connections with limbic system and was correlated with both nocturnal sleep disruption and CSF AD biomarkers.

Diuretic drug binding to human glutathione transferase P1-1: potential role of Cys-101 revealed in the double mutant C47S/Y108V.

The diuretic drug ethacrynic acid (EA), both an inhibitor and substrate of pi class glutathione S-transferase (GST P1-1), has been tested in clinical trials as an adjuvant in chemotherapy. We recently studied the role of the active site residue Tyr-108 in binding EA to the enzyme and found that the analysis was complicated by covalent binding of this drug to the highly reactive Cys-47. Previous attempts to eliminate this binding by chemical modification yielded ambiguous results and therefore we decided here to produce a double mutant C47S/Y108V by site directed mutagenesis and further expression in Escherichia coli and the interaction of EA and its GSH conjugate (EASG) examined by calorimetric studies and X-ray diffraction. Surprisingly, in the absence of Cys-47, Cys-101 (located at the dimer interface) becomes a target for modification by EA, albeit at a lower conjugation rate than Cys-47. The Cys-47 → Ser mutation in the double mutant enzyme induces a positive cooperativity between the two subunits when ligands with affinity to G-site bind to enzyme. However, this mutation does not seem to affect the thermodynamic properties of ligand binding to the electrophilic binding site (H-site) and the thermal or chemical stability of this double mutant does not significantly affect the unfolding mechanism in either the absence or presence of ligand. Crystal structures of apo and an EASG complex are essentially identical with a few exceptions in the H-site and in the water network at the dimer interface.

Novel IgE recognized components of Lolium perenne pollen extract: comparative proteomics evaluation of allergic patients sensitization profiles.

In the last years, proteomic investigation provided a powerful tool in molecular characterization of complex allergen sources with relevant implications in both diagnosis and immunotherapic treatment of allergies. We followed a proteomic approach to characterize ryegrass (Lolium perenne) pollen, a common cause of seasonal allergic diseases affecting an increasing part of world population. Peptide shotgun experiments performed on nanoLiquid Ultra Pressure Chromatography coupled with fast Q-TOF MS-MS/MS acquisition protocols (MS(E)) and 2-DE immunoblot combined with MALDI-TOF-TOF analysis allowed the detection of all previously identified ryegrass allergens. Comparative analysis of immunoblot highlighted a class of patients characterized by a more complex 2-DE pattern associated with increased levels of IgE antibodies and by higher susceptibility to multiple sensitization toward different allergen sources. Cluster analysis revealed that all these patients recognized profilin, considered the main cross-reactive allergen in grass pollen. Furthermore, mass spectrometry analysis revealed the presence of other IgE reactive components in ryegrass pollen that might be involved in polysensitization, such as cyclophilin, fructosyltransferase and legumin-like protein.