Antiviral mechanisms of two broad-spectrum monoclonal antibodies for rabies prophylaxis and therapy

Rabies is an acute and lethal encephalomyelitis caused by lyssaviruses, among which rabies virus (RABV) is the most prevalent and important for public health. Although preventable through the post-exposure administration of rabies vaccine and immunoglobulins (RIGs), the disease is almost invariably fatal since the onset of clinical signs. Two human neutralizing monoclonal antibodies (mAbs), RVC20 and RVC58, have been shown to be effective in treating symptomatic rabies. To better understand how these mAbs work, we conducted structural modeling and in vitro assays to analyze their mechanisms of action, including their ability to mediate Fc-dependent effector functions. Our results indicate that both RVC20 and RVC58 recognize and lock the RABV-G protein in its pre-fusion conformation. RVC58 was shown to neutralize more potently the extra-cellular virus, while RVC20 mainly acts by reducing viral spreading from infected cells. Importantly, RVC20 was more effective in promoting effector functions compared to RVC58 and 17C7-RAB1 mAbs, the latter of which is approved for human rabies post-exposure treatment. These results provide valuable insights into the multiple mechanisms of action of RVC20 and RVC58 mAbs, offering relevant information for the development of these mAbs as treatment for human rabies

Alternative Methods to Current In Vivo Procedures to Address the 3Rs Tenet in Rabies Proficiency Testing

Canine rabies is responsible for an estimated 59,000 human deaths every year. In an attempt to reach the ZeroBy30 goal, robust disease surveillance coupled with improved diagnostics play a paramount role in ensuring reliable data and gradually attesting rabies control advancements. In this context, proficiency testing is organized to harmonize rabies diagnostic capacities. In most exercises, rabies-positive samples consist of brains collected from intracerebrally inoculated mice. This procedure causes distress and severe suffering to animals, raising important ethical concerns that can no longer be ignored. In the last decades, the 3Rs tenet (Replace, Reduce, Refine) has been successfully implemented in several scientific areas, and we strongly support its application in the framework of rabies proficiency testing. Here, we discuss cell-based technologies as innovative sustainable in vitro candidate systems to replace in vivo experiments for the production of proficiency testing samples. The application of these alternative methods can allow completely in vitro or ex vivo production of rabies proficiency testing panels, which would represent an important replacement or reduction/refinement for current in vivo procedures

Sisymbrium officinale, the Plant of Singers: A Review of Its Properties and Uses

Sisymbrium officinale (hedge mustard and formerly called Erysimum officinale) is a common plant in wild lands of Europe and Africa. It is also cultivated for its seeds and leaves to be used in salad or mustard. Sisymbrium officinale is useful not only in culinary preparations, but it also seems to possess interesting therapeutic properties, especially for throat diseases such as aphonia and hoarseness. For this reason, it is commonly called “herb of singers” (in Italian, “Erba dei cantanti”). Indeed a cup of Sisymbrium officinale infusion is frequently consumed by singers before artistic performance, even if its beneficial ability still needs to be scientifically demonstrated. Some preliminary data can be analyzed, but new efforts and resources should be devoted to study and investigate a plant with valuable therapeutic potential. This review summarizes the data available for Sisymbrium officinale

Targeting the Regulatory Subunit R2Alpha of Protein Kinase A in Human Glioblastoma through shRNA-Expressing Lentiviral Vectors

Glioblastoma is the most malignant and most common form of brain tumor, still today associated with a poor 14-months median survival from diagnosis. Protein kinase A, particularly its regulatory subunit R2Alpha, presents a typical intracellular distribution in glioblastoma cells compared to the healthy brain parenchyma and this peculiarity might be exploited in a therapeutic setting. In the present study, a third-generation lentiviral system for delivery of shRNA targeting the regulatory subunit R2Alpha of protein kinase A was developed. Generated lentiviral vectors are able to induce an efficient and stable downregulation of R2Alpha in different cellular models, including non-stem and stem-like glioblastoma cells. In addition, our data suggest a potential correlation between silencing of the regulatory subunit of protein kinase A and reduced viability of tumor cells, apparently due to a reduction in replication rate. Thus, our findings support the role of protein kinase A as a promising target for novel anti-glioma therapies

Biological activities of the aerial and undergound parts of Gymnadenia nigra Rchb.f. (syn. Nigritella nigra (L.) Rchb. f.) from the Italian Alps

This study investigated the bioactivity of both aerial (GNAR) and underground (GNUG) parts of Gymnadenia nigra Rchb.f. (syn. Nigritella nigra (L.) Rchb. f.) (Orchidaceae). The obtained data proved interesting when the samples were tested in two adrenocortical cancer cell lines (SW13 and H295R). In particular, the GNAR 80% methanol extract distinctly inhibited their viability after 24 h at a concentration of 1 μg/μL by MTT assay and trypan blue dye exclusion method. Cell morphology evaluation by means Wright’s staining also showed significant results, particularly in SW13 cells under the effect of both extracts. GNAR extract was able to scavenge the DPPH radical better than GNUG extract. It also was more active in albumin denaturation (a maximum % denaturation equal to 463.0 ± 8.3 vs 77.3 ± 13.3) and protease inhibition (a maximum % inhibition equal to 138.5 ± 7.0 vs 2.1 ± 2.0) tests. The results highlighted an important antitumor activity of G. nigra in vitro that deserves to be further studied

Biological activities of the aerial and undergound parts of Gymnadenia nigra Rchb.f. (syn. Nigritella nigra (L.) Rchb. f.) from the Italian Alps

: This study investigated the bioactivity of both aerial (GNAR) and underground (GNUG) parts of Gymnadenia nigra Rchb.f. (syn. Nigritella nigra (L.) Rchb. f.) (Orchidaceae). The obtained data proved interesting when the samples were tested in two adrenocortical cancer cell lines (SW13 and H295R). In particular, the GNAR 80% methanol extract distinctly inhibited their viability after 24 h at a concentration of 1 µg/µL by MTT assay and trypan blue dye exclusion method. Cell morphology evaluation by means Wright's staining also showed significant results, particularly in SW13 cells under the effect of both extracts. GNAR extract was able to scavenge the DPPH radical better than GNUG extract. It also was more active in albumin denaturation (a maximum % denaturation equal to 463.0 ± 8.3 vs 77.3 ± 13.3) and protease inhibition (a maximum % inhibition equal to 138.5 ± 7.0 vs 2.1 ± 2.0) tests. The results highlighted an important antitumor activity of G. nigra in vitro that deserves to be further studied

Digestive and gastroprotective effects of Achillea erba-rotta subsp. moschata (Wulfen) I.Richardson (syn. A. moschata Wulfen) (Asteraceae): From traditional uses to preclinical studies

Ethnopharmacological relevance: Achillea erba-rotta subsp. moschata (Wulfen) I.Richardson (syn. A. moschata Wulfen) (Asteraceae) is an alpine endemic plant whose aerial parts are harvested by the locals mainly for the digestive properties. Despite its widespread use, few studies have been conducted to date to verify its bioactivity. Aim of the study: The purpose of the work was to meet the tradition confirming with experimental data the popular belief that the consumption of this species offers beneficial effects to the gastrointestinal system. Materials and methods: Using Soxhlet apparatus, the dried aerial parts of A. erba-rotta subsp. moschata were successively extracted with petroleum ether (PET), dichloromethane (DCM) and methanol (MeOH). The essential oil (EO) was obtained by hydrodistillation using a Clevenger apparatus while infusion (AE) was prepared following the traditional local recipe. Their chemical characterization was performed by various techniques including SPME-GC/MS, GC/MS and HPLC/MS-MS. An in vitro biological screening was carried out. The influence of AE on lipid digestion was monitored by titration of free fatty acids (FFA) during pancreatic lipase activity with the pH-stat method. For all extracts and EO, the anti-Helicobacter pylori activity was assessed by the broth microdilution method, the influence on cell viability was evaluated against NCI–N87, OE21 and Caco-2 cell lines and a preliminary toxicity evaluation was done using Brine Shrimp lethality (BSL) assay. The anti-inflammatory potential was evidenced by interleukin IL-1- induced IL8 expression on Caco-2 cells. Results: AE increased by 15% the FFA releasing compared to the pancreatic lipase alone. PET, DCM and MeOH extracts as well as AE and EO were considered active against the growth of both antimicrobial susceptible and resistant strains of H. pylori with MIC values starting from 16 μg/mL. PET and DCM (IC50 = 89 μg/mL and 96 μg/mL, respectively, against Caco-2 cell line) extracts showed the high effect on cell viability while the EO reduced in 50% of cell viability at 1.48 μL/mL (NCI–N87 cells), 1.42 μL/mL (OE21 cells), and 3.44 μL/mL (Caco-2 cells) corroborating the BSL results. In different degrees, all extracts and EO inhibited the IL-1β-stimulated IL-8 production in Caco-2 cells.Conclusions: The obtained data are encouraging and provide a scientific basis for the traditional use of A. erbarotta subsp. moschata as a digestive agent although they need to be further corroborated by studies involving the investigation of both the in vivo activities and the role of the compounds detected in the extracts

Digestive and gastroprotective effects of Achillea erba-rotta subsp. moschata (Wulfen) I.Richardson (syn. A. moschata Wulfen) (Asteraceae): From traditional uses to preclinical studies

Ethnopharmacological relevance: Achillea erba-rotta subsp. moschata (Wulfen) I.Richardson (syn. A. moschata Wulfen) (Asteraceae) is an alpine endemic plant whose aerial parts are harvested by the locals mainly for the digestive properties. Despite its widespread use, few studies have been conducted to date to verify its bioactivity. Aim of the study: The purpose of the work was to meet the tradition confirming with experimental data the popular belief that the consumption of this species offers beneficial effects to the gastrointestinal system. Materials and methods: Using Soxhlet apparatus, the dried aerial parts of A. erba-rotta subsp. moschata were successively extracted with petroleum ether (PET), dichloromethane (DCM) and methanol (MeOH). The essential oil (EO) was obtained by hydrodistillation using a Clevenger apparatus while infusion (AE) was prepared following the traditional local recipe. Their chemical characterization was performed by various techniques including SPME-GC/MS, GC/MS and HPLC/MS-MS. An in vitro biological screening was carried out. The influence of AE on lipid digestion was monitored by titration of free fatty acids (FFA) during pancreatic lipase activity with the pH-stat method. For all extracts and EO, the anti-Helicobacter pylori activity was assessed by the broth microdilution method, the influence on cell viability was evaluated against NCI–N87, OE21 and Caco-2 cell lines and a preliminary toxicity evaluation was done using Brine Shrimp lethality (BSL) assay. The anti-inflammatory potential was evidenced by interleukin IL-1- induced IL8 expression on Caco-2 cells. Results: AE increased by 15% the FFA releasing compared to the pancreatic lipase alone. PET, DCM and MeOH extracts as well as AE and EO were considered active against the growth of both antimicrobial susceptible and resistant strains of H. pylori with MIC values starting from 16 μg/mL. PET and DCM (IC50 = 89 μg/mL and 96 μg/mL, respectively, against Caco-2 cell line) extracts showed the high effect on cell viability while the EO reduced in 50% of cell viability at 1.48 μL/mL (NCI–N87 cells), 1.42 μL/mL (OE21 cells), and 3.44 μL/mL (Caco-2 cells) corroborating the BSL results. In different degrees, all extracts and EO inhibited the IL-1β-stimulated IL-8 production in Caco-2 cells

A single amino acid substitution confers B-cell clonogenic activity to the HIV-1 matrix protein p17

Recent data highlight the presence, in HIV-1-seropositive patients with lymphoma, of p17 variants (vp17s) endowed with B-cell clonogenicity, suggesting a role of vp17s in lymphomagenesis. We investigated the mechanisms responsible for the functional disparity on B cells between a wild-type p17 (refp17) and a vp17 named S75X. Here, we show that a single Arginine (R) to Glycine (G) mutation at position 76 in the refp17 backbone (p17R76G), as in the S75X variant, is per se sufficient to confer a B-cell clonogenic potential to the viral protein and modulate, through activation of the PTEN/PI3K/Akt signaling pathway, different molecules involved in apoptosis inhibition (CASP-9, CASP-7, DFF-45, NPM, YWHAZ, Src, PAX2, MAPK8), cell cycle promotion and cancer progression (CDK1, CDK2, CDK8, CHEK1, CHEK2, GSK-3 beta, NPM, PAK1, PP2C-alpha). Moreover, the only R to G mutation at position 76 was found to strongly impact on protein folding and oligomerization by altering the hydrogen bond network. This generates a conformational shift in the p17 R76G mutant which enables a functional epitope(s), masked in refp17, to elicit B-cell growth-promoting signals after its interaction with a still unknown receptor(s). Our findings offer new opportunities to understand the molecular mechanisms accounting for the B-cell growth-promoting activity of vp17s

Biological activities of the aerial and undergound parts of <i>Gymnadenia nigra</i> Rchb.f. (syn. <i>Nigritella nigra</i> (L.) Rchb. f.) from the Italian Alps

This study investigated the bioactivity of both aerial (GNAR) and underground (GNUG) parts of Gymnadenia nigra Rchb.f. (syn. Nigritella nigra (L.) Rchb. f.) (Orchidaceae). The obtained data proved interesting when the samples were tested in two adrenocortical cancer cell lines (SW13 and H295R). In particular, the GNAR 80% methanol extract distinctly inhibited their viability after 24 h at a concentration of 1 µg/µL by MTT assay and trypan blue dye exclusion method. Cell morphology evaluation by means Wright’s staining also showed significant results, particularly in SW13 cells under the effect of both extracts. GNAR extract was able to scavenge the DPPH radical better than GNUG extract. It also was more active in albumin denaturation (a maximum % denaturation equal to 463.0 ± 8.3 vs 77.3 ± 13.3) and protease inhibition (a maximum % inhibition equal to 138.5 ± 7.0 vs 2.1 ± 2.0) tests. The results highlighted an important antitumor activity of G. nigra in vitro that deserves to be further studied.</p