The strongest protein binder is surprisingly labile.

Bacterial adhesins are cell-surface proteins that anchor to the cell wall of the host. The first stage of infection involves the specific attachment to fibrinogen (Fg), a protein found in human blood. This attachment allows bacteria to colonize tissues causing diseases such as endocarditis. The study of this family of proteins is hence essential to develop new strategies to fight bacterial infections. In the case of the Gram-positive bacterium Staphylococcus aureus, there exists a class of adhesins known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Here, we focus on one of them, the clumping factor A (ClfA), which has been found to bind Fg through the dock-lock-latch mechanism. Interestingly, it has recently been discovered that MSCRAMM proteins employ a catch-bond to withstand forces exceeding 2 nN, making this type of interaction as mechanically strong as a covalent bond. However, it is not known whether this strength is an evolved feature characteristic of the bacterial protein or is typical only of the interaction with its partner. Here, we combine single-molecule force spectroscopy, biophysical binding assays, and molecular simulations to study the intrinsic mechanical strength of ClfA. We find that despite the extremely high forces required to break its interactions with Fg, ClfA is not by itself particularly strong. Integrating the results from both theory and experiments we dissect contributions to the mechanical stability of this protein.

Unveiling the immunomodulatory effect of the novel probiotic Akkermansia muciniphila and its protective effect in vitro.

Akkermansia muciniphila, a bacterium found in the human microbiota, has gained interest due to its potential health benefits. Previous studies have linked its absence to inflammatory disorders, while also suggesting its role in maintaining a healthy gut barrier. However, there is limited information on its specific effects on the immune system. Therefore, the aim of this research was to analyze the in vitro response triggered by A. muciniphila employing RAW 264.7 macrophages. The study focused on investigating the production of cytokines and nitric oxide, along with evaluating the expression of inflammatory surface cellular markers. Additionally, we assessed its potential to protect against intestinal infections, using Salmonella enterica serovar Enteritidis as a model. Our findings reveal a modulation effect of A. muciniphila with pro-inflammatory features, including the release of pro-inflammatory cytokines and upregulation of CD40 and CD80 surface markers, in contrast with previous reported data. Importantly, A. muciniphila could protect against Salmonella infection by promoting macrophage activation, appearing as a promising probiotic candidate for the control of intestinal infections.

High-throughput virtual search of small molecules for controlling the mechanical stability of human CD4.

Protein mechanical stability determines the function of a myriad of proteins, especially proteins from the extracellular matrix. Failure to maintain protein mechanical stability may result in diseases and disorders such as cancer, cardiomyopathies, or muscular dystrophy. Thus, developing mutation-free approaches to enhance and control the mechanical stability of proteins using pharmacology-based methods may have important implications in drug development and discovery. Here, we present the first approach that employs computational high-throughput virtual screening and molecular docking to search for small molecules in chemical libraries that function as mechano-regulators of the stability of human cluster of differentiation 4, receptor of HIV-1. Using single-molecule force spectroscopy, we prove that these small molecules can increase the mechanical stability of CD4D1D2 domains over 4-fold in addition to modifying the mechanical unfolding pathways. Our experiments demonstrate that chemical libraries are a source of mechanoactive molecules and that drug discovery approaches provide the foundation of a new type of molecular function, that is, mechano-regulation, paving the way toward mechanopharmacology.

Refining Immunogenicity through Intradermal Delivery of Outer Membrane Vesicles against Shigella flexneri in Mice.

Shigellosis remains a global health concern, especially in low- and middle-income countries. Despite improvements in sanitation, the absence of a licensed vaccine for human use has prompted global health organizations to support the development of a safe and effective multivalent vaccine that is cost-effective and accessible for limited-resource regions. Outer Membrane Vesicles (OMVs) have emerged in recent years as an alternative to live attenuated or whole-inactivated vaccines due to their immunogenicity and self-adjuvating properties. Previous works have demonstrated the safety and protective capacity of OMVs against Shigella flexneri infection in mouse models when administered through mucosal or intradermal routes. However, some immunological properties, such as the cellular response or cross-protection among different Shigella strains, remained unexplored. In this study, we demonstrate that intradermal immunization of OMVs with needle-free devices recruits a high number of immune cells in the dermis, leading to a robust cellular response marked by antigen-specific cytokine release and activation of effector CD4 T cells. Additionally, functional antibodies are generated, neutralizing various Shigella serotypes, suggesting cross-protective capacity. These findings highlight the potential of OMVs as a promising vaccine platform against shigellosis and support intradermal administration as a simple and painless vaccination strategy to address this health challenge.

Oral Immunogenicity of Enterotoxigenic Escherichia coli Outer Membrane Vesicles Encapsulated into Zein Nanoparticles Coated with a Gantrez® AN-Mannosamine Polymer Conjugate.

Enterotoxigenic Escherichia coli (ETEC) represents a major cause of morbidity and mortality in the human population. In particular, ETEC infections affect children under the age of five from low-middle income countries. However, there is no licensed vaccine against this pathogen. ETEC vaccine development is challenging since this pathotype expresses a wide variety of antigenically diverse virulence factors whose genes can be modified due to ETEC genetic plasticity. To overcome this challenge, we propose the use of outer membrane vesicles (OMVs) isolated from two ETEC clinical strains. In these OMVs, proteomic studies revealed the presence of important immunogens, such as heat-labile toxin, colonization factors, adhesins and mucinases. Furthermore, these vesicles proved to be immunogenic after subcutaneous administration in BALB/c mice. Since ETEC is an enteropathogen, it is necessary to induce both systemic and mucosal immunity. For this purpose, the vesicles, free or encapsulated in zein nanoparticles coated with a Gantrez®-mannosamine conjugate, were administered orally. Biodistribution studies showed that the encapsulation of OMVs delayed the transit through the gut. These results were confirmed by in vivo study, in which OMV encapsulation resulted in higher levels of specific antibodies IgG2a. Further studies are needed to evaluate the protection efficacy of this vaccine approach.

Effect of topical berberine in murine cutaneous leishmaniasis lesions.

OBJECTIVES: More effective topical treatments remain an unmet need for the localized forms of cutaneous leishmaniasis (CL). The aim of this study was to evaluate the efficacy and safety of a topical berberine cream in BALB/c mice infected with Leishmania major parasites. METHODS: A cream containing 0.5% berberine-ß-glycerophosphate salt and 2.5% menthol was prepared. Its physicochemical and stability properties were determined. The cream was evaluated for its capacity to reduce lesion size and parasitic load as well as to promote wound healing after twice-a-day administration for 35 days. Clinical biochemical profile was used for estimating off-target effects. In vitro time-to-kill curves in L. major-infected macrophages and skin and plasma pharmacokinetics were determined, aiming to establish pharmacokinetic/pharmacodynamic relationships. RESULTS: The cream was stable at 40°C for 3 months and at 4°C for at least 8 months. It was able to halt lesion progression in all treated mice. At the end of treatment, parasite load in the skin was reduced by 99.9% (4 log) and genes involved in the wound healing process were up-regulated compared with untreated mice.The observed effects were higher than expected from in vitro time-to-kill kinetic and plasma berberine concentrations, which ranged between 0.07 and 0.22 µM. CONCLUSIONS: The twice-a-day administration of a topical berberine cream was safe, able to stop parasite progression and improved the appearance of skin CL lesions. The relationship between drug plasma levels and in vivo effect was unclear.

Suicide Presentations to an Emergency Department Pre and During the COVID Lockdown, March-May 2020, in Spain.

The impact of the COVID-19 pandemic and lockdown may affect suicide risk and behavior. We compared suicide-related presentations during the national quarantine with pre-COVID data. This observational incidence study compared all suicide-related presentations at the University Hospital Reina Sofia emergency department (ED) in Cordoba, Spain, for the lockdown period from 15 March to 15 May 2020, with the same period in 2019. Descriptive and inferential analyses of case characteristics between 2019 and 2020 periods were conducted, and a multivariable logistic regression model developed. Results: Despite a decrease in overall ED cases during the lockdown, the number of suicide-related presentations stayed the same and represented a significantly larger proportion of cases in comparison to the pre-COVID period (0.42 vs. 0.87%, in 2019 and 2020, respectively; p < .001). The number presenting alone more than doubled during the COVID lockdown, increasing to 42.9% compared with 19.4% in 2019 (p = .002). An increase in presentations with a family history of suicide was also found. Conclusions: Suicide cases represented an increased proportion of ED cases during the lockdown. Presenting to ED alone, having a family history of suicide, habitual drug consumption, and hospital admission to ICU differentiated cases between pre and during COVID periods. These findings should be considered in light of the second wave and further implementation of lockdown measures.

Oral Efficacy of a Diselenide Compound Loaded in Nanostructured Lipid Carriers in a Murine Model of Visceral Leishmaniasis.

Leishmaniasis urgently needs new oral treatments, as it is one of the most important neglected tropical diseases that affects people with poor resources. The drug discovery pipeline for oral administration currently discards entities with poor aqueous solubility and permeability (class IV compounds in the Biopharmaceutical Classification System, BCS) such as the diselenide 2m, a trypanothione reductase (TR) inhibitor. This work was assisted by glyceryl palmitostearate and diethylene glycol monoethyl ether-based nanostructured lipid carriers (NLC) to render 2m bioavailable and effective after its oral administration. The loading of 2m in NLC drastically enhanced its intestinal permeability and provided plasmatic levels higher than its effective concentration (IC50). In L. infantum-infected BALB/c mice, 2m-NLC reduced the parasite burden in the spleen, liver, and bone marrow by at least 95% after 5 doses, demonstrating similar efficacy as intravenous Fungizone. Overall, compound 2m and its formulation merit further investigation as an oral treatment for visceral leishmaniasis.

Changes in the nanoparticle uptake and distribution caused by an intramacrophagic parasitic infection.

This study investigates if visceral leishmaniasis (VL) infection has some effects on the organ and cellular uptake and distribution of 100-200 nm near-infrared fluorescently labelled non-biodegradable polystyrene latex beads (PS NPs) or biodegradable polylactic-co-glycolic nanoparticles (PLGA NPs), as this parasitic infection produces morphological alterations in liver, spleen and bone marrow, organs highly involved in NP sequestration. The results showed that the magnitude of the effect was specific for each organ and type of NP. With the exception of the liver, the general trend was a decrease in NP organ and cellular uptake, mostly due to immune cell mobilization and/or weight organ gain, as vascular permeability was increased. Moreover, NPs redistributed among different phagocytic cells to adapt infection associated changes and cellular alterations. In the liver, it is noteworthy that only isolated Kuffer cells (KCs) captured NPs, whereas they were not taken up by KC forming granulomas. In the spleen, NPs redistributed from macrophages and dendritic cells towards B cells and inflammatory monocytes although they maintained their preferential accumulation in the marginal zone and red pulp. Comparatively, the infection rarely affected the NP cellular distribution in the bone marrow. NP cellular target changes in VL infection could affect their therapeutic efficacy and should be considered for more efficient drug delivery.

Berberine-Loaded Liposomes for the Treatment of Leishmania infantum-Infected BALB/c Mice.

Berberine (BER)-an anti-inflammatory quaternary isoquinoline alkaloid extracted from plants-has been reported to have a variety of biologic properties, including antileishmanial activity. This work addresses the preparation of BER-loaded liposomes with the aim to prevent its rapid liver metabolism and improve the drug selective delivery to the infected organs in visceral leishmaniasis (VL). BER liposomes (LP-BER) displayed a mean size of 120 nm, negative Z-potential of -38 mV and loaded 6 nmol/µmol lipid. In vitro, the loading of BER in liposomes enhanced its selectivity index more than 7-fold by decreasing its cytotoxicity to macrophages. In mice, LP-BER enhanced drug accumulation in the liver and the spleen. Consequently, the liposomal delivery of the drug reduced parasite burden in the liver and spleen by three and one logarithms (99.2 and 93.5%), whereas the free drug only decreased the infection in the liver by 1-log. The organ drug concentrations-far from IC50 values- indicate that BER immunomodulatory activity or drug metabolites also contribute to the efficacy. Although LP-BER decreased 10-fold-an extremely rapid clearance of the free drug in mice-the value remains very high. Moreover, LP-BER reduced plasma triglycerides levels.

Evaluation of Skin Permeation and Retention of Topical Dapsone in Murine Cutaneous Leishmaniasis Lesions.

The oral administration of dapsone (DAP) for the treatment of cutaneous leishmaniasis (CL) is effective, although serious hematological side effects limit its use. In this study, we evaluated this drug for the topical treatment of CL. As efficacy depends on potency and skin penetration, we first determined its antileishmanial activity (IC50 = 100 µM) and selectivity index in vitro against Leishmania major-infected macrophages. In order to evaluate the skin penetration ex vivo, we compared an O/W cream containing DAP that had been micronized with a pluronic lecithin emulgel, in which the drug was solubilized with diethylene glycol monoethyl ether. For both formulations we obtained similar low flux values that increased when the stratum corneum and the epidermis were removed. In vivo efficacy studies performed on L. major-infected BALB/c mice revealed that treatment not only failed to cure the lesions but made their evolution and appearance worse. High plasma drug levels were detected and were concomitant with anemia and iron accumulation in the spleen. This side effect was correlated with a reduction of parasite burden in this organ. Our results evidenced that DAP in these formulations does not have an adequate safety index for use in the topical therapy of CL.

Combination of paromomycin plus human anti-TNF-α antibodies to control the local inflammatory response in BALB/ mice with cutaneous leishmaniasis lesions.

BACKGROUND: Cutaneous leishmaniasis (CL) skin lesions are the result of a deregulated immune response, which is unable to eliminate Leishmania parasites. The control of both, parasites and host immune response, is critical to prevent tissue destruction. The skin ulceration has been correlated with high TNF-α level. OBJECTIVE: Because human anti-TNF-α antibodies (Ab) have been successfully assayed in several mice inflammatory diseases, we hypothesized that their anti-inflammatory effect could optimize the healing of CL lesions achieved after topical application of paromomycin (PM), the current chemotherapy against CL. METHODS AND RESULTS: We first compared the in vitro efficacy of PM and Ab alone and the drug given in combination with Ab to assess if the Ab could interfere with PM leishmanicidal activity in L. major-infected bone marrow-derived macrophages. The combination therapy had similar antileishmanial activity to the drug alone and showed no influence on NO production, which allows macrophage-mediated parasite killing. Next, we demonstrated in an in vivo model of Imiquimod®-induced inflammation that topical Ab and PM inhibit the infiltration of inflammatory cells in the skin. In the efficacy studies in L. major-infected BALB/c mice, PM combined with Ab led to a sharp infection reduction and showed a stronger anti-inflammatory activity than PM alone. This was confirmed by the down-regulation of TNF-α, IL-1ß, iNOS, IL-17, and CCL3 as well as by a decrease of the neutrophilic infiltrate during infection upon treatment with the Ab. CONCLUSIONS: In terms of parasite elimination and inflammation reduction, topical application of Ab in combination with PM was more effective than the drug alone.

Skin vaccination using microneedles coated with a plasmid DNA cocktail encoding nucleosomal histones of Leishmania spp.

Vaccine delivery using microneedles (MNs) represents a safe, easily disposable and painless alternative to traditional needle immunizations. The MN delivery of DNA vaccines to the dermis may result in a superior immune response and/or an equivalent immune response at a lower vaccine dose (dose-sparing). This could be of special interest for immunization programs against neglected tropical diseases such as leishmaniasis. In this work, we loaded a MN device with 60µg of a plasmid DNA cocktail encoding the Leishmania infantum nucleosomal histones H2A, H2B, H3 and H4 and compared its immunogenicity and protective capacity against conventional s.c. or i.d. injection of the plasmid. Mice immunized with MNs showed increased ratios of IFN-γ/IL-10, IFN-γ/IL-13, IFN-γ/IL-4, and IFN-γ/TGF-ß in the spleens and lymph nodes compared with mice immunized by s.c. and i.d. routes. Furthermore, CCXCL9, CXCL10 and CCL2 levels were also higher. These data suggest that the nucleic acid immunization using MNs produced a better bias towards a Th1 response. However, none of the immunizations strategies were able to control Leishmania major infection in BALB/c mice, as illustrated by an increase in lesion size and parasite burden.