Metformin and Trehalose-Modulated Autophagy Exerts a Neurotherapeutic Effect on Parkinson's Disease.

Since the number of aged people will increase in the next years, neurodegenerative diseases, including Parkinson's Disease (PD), will also rise. Recently, we demonstrated that autophagy stimulation with rapamycin decreases dopaminergic neuronal death mediated by oxidative stress in the paraquat (PQ)-induced PD model. Assessing the neurotherapeutic efficacy of autophagy-inducing molecules is critical for preventing or delaying neurodegeneration. Therefore, we evaluated the autophagy inducers metformin and trehalose effect in a PD model. Autophagy induced by both molecules was confirmed in the SH-SY5Y dopaminergic cells by detecting increased LC3-II marker and autophagosome number compared to the control by western blot and transmission electron microscopy. Both autophagy inducers showed an antioxidant effect, improved mitochondrial activity, and decreased dopaminergic cell death induced by PQ. Next, we evaluated the effect of both inducers in vivo. C57BL6 mice were pretreated with metformin or trehalose before PQ administration. Cognitive and motor deteriorated functions in the PD model were evaluated through the nest building and the gait tests and were prevented by metformin and trehalose. Both autophagy inducers significantly reduced the dopaminergic neuronal loss, astrocytosis, and microgliosis induced by PQ. Also, cell death mediated by PQ was prevented by metformin and trehalose, assessed by TUNEL assay. Metformin and trehalose induced autophagy through AMPK phosphorylation and decreased α-synuclein accumulation. Therefore, metformin and trehalose are promising neurotherapeutic autophagy inducers with great potential for treating neurodegenerative diseases such as PD.

Pursuing Multiple Biomarkers for Early Idiopathic Parkinson's Disease Diagnosis.

Parkinson's disease (PD) ranks first in the world as a neurodegenerative movement disorder and occurs most commonly in an idiopathic form. PD patients may have motor symptoms, non-motor symptoms, including cognitive and behavioral changes, and symptoms related to autonomic nervous system (ANS) failures, such as gastrointestinal, urinary, and cardiovascular symptoms. Unfortunately, the diagnostic accuracy of PD by general neurologists is relatively low. Currently, there is no objective molecular or biochemical test for PD; its diagnosis is based on clinical criteria, mainly by cardinal motor symptoms, which manifest when patients have lost about 60-80% of dopaminergic neurons. Therefore, it is urgent to establish a panel of biomarkers for the early and accurate diagnosis of PD. Once the disease is accurately diagnosed, it may be easier to unravel idiopathic PD's pathogenesis, and ultimately, finding a cure. This review discusses several biomarkers' potential to set a panel for early idiopathic PD diagnosis and future directions.

4-1BBL as a Mediator of Cross-Talk between Innate, Adaptive, and Regulatory Immunity against Cancer.

The ability of tumor cells to evade the immune system is one of the main challenges we confront in the fight against cancer. Multiple strategies have been developed to counteract this situation, including the use of immunostimulant molecules that play a key role in the anti-tumor immune response. Such a response needs to be tumor-specific to cause as little damage as possible to healthy cells and also to track and eliminate disseminated tumor cells. Therefore, the combination of immunostimulant molecules and tumor-associated antigens has been implemented as an anti-tumor therapy strategy to eliminate the main obstacles confronted in conventional therapies. The immunostimulant 4-1BBL belongs to the tumor necrosis factor (TNF) family and it has been widely reported as the most effective member for activating lymphocytes. Hence, we will review the molecular, pre-clinical, and clinical applications in conjunction with tumor-associated antigens in antitumor immunotherapy, as well as the main molecular pathways involved in this association.

An Oncolytic Adenovirus Encoding SA-4-1BBL Adjuvant Fused to HPV-16 E7 Antigen Produces a Specific Antitumor Effect in a Cancer Mouse Model.

Human papillomaviruses (HPVs) are responsible for about 25% of cancer cases worldwide. HPV-16 E7 antigen is a tumor-associated antigen (TAA) commonly expressed in HPV-induced tumors; however, it has low immunogenicity. The interaction of 4-1BBL with its receptor induces pleiotropic effects on innate, adaptive, and regulatory immunity and, if fused to TAAs in DNA vaccines, can improve the antitumor response; however, there is low transfection and antitumor efficiency. Oncolytic virotherapy is promising for antitumor gene therapy as it can be selectively replicated in tumor cells, inducing cell lysis, and furthermore, tumor cell debris can be taken in by immune cells to potentiate antitumor responses. In this study, we expressed the immunomodulatory molecule SA-4-1BBL fused to E7 on an oncolytic adenovirus (OAd) system. In vitro infection of TC-1 tumor cells and NIH-3T3 non-tumor cells with SA/E7/4-1BBL OAd demonstrated that only tumor cells are selectively destroyed. Moreover, protein expression is targeted to the endoplasmic reticulum in both cell lines when a signal peptide (SP) is added. Finally, in an HPV-induced cancer murine model, the therapeutic oncolytic activity of OAd can be detected, and this can be improved when fused to E7 and SP.

Advances in gene therapy for hemophilia.

Hemophilia is a hereditary disorder that can be life-threatening in individuals who have severe spontaneous bleeding resulting from minor trauma or surgery. Although replacement therapy of the missing exogenous factor has improved patients' quality of life, it has not been possible to establish a long-term treatment. Due to the severity of the disease and the need for repetitive doses throughout the patient's life, replacement therapy has become a high-cost treatment option; therefore, the development of self-sustainable long-term therapies is critical. Hemophilia is a good candidate for gene therapy because it is a monogenic disease that can be counteracted by expression of the missing factor. In this article, we review some of the most relevant advances in gene therapy for this illness.

Autophagy Stimulation Decreases Dopaminergic Neuronal Death Mediated by Oxidative Stress.

The neurodegenerative process of Parkinson's disease (PD) involves autophagy impairment and oxidative stress. Therefore, we wanted to determine whether stimulation of autophagy protects dopaminergic cell death induced by oxidative stress in a PD model. Since environmental exposure to herbicides increases the risk to develop PD, the experimental model was established using the herbicide paraquat, which induces autophagy disruption, oxidative stress, and cell death. Rapamycin-stimulated autophagy inhibited calpain-dependent and independent apoptosis induced by paraquat. Autophagy stimulation decreased oxidative stress and peroxiredoxins (PRXs) hyperoxidation induced by paraquat. Cells exposed to paraquat displayed abnormally large autophagosomes enclosing mitochondria, which correlates with an increase of p62, an essential mitophagy regulator. Interestingly, when autophagy was stimulated before paraquat treatment, autophagosome size and number were similar to that observed in control cells. Motor and cognitive function impairment induced by paraquat showed an improvement when preceded by autophagy stimulation. Importantly, dopaminergic neuronal death and microglial activation mediated by paraquat were significantly reduced by rapamycin-induced autophagy. Our results indicate that autophagy stimulation has a protective effect on dopaminergic neurons and may have a promising potential to prevent or delay PD progression.

A DNA Vaccine Encoding SA-4-1BBL Fused to HPV-16 E7 Antigen Has Prophylactic and Therapeutic Efficacy in a Cervical Cancer Mouse Model.

The SA-4-1BBL, an oligomeric novel form of the natural ligand for the 4-1BB co-stimulatory receptor of the tumor necrosis factor (TNF) superfamily, as a recombinant protein has potent pleiotropic effects on cells of innate, adaptive, and regulatory immunity with demonstrated therapeutic efficacy in several tumor models. However, the production of soluble form of SA-4-1BBL protein and quality control is time and resource intensive and face various issues pertinent to clinical development of biologics. The present study sought to take advantage of the simplicity and translatability of DNA-based vaccines for the production and delivery of SA-4-1BBL for cancer immune prevention and therapy. A chimeric HPV-16 E7 DNA vaccine (SP-SA-E7-4-1BBL) was constructed that contains the signal peptide (SP) of calreticulin (CRT), streptavidin (SA) domain of SA-4-1BBL, HPV-16 E7 double mutant gene, and the extracellular domain of mouse 4-1BBL. Immunization by gene gun with SP-SA-E7-4-1BBL induced greater prophylactic as well as therapeutic effects in C57BL/6 mice against TC-1 tumor model compared with immunization with E7wt, SP-SA-4-1BBL or reference-positive control CRT-E7wt. The therapeutic efficacy of the DNA vaccine was associated with increased frequency of E7-specific T cells producing interferon (IFN)-γ. Overall, our data suggest that this DNA-based vaccine strategy might represent a translational approach because it provides a simpler and versatile alternative to a subunit vaccine based on SA-4-1BBL and E7 proteins.

Group X hybrid histidine kinase Chk1 is dispensable for stress adaptation, host-pathogen interactions and virulence in the opportunistic yeast Candida guilliermondii.

Hybrid histidine kinases (HHKs) progressively emerge as prominent sensing proteins in the fungal kingdom and as ideal targets for future therapeutics. The group X HHK is of major interest, since it was demonstrated to play an important role in stress adaptation, host-pathogen interactions and virulence in some yeast and mold models, and particularly Chk1, that corresponds to the sole group X HHK in Candida albicans. In the present work, we investigated the role of Chk1 in the low-virulence species Candida guilliermondii, in order to gain insight into putative conservation of the role of group X HHK in opportunistic yeasts. We demonstrated that disruption of the corresponding gene CHK1 does not influence growth, stress tolerance, drug susceptibility, protein glycosylation or cell wall composition in C. guilliermondii. In addition, we showed that loss of CHK1 does not affect C. guilliermondii ability to interact with macrophages and to stimulate cytokine production by human peripheral blood mononuclear cells. Finally, the C. guilliermondii chk1 null mutant was found to be as virulent as the wild-type strain in the experimental model Galleria mellonella. Taken together, our results demonstrate that group X HHK function is not conserved in Candida species.

Disruption of Protein Mannosylation Affects Candida guilliermondii Cell Wall, Immune Sensing, and Virulence.

The fungal cell wall contains glycoproteins that interact with the host immune system. In the prominent pathogenic yeast Candida albicans, Pmr1 acts as a Golgi-resident ion pump that provides cofactors to mannosyltransferases, regulating the synthesis of mannans attached to glycoproteins. To gain insight into a putative conservation of such a crucial process within opportunistic yeasts, we were particularly interested in studying the role of the PMR1 homolog in a low-virulent species that rarely causes candidiasis, Candida guilliermondii. We disrupted C. guilliermondii PMR1 and found that loss of Pmr1 affected cell growth and morphology, biofilm formation, susceptibility to cell wall perturbing agents, mannan levels, and the wall composition and organization. Despite the significant increment in the amount of ß1,3-glucan exposed at the wall surface, this positively influenced only the ability of the mutant to stimulate IL-10 production by human monocytes, suggesting that recognition of both mannan and ß1,3-glucan, is required to stimulate strong levels of pro-inflammatory cytokines. Accordingly, our results indicate C. guilliermondii sensing by monocytes was critically dependent on the recognition of N-linked mannans and ß1,3-glucan, as reported in other Candida species. In addition, chemical remotion of cell wall O-linked mannans was found to positively influence the recognition of C. guilliermondii by human monocytes, suggesting that O-linked mannans mask other cell wall components from immune cells. This observation contrasts with that reported in C. albicans. Finally, mice infected with C. guilliermondii pmr1Δ null mutant cells had significantly lower fungal burdens compared to animals challenged with the parental strain. Accordingly, the null mutant showed inability to kill larvae in the Galleria mellonella infection model. This study thus demonstrates that mannans are relevant for the C. guilliermondii-host interaction, with an atypical role for O-linked mannans.

Comparative in vitro study of the antimicrobial activities of different commercial antibiotic products for intravenous administration.

BACKGROUND: The antimicrobial resistance is a global problem, probably due to the indiscriminate and irrational use of antibiotics, prescriptions for incorrect medicines or incorrect determinations of dose, route and/or duration. Another consideration is the uncertainty of patients receiving antibiotics about whether the quality of a generic medicine is equal to, greater than or less than its equivalent brand-name drug. The antibiotics behaviors must be evaluated in vitro and in vivo in order to confirm their suitability for therapeutic use. METHODS: The antimicrobial activities of Meropenem and Piperacillin/Tazobactam were studied by microbiological assays to determine their potencies (content), minimal inhibitory concentrations (MICs), critical concentrations and capacity to produce spontaneous drug-resistant mutants. RESULTS: With respect to potency (content) all the products fulfill USP requirements, so they should all be considered pharmaceutical equivalents. The MIC values of the samples evaluated (trade marks and generics) were the same for each strain tested, indicating that all products behaved similarly. The critical concentration values were very similar for all samples, and the ratios between the critical concentration of the standard and those of each sample were similar to the ratios of their specific antibiotic contents. Overall, therefore, the results showed no significant differences among samples. Finally, the production of spontaneous mutants did not differ significantly among the samples evaluated. CONCLUSIONS: All the samples are pharmaceutical equivalents and the products can be used in antimicrobial therapy.