Denervation of skeletal muscles induces severe muscle atrophy, which is preceded by cellular alterations such as increased plasma membrane permeability, reduced resting membrane potential and accelerated protein catabolism. The factors that induce these changes remain unknown. Conversely, functional recovery following denervation depends on successful reinnervation. Here, we show that activation of nicotinic acetylcholine receptors (nAChRs) by quantal release of acetylcholine (ACh) from motoneurons is sufficient to prevent changes induced by denervation. Using in vitro assays, ACh and non-hydrolysable ACh analogs repressed the expression of connexin43 and connexin45 hemichannels, which promote muscle atrophy. In co-culture studies, connexin43/45 hemichannel knockout or knockdown increased innervation of muscle fibers by dorsal root ganglion neurons. Our results show that ACh released by motoneurons exerts a hitherto unknown function independent of myofiber contraction. nAChRs and connexin hemichannels are potential molecular targets for therapeutic intervention in a variety of pathological conditions with reduced synaptic neuromuscular transmission.

Group B Streptococcus (GBS) is the primary etiological agent of sepsis and meningitis in newborns and is associated with premature birth and stillbirth. The development of a licensed vaccine is one of the pending challenges for the World Health Organization. Previously, we showed that oral immunization with surface immune protein (SIP) decreases vaginal colonization of GBS and generates functional opsonizing antibodies, which was determined by opsonophagocytic assays (OPA) in vitro. We also showed that the protein has an adjuvant vaccine profile. Therefore, an oral vaccine based on SIP may be an attractive alternative to employ in the development of new vaccines against GBS. Lactococcus lactis is a highlighted oral vaccine probiotic inducer of the mucosal immune response. This bacterium could serve as an antigen-delivering vehicle for the development of an edible vaccine and has been used in clinical trials. In this study, we showed that an oral vaccine with a recombinant L. lactis strain secreting SIP from GBS (rL. lactis-SIP) can induce protective humoral and cellular immunity in an experimental model of GBS vaginal colonization in C57BL/6 mice. Mice immunized with rL. lactis-SIP were protected against clinical symptoms and bacterial colonization after GBS vaginal colonization. Our rL. lactis-SIP vaccine also induces an increase of immunoglobulin G (IgG) and immunoglobulin A (IgA) specifically against SIP. The adoptive transfer of serum from vaccinated mice to naïve mice generated protection against GBS vaginal colonization. Moreover, the rL. lactis-SIP strain induces the activation of SIP-specific T cells, which could decrease GBS vaginal colonization and generate protective antibodies when transferred to other mice. Our experimental observations strongly support the notion that rL. lactis-SIP induces protective humoral and cellular immunity and could be considered as a novel alternative in the development of vaccines for GBS.

Background: Chile is one of the South American countries certified as malaria-free since 1945. However, the recent increase of imported malaria cases and the presence of the vector Anopheles pseudopunctipennis in previously endemic areas in Chile require an active malaria surveillance programme. Methods: Specimens from 268 suspected malaria cases-all imported-collected between 2015 and 2018 at the Public Health Institute of Chile (ISP), were diagnosed by microscopy and positive cases were included for epidemiological analysis. A photo-induced electron transfer fluorogenic primer real-time PCR (PET-PCR) was used to confirm the presence of malaria parasites in available blood samples. Sanger sequencing of drug resistance molecular markers (pfk13, pfcrt and pfmdr1) and microsatellite (MS) analysis were performed in confirmed Plasmodium falciparum samples and results were related to origin of infection. Results: Out of the 268 suspected cases, 65 were Plasmodium spp. positive by microscopy. A total of 63% of the malaria patients were male and 37% were female; 43/65 of the patients acquired infections in South American endemic countries. Species confirmation of available blood samples by PET-PCR revealed that 15 samples were positive for P. falciparum, 27 for Plasmodium vivax and 4 were mixed infections. The P. falciparum samples sequenced contained four mutant pfcrt genotypes (CVMNT, CVMET, CVIET and SVMNT) and three mutant pfmdr1 genotypes (Y184F/ S1034C/N1042D/D1246Y, Y184F/N1042D/D1246Y and Y184F). MS analysis confirmed that all P. falciparum samples presented different haplotypes according to the suspected country of origin. Four patients with P. vivax infection returned to the health facilities due to relapses. Conclusion: The timely detection of polymorphisms associated with drug resistance will contribute to understanding if current drug policies in the country are appropriate for treatment of imported malaria cases and provide information about the most frequent resistant genotypes entering Chile.

Vaccine-induced protection against pathogens, especially subunit-based vaccines, are related to antigen properties but mainly in their ability to stimulate the immune system by the use of an adjuvant. Modern vaccines are formulated with a high level of antigen purity, where an efficient adjuvant is necessary. In this context, the use of protein Toll-Like Receptor (TLR) agonists as vaccine adjuvants has been highlighted because of their optimal immunogenicity and minimal toxicity. The Surface Immunogenic Protein (SIP) from Group B Streptococcus (GBS) has gained importance as a new potential protein-based vaccine. Recently, we reported that recombinant SIP (rSIP) expressed by E. coli and purified by High Performance Liquid Chromatography (HPLC) alone induces a protective humoral immune response. In this study, we present the immunomodulatory properties of rSIP as a protein-based adjuvant, as an agonist of TLR. To this end, we showed that C57BL/6 bone marrow-derived dendritic cells pulsed by rSIP resulted in enhanced CD40, CD80, CD86, and Major Histocompatibility Complex (MHC) class II as well as increased secretion proinflammatory cytokines Interleukin (IL)-6, Interferon (IFN)-γ, Tumor Necrosis Factor (TNF)-α, and IL-10. Next, we investigated the in vivo effect of rSIP in the absence or presence of ovalbumin (OVA) on antigen-specific antibody secretion in C57BL/6 mice. Immunization with rSIP plus OVA showed that anti-OVA IgG2a and IgG1a increased significantly compared with OVA alone in C57BL/6 mice. Also, the immunization of rSIP plus OVA generates increased serum cytokines levels characterized by IL-12p70, IL-10, IL-4, and IFN-γ. Interestingly, we observed that rSIP stimulate Toll Like Receptor (TLR)2 and TLR4, individually expressed by Human embryonic kidney (HEK) 293-derived TLR reporter cells. These findings suggest that rSIP is a new potential protein TLR agonist adjuvant and may be employed in the development of new vaccines.

Background: Group B Streptococcus (GBS) is the leading cause of invasive neonatal infection. In this study, we aimed to evaluate the analytical validation of qualitative real-time polymerase chain reaction (qPCR) as a means to detect GBS. Methods: Genomic DNA (gDNA) was purified from 12 ATCC bacterial strains, two belonging to GBS and the remainder acting as negative controls. Additionally, gDNA was isolated from 21 strains of GBS from various serotypes (Ia, Ib and II-VIII). All gDNA was used to evaluate the analytical validation of the qPCR method employing a specific Taqman probe. Inclusivity, exclusivity, anticipated reportable range, the limit of detection and robustness were evaluated. The methods used are described in international guidelines and other existing reports. The performance of this qPCR method for detecting GBS was compared to other microbiological methods used with vaginal-rectal samples from pregnant women. Results: Our qPCR method for detecting GBS was analytically validated. It has a limit of detection of 0.7 GE/μL and 100% analytical specificity. It detects all strains of GBS with the same level of performance as microbiological methods. Conclusion: Data suggest that this qPCR method performs adequately as a means to detect GBS in vaginal-rectal swabs from pregnant women.