Non-coding RNAs in Alzheimer's disease: perspectives from omics studies.

Neurodegenerative diseases such as Alzheimer's disease (AD) are characterized by the progressive loss of neurons in the brain and the spinal cord. The pathophysiology of AD is multifactorial with heterogeneous molecular manifestations. The lack of efficacious therapies for AD reinforces the importance of exploring in depth multifaceted disease mechanisms. Recent progresses on AD have generated a large amount of RNA-sequencing data at both bulk and single cell levels and revealed thousands of genes with expression changes in AD. However, the upstream regulators of such gene expression changes are largely unknown. Non-coding RNAs (ncRNAs) represent the majority of the human transcriptome, and regulatory ncRNAs have been found to play an important role in regulating gene expression. A single miRNA usually targets a number of mRNAs and thus such ncRNAs are particular important for understanding disease mechanisms and developing novel therapeutics. This review aims to summarize the recent findings on the roles of ncRNAs in AD from ncRNA-omics studies with a focus on ncRNA signatures, interactions between ncRNAs and mRNAs, and ncRNA-regulated pathways in AD. We also review the potential of specific ncRNAs to serve as biomarkers and therapeutic targets for AD. In the end, we point out future directions for studying ncRNAs in AD.

New synthetic molecules incorporated into polymeric micelles used for treatment against visceral leishmaniasis.

Treatment against visceral leishmaniasis (VL) presents problems, mainly related to drug toxicity, high cost and/or by emergence of resistant strains. In the present study, two vanillin synthetic derivatives, 3 s [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] and 3 t [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde], were evaluated as therapeutic candidates in a murine model against Leishmania infantum infection. Molecules were used pure (3 s and 3 t) or incorporated into Poloxamer 407-based micelles (3 s/M and 3 t/M) in the infected animals, which also received amphotericin B (AmpB) or Ambisome® as control. Results showed that 3 s/M and 3 t/M compositions induced a Th1-type immune response in treated animals, with higher levels of IFN-γ, IL-2, TNF-α, IL-12, nitrite, and IgG2a antibodies. Animals presented also low toxicity and significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, as compared as control groups mice, with the evaluations performed one and 30 days after the application of the therapeutics. In conclusion, preliminary data suggest that 3 s/M and 3 t/M could be considered for future studies as therapeutic agents against VL.

Angiotensin-converting enzymes 1 and 2 in the feces: presence and catalytic activity in the rat 2,4,6-trinitrobenzene sulfonic acid-induced model of colitis.

BACKGROUND AND AIM: Inflammatory bowel disease is challenging to diagnose. Fecal biomarkers offer noninvasive solutions. The renin-angiotensin-aldosterone system is implicated in intestinal inflammation. Angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) regulate its activity, but conflicting findings on these enzymes in colitis require further investigation. We aimed to assess ACE and ACE2 presence and activities in the feces, serum, and colon of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced rats. METHODS: Colitis was induced in male rats by rectal instillation of a 21% ethanolic TNBS solution. After rats' sacrifice, colonic portions, serum, and feces were collected. ACE and ACE2 presence in the feces was analyzed by western Blot, and colonic and serum enzymes' concentrations were quantified using ELISA kits. ACE activity was assessed using Hippuryl-His-Leu and Z-Phe-His-Leu as substrates. ACE2 activity was assessed using Mca-APK (Dnp) as a substrate in the presence and absence of DX600 (ACE2 inhibitor). RESULTS: An ACE isoform of ~70 kDa was found only in the feces of TNBS-induced rats. ACE concentration was higher than that of ACE2 in the serum and the inflamed colon. ACE N-domain activity was higher than that of the C-domain in all matrices. ACE2 activity was higher in the feces of TNBS-induced animals compared to controls. CONCLUSION: A 70 kDa ACE isoform only detected in the feces of TNBS-induced rats may have translational relevance. ACE N-domain seems to play a significant role in regulating colonic lesions. Further research using human samples is necessary to validate these findings.

Treatment using vanillin-derived synthetic molecules incorporated into polymeric micelles is effective against infection caused by Leishmania amazonensis species.

Treatment against leishmaniasis presents problems, mainly due to the toxicity of the drugs, high cost, and the emergence of resistant strains. A previous study showed that two vanillin-derived synthetic molecules, 3s [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] and 3t [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde], presented antileishmanial activity against Leishmania infantum, L. amazonensis, and L. braziliensis species. In the present work, 3s and 3t were evaluated to treat L. amazonensis-infected mice. Molecules were used pure or incorporated into Poloxamer 407-based micelles. In addition, amphotericin B (AmpB) and its liposomal formulation, Ambisome®, were used as control. Animals received the treatment and, one and 30 days after, they were euthanized to evaluate immunological, parasitological, and biochemical parameters. Results showed that the micellar compositions (3s/Mic and 3t/Mic) induced significant reductions in the lesion mean diameter and parasite load in the infected tissue and distinct organs, as well as a specific and significant antileishmanial Th1-type immune response, which was based on significantly higher levels of IFN-γ, IL-12, nitrite, and IgG2a isotype antibodies. Drug controls showed also antileishmanial action; although 3s/Mic and 3t/Mic have presented better and more significant parasitological and immunological data, which were based on significantly higher IFN-γ production and lower parasite burden in treated animals. In addition, significantly lower levels of urea, creatinine, alanine transaminase, and aspartate transaminase were found in mice treated with 3s/Mic and 3t/Mic, when compared to the others. In conclusion, results suggest that 3s/Mic and 3t/Mic could be considered as therapeutic candidates to treat against L. amazonensis infection.

Multiomics Analyses Identify Proline Endopeptidase-Like Protein As a Key Regulator of Protein Trafficking, a Pathway Underlying Alzheimer's Disease Pathogenesis.

Current treatments for Alzheimer's disease (AD) help reduce symptoms for a limited time but do not treat the underlying pathology. To identify potential therapeutic targets for AD, an integrative network analysis was previously carried out using 364 human postmortem control, mild cognitive impairment, and AD brains. This analysis identified proline endopeptidase-like protein (PREPL), an understudied protein, as a downregulated protein in late-onset AD patients. In this study we investigate the role of PREPL. Analyses of data from human postmortem samples and PREPL knockdown (KD) cells suggest that PREPL expression modulates pathways associated with protein trafficking, synaptic activities, and lipid metabolism. Furthermore, PREPL KD impairs cell proliferation and modulates the structure of vesicles, levels of neuropeptide-processing enzymes, and secretion of neuropeptides. In addition, decrease in PREPL levels leads to changes in the levels of a number of synaptic proteins as well as changes in the levels of secreted amyloid beta (Aß) 42 peptide and Tau phosphorylation. Finally, we report that local decrease in PREPL levels in mouse hippocampus attenuates long-term potentiation, suggesting a role in synaptic plasticity. Together, our results indicate that PREPL affects neuronal function by modulating protein trafficking and synaptic function, an important mechanism of AD pathogenesis. SIGNIFICANCE STATEMENT: Integrative network analysis reveals proline endopeptidase-like protein (PREPL) to be downregulated in human sporadic late-onset Alzheimer's disease brains. Down regulation of PREPL leads to increases in amyloid beta secretion, Tau phosphorylation, and decreases in protein trafficking and long-term potentiation.

Immunization with recombinant LiHyp1 protein plus adjuvant is protective against tegumentary leishmaniasis.

Tegumentary leishmaniasis (TL) is the main clinical manifestation of leishmaniasis, and it can cause the infected hosts to self-healing cutaneous lesions until mutilating scars in mucosal membranes, particularly in the nose and throat. The treatment against disease presents problems, and the diagnosis is hampered by variable sensitivity and/or specificity of the tests. In this context, the development of prophylactic vaccines could be considered as a strategy to control the disease. Previously, we showed that the recombinant LiHyp1 protein plus adjuvant protected mice from infection with Leishmania infantum, which causes visceral leishmaniasis. In the present study, we tested whether rLiHyp1 could induce protection against infection with L. amazonensis, a parasite species able to cause TL. We immunized BALB/c mice with rLiHyp1 plus saponin (rLiHyp1/S) or incorporated in micelles (rLiHyp1/M) as adjuvants and performed parasitological and immunological evaluations before and after infection. Results showed that after in vitro stimulation from spleen cell cultures using rLiHyp1 or a Leishmania antigenic extract (SLA), rLiHyp1/S and rLiHyp1/M groups developed a Th1-type immune response, which was characterized by high levels of IFN-γ, IL-2, TNF-α and IL-12 cytokines, nitrite, and IgG2a isotype antibodies when compared to values found in the control (saline, saponin, micelles alone) groups, which showed higher levels of anti-SLA IL-4, IL-10, and IgG1 antibodies before and after challenge. In addition, mice receiving rLiHyp1/S or rLiHyp1/M presented significant reductions in the lesion average diameter and parasite load in the infected tissue and internal organs. Blood samples were collected from healthy subjects and TL patients to obtain PBMC cultures, which were in vitro stimulated with rLiHyp1 or SLA, and results showed higher lymphoproliferation and IFN-γ production after stimulus using rLiHyp1, as compared to values found using SLA. These results suggest that rLiHyp1 plus adjuvant was protective against experimental TL and could also be considered for future studies as a vaccine candidate against human disease.

GPR83 engages endogenous peptides from two distinct precursors to elicit differential signaling.

PEN is an abundant neuropeptide that activates GPR83, a G protein-coupled receptor that is considered a novel therapeutic target due to its roles in regulation of feeding, reward, and anxiety-related behaviors. The major form of PEN in the brain is 22 residues in length. Previous studies have identified shorter forms of PEN in mouse brain and neuroendocrine cells; these shorter forms were named PEN18, PEN19 and PEN20, with the number reflecting the length of the peptide. The C-terminal five residues of PEN20 are identical to the C-terminus of a procholecystokinin (proCCK)-derived peptide, named proCCK56-62, that is present in mouse brain. ProCCK56-62 is highly conserved across species although it has no homology to the bioactive cholecystokinin domain. ProCCK56-62 and a longer form, proCCK56-63 were tested for their ability to engage GPR83. Both peptides bind GPR83 with high affinity, activate second messenger pathways, and induce ligand-mediated receptor endocytosis. Interestingly, the shorter PEN peptides, ProCC56-62, and ProCCK56-63 differentially activate signal transduction pathways. Whereas PEN22 and PEN20 facilitate receptor coupling to Gai, PEN18, PEN19 and ProCCK peptides facilitate coupling to Gas. Furthermore, the ProCCK peptides exhibit dose dependent Ga subtype selectivity in that they faciliate coupling to Gas at low concentrations and Gai at high concentrations. These data demonstrate that peptides derived from two distinct peptide precursors can differentially activate GPR83, and that GPR83 exhibits Ga subtype preference depending on the nature and concentration of the peptide. These results are consistent with the emerging idea that endogenous neuropeptides function as biased ligands. Significance Statement We found that peptides derived from proCCK bind and activate GPR83, a G protein-coupled receptor that is known to bind peptides derived from proSAAS. Different forms of the proCCK- and proSAAS-derived peptides show biased agonism, activating Gas or Gai depending on the length of the peptide and/or its concentration.

The ambiguous role of obesity in oncology by promoting cancer but boosting antitumor immunotherapy.

Obesity is nowadays considered a pandemic which prevalence's has been steadily increasingly in western countries. It is a dynamic, complex, and multifactorial disease which propitiates the development of several metabolic and cardiovascular diseases, as well as cancer. Excessive adipose tissue has been causally related to cancer progression and is a preventable risk factor for overall and cancer-specific survival, associated with poor prognosis in cancer patients. The onset of obesity features a state of chronic low-grade inflammation and secretion of a diversity of adipocyte-derived molecules (adipokines, cytokines, hormones), responsible for altering the metabolic, inflammatory, and immune landscape. The crosstalk between adipocytes and tumor cells fuels the tumor microenvironment with pro-inflammatory factors, promoting tissue injury, mutagenesis, invasion, and metastasis. Although classically established as a risk factor for cancer and treatment toxicity, recent evidence suggests mild obesity is related to better outcomes, with obese cancer patients showing better responses to treatment when compared to lean cancer patients. This phenomenon is termed obesity paradox and has been reported in different types and stages of cancer. The mechanisms underlying this paradoxical relationship between obesity and cancer are still not fully described but point to systemic alterations in metabolic fitness and modulation of the tumor microenvironment by obesity-associated molecules. Obesity impacts the response to cancer treatments, such as chemotherapy and immunotherapy, and has been reported as having a positive association with immune checkpoint therapy. In this review, we discuss obesity's association to inflammation and cancer, also highlighting potential physiological and biological mechanisms underlying this association, hoping to clarify the existence and impact of obesity paradox in cancer development and treatment.

Leishmania LiHyC protein is immunogenic and induces protection against visceral leishmaniasis.

Treatment against visceral leishmaniasis (VL) presents problems by the toxicity of drugs, high cost and/or emergence of resistant strains. The diagnosis is hampered by variable sensitivity and/or specificity of tests. In this context, prophylactic vaccination could represent a control measure against disease. In this study, the protective efficacy of Leishmania LiHyC protein was evaluated in a murine model against Leishmania infantum infection. LiHyC was used as recombinant protein (rLiHyC) associated with saponin (rLiHyC/S) or Poloxamer 407-based polymeric micelles (rLiHyC/M) to immunize mice. Animals received also saline, saponin or empty micelles as controls. The immunogenicity was evaluated before and after the challenge, and results showed that vaccination with rLiHyC/S or rLiHyC/M induced the production of high levels of interferon-gamma (IFN-γ), interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor in cell culture supernatants, as well as higher IFN-γ expression evaluated by RT-qPCR and involvement from CD4+ and CD8+ T-cell subtypes producing IFN-γ, tumor necrosis factor-α and IL-2. A positive lymphoproliferative response was also found in cell cultures from vaccinated animals, besides high levels of rLiHyC- and parasite-specific nitrite and IgG2a antibodies. Immunological assays correlated with significant reductions in the parasite load in the spleens, livers, bone marrows and draining lymph nodes from vaccinated mice, when compared to values found in the controls. The micellar composition showed slightly better immunological and parasitological data, as compared to rLiHyC/S. Results suggest that rLiHyC associated with adjuvants could be considered for future studies as a vaccine candidate against VL.

Development of an immunogen containing CD4+/CD8+ T-cell epitopes for the prophylaxis of tegumentary leishmaniasis.

Tegumentary leishmaniasis (TL) is a disease of high severity and incidence in Brazil, and Leishmania braziliensis is its main etiological agent. The inefficiency of control measures, such as high toxicity and costs of current treatments and the lack of effective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present work developed a gene encoding multiple T-cell (CD4+/CD8+) epitope, derived from conserved proteins found in Leishmania species and associated with TL, to generate a chimeric protein (rMEP/TL) and compose a vaccine formulation. For this, six T-cell epitopes were selected by immunoinformatics approaches from proteins present in the amastigote stage and associated with host-parasite interactions. The following formulations were then tested in an L. braziliensis murine infection model: rMEP/TL in saline or associated with MPLA-PHAD®. Our data revealed that, after immunization (three doses; 14-day intervals) and subsequent challenging, rMEP/TL and rMEP/TL + MPLA-vaccinated mice showed an increased production of key immunological biomarkers of protection, such as IgG2a, IgG2a/IgG1, NO, CD4+, and CD8+ T-cells with IFN-γ and TNF-α production, associated with a reduction in CD4+IL-10+ and CD8+IL-10+ T-cells. Vaccines also induced the development of central (CD44highCD62Lhigh) and effector (CD44highCD62Llow) memory of CD4+ and CD8+ T-cells. These findings, associated with the observation of lower rates of parasite burdens in the vaccinated groups, when compared to the control groups, suggest that immunization with rMEP/TL and, preferably, associated with an adjuvant, may be considered an effective tool to prevent TL. KEY POINTS: • Rational design approaches for vaccine development. • Central and effector memory of CD4+ and CD8+ T-cells. • Vaccine comprised of rMEP/TL plus MPLA as an effective tool to prevent TL.

Flau-A, a naphthoquinone derivative, is a promising therapeutic candidate against visceral leishmaniasis: A preliminary study.

Visceral leishmaniasis (VL) is a neglected tropical disease found in tropical and subtropical regions in the world. The therapeutics used for the treatment against disease presents problems, mainly related to drug toxicity, route of administration, high cost and/or by emergence of resistant strains. In this context, the search for alternative antileishmanial candidates is desirable. Recently, a naphthoquinone derivative namely 2-(2,3,4-tri-O-acetyl-6-deoxy-ß-L-galactopyranosyloxy)-1,4-naphthoquinone or Flau-A showed an effective in vitro biological action against Leishmania infantum. In the present study, the efficacy of this naphthoquinone derivative was evaluated in an in vivo infection model. BALB/c mice (n = 12 per group) were infected and later received saline or were treated with empty micelles (B/Mic), free Flau-A or it incorporated in Poloxamer 407-based micelles (Flau-A/Mic). The products were administered subcutaneously in the infected animals, which were then euthanized one (n = 6 per group) and 15 (n = 6 per group) days post-therapy, when immunological and parasitological evaluations were performed. Results showed that animals treated with Flau-A or Flau-A/Mic produced significantly higher levels of antileishmanial IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and IgG2a isotype antibody, when compared to data found in the control (saline and B/Mic) groups; which showed significantly higher levels of parasite-specific IL-4, IL-10 and IgG1 antibody. In addition, animals receiving free Flau-A or Flau-A/Mic presented also significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, when compared to the controls. A low hepatic and renal toxicity was also found. Overall, Flau-A/Mic showed better immunological and parasitological results, when compared to the use of free molecule. In conclusion, preliminary data suggest that this composition could be considered in future studies as promising therapeutic candidate against VL.

Osteoarticular infections in infants under 3 months of age.

BACKGROUND: Acute osteoarticular infections (OAI) in infants under 3 months of age (≤3M) are rare and remain a diagnostic challenge. Orthopedic complications and functional sequelae have been less well described in this age group. Our aims were to evaluate trends in aetiology, management, and outcomes of OAI ≤ 3M, and to compare these younger children who have OAI with older children. METHODS: A longitudinal observational study was conducted of OAI cases admitted to tertiary care pediatric hospital from 2008 to 2018. OAI ≤ 3M was compared with children above 3 months. Clinical, microbiological, imaging, and outcome data were analyzed. RESULTS: We identified 24 (9.1%) of the 263 OAI in children under 3 months. Analyzing OAI ≤ 3M there was a twofold increase since 2014; 54% were males with a median age of 28 days (IQR: 13.5-60.0), 10 (41.7%) were premature and nine (37.5%) had healthcare-associated infections. Microbiological causes were identified in 87.5%, mostly Staphylococcus aureus (57.1%) and Group B Streptococcus (23.8%), and 25% were multidrug-resistant (5 methicillin-resistant S. aureus and 1 Enterobacter cloacae). Bacteremia (100% vs 36.8%, P = 0.037), multidrug resistant bacteria (75% vs 16, P = 0.04), and healthcare-associated infections (100% vs 26.3%, P = 0.014) were associated with sequelae. Comparing OAI ≤ 3M with older children, OAI ≤ 3M were treated with longer antibiotic courses, had more complications and sequelae (17.4% vs 3.2%, P = 0.002). CONCLUSIONS: S. aureus is still the most common cause of OAI ≤ 3M, and 25% of causative bacteria were multidrug-resistant bacteria. Complications and sequelae were more frequent in OAI ≤ 3M when compared with older children.

Parasitological and immunological evaluation of a quinoline derivative salt incorporated into a polymeric micelle formulation against Leishmania infantum infection.

Leishmaniasis is a parasitic disease caused by Leishmania protozoa, which presents a large spectrum of clinical manifestations. In the present study, a quinoline derivative salt named N-(2-((7-chloroquinolin-4-yl)amino)ethyl)-N-(prop-2-yn-1-yl)prop-2-yn-1-aminium chloride or QDS3 was in vitro and in vivo tested against L. infantum by means of its incorporation in Poloxamer 407-based polymeric micelles (QDS3/M). The in vitro antileishmanial activity of QDS3 and QDS3/M was investigated in L. infantum promastigotes, axenic amastigotes and infected macrophages. BALB/c mice were infected with L. infantum, and parasitological parameters were evaluated 1 and 15 days post-treatment by determining the parasite load by a limiting dilution assay, besides a quantitative PCR (qPCR) method. Immunological response was assessed based on production of cellular cytokines, as well as by quantification of nitrite levels and specific antibodies. In vitro results showed that QDS3 free or in micelles presented effective antileishmanial action against both parasite stages, being more effective in amastigotes. In vivo data showed that treatment using QDS3 or QDS3/M reduced the parasite load in the livers, spleens, draining lymph nodes (dLN) and bone marrows of the treated animals, 1 and 15 days after treatment, when compared to values found in the control groups. Additionally, treated mice developed a polarized Th1-type immune response, with higher levels of IL-12, IFN-γ, GM-CSF and nitrite, besides high production of specific IgG2a antibodies, when compared to the controls. Parasitological and immunological data obtained using the micellar composition were better than the others. In conclusion, QDS3, mainly when applied in a delivery adjuvant system, could be considered for future studies as therapeutic candidate against VL.

Histomorphometry Changes and Decreased Reactivity to Angiotensin II in the Ileum and Colon of Streptozotocin-Induced Diabetic Rats.

Diabetes mellitus (DM) is a chronic progressive metabolic disorder associated with several gastrointestinal complications, affecting up to 75% of patients. Knowing that Angiotensin II (AngII) also regulates intestinal contraction, we decided to evaluate changes in ileum and colon histomorphometry and AngII reactivity in a rat model of DM. Streptozotocin (STZ, 55 mg/kg) was administered to induce DM to 24 adult male Wistar rats. Diabetic rats displayed all the characteristic signs of type 1 DM (T1DM) and fecal excretion increased about 4-fold over 14 days, while the excretion of controls remained unaltered. Compared to controls, diabetic ileum and colon presented an increase in both macroscopic (length, perimeter and weight) and microscopic (muscular wall thickness) parameters. Functionally, AngII-induced smooth muscle contraction was lower in diabetic rats, except in the distal colon. These differences in the contractile response to AngII may result from an imbalance between AngII type 1 (antagonized by candesartan, 10 nM) and type 2 receptors activation (antagonized by PD123319, 100 nM). Taken together, these results indicate that an early and refined STZ-induced T1DM rat model already shows structural remodelling of the gut wall and decreased contractile response to AngII, findings that may help to explain diabetic dysmotility.

Fetal Undernutrition Modifies Vascular RAS Balance Enhancing Oxidative Damage and Contributing to Remodeling.

Fetal stress is known to increase susceptibility to cardiometabolic diseases and hypertension in adult age in a process known as fetal programming. This study investigated the relationship between vascular RAS, oxidative damage and remodeling in fetal programming. Six-month old Sprague-Dawley offspring from mothers that were fed ad libitum (CONTROL) or with 50% intake during the second half of gestation (maternal undernutrition, MUN) were used. qPCR or immunohistochemistry were used to obtain the expression of receptors and enzymes. Plasma levels of carbonyls were measured by spectrophotometry. In mesenteric arteries from MUN rats we detected an upregulation of ACE, ACE2, AT1 receptors and NADPH oxidase, and lower expression of AT2, Mas and MrgD receptors compared to CONTROL. Systolic and diastolic blood pressure and plasma levels of carbonyls were higher in MUN than in CONTROL. Vascular morphology evidenced an increased media/lumen ratio and adventitia/lumen ratio, and more connective tissue in MUN compared to CONTROL. In conclusion, fetal undernutrition indices RAS alterations and oxidative damage which may contribute to the remodeling of mesenteric arteries, and increase the risk of adverse cardiovascular events and hypertension.

Acarbose presents in vitro and in vivo antileishmanial activity against Leishmania infantum and is a promising therapeutic candidate against visceral leishmaniasis.

Treatment against visceral leishmaniasis (VL) is mainly hampered by drug toxicity, long treatment regimens and/or high costs. Thus, the identification of novel and low-cost antileishmanial agents is urgent. Acarbose (ACA) is a specific inhibitor of glucosidase-like proteins, which has been used for treating diabetes. In the present study, we show that this molecule also presents in vitro and in vivo specific antileishmanial activity against Leishmania infantum. Results showed an in vitro direct action against L. infantum promastigotes and amastigotes, and low toxicity to mammalian cells. In addition, in vivo experiments performed using free ACA or incorporated in a Pluronic® F127-based polymeric micelle system called ACA/Mic proved effective for the treatment of L. infantum-infected BALB/c mice. Treated animals presented significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes when compared to the controls, as well as the development of antileishmanial Th1-type humoral and cellular responses based on high levels of IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and IgG2a isotype antibodies. In addition, ACA or ACA-treated animals suffered from low organ toxicity. Treatment with ACA/Mic outperformed treatments using either Miltefosine or free ACA based on parasitological and immunological evaluations performed one and 15 days post-therapy. In conclusion, data suggest that the ACA/Mic is a potential therapeutic agent against L. infantum and merits further consideration for VL treatment.

Development of a chimeric protein based on a proteomic approach for the serological diagnosis of human tegumentary leishmaniasis.

Leishmania braziliensis is responsible for most cases of human tegumentary leishmaniasis (HTL) and has caused a wide range of clinical manifestations, including cutaneous (CL) and mucosal leishmaniasis (ML). The diagnosis is based on criteria that consider epidemiological data, clinical findings, and laboratory tests and is hard to establish. For laboratory tests, none of the assays available can be considered gold standards for disease detection. In addition, the Montenegro skin test, essential to supporting infectologists in the clinical management of the disease, is no longer available in Brazil. Thus, the aim of this study was to develop new targets to be used in diagnostic tests for HTL. In the first step, we carried out two-dimensional gel electrophoresis, followed by mass spectrometry, combined with heat map analysis and immunoproteomics approach, and disclosed eight proteins expressed in the amastigote stage specifically recognized by serum from CL and ML patients. A chimeric protein was designed based on the combination of thirteen linear B-cell epitopes, identified by immunoinformatics analysis, from L. braziliensis proteins. Our results showed that the strategy used in this work was successful in developing an antigen to be used in immunological assays (100.0% sensitivity and specificity) in the detection of HTL cases and in comparison with results obtained from an ELISA using soluble L. braziliensis antigen (SLb-Antigen) and immunofluorescence assay (Bio-Manguinhos/FIOCRUZ). The present technology opens the door for its use in field exams by means of an immunochromatographic test, which will be even more helpful in regions without laboratory structures.Key points• Rational strategy to develop antigens.• Integration between immunoproteomic and immunoinformatics analysis.• Chimeric protein shows high performance in HTL diagnosis.

Interaction between the Renin-Angiotensin System and Enteric Neurotransmission Contributes to Colonic Dysmotility in the TNBS-Induced Model of Colitis.

Angiotensin II (Ang II) regulates colon contraction, acting not only directly on smooth muscle but also indirectly, interfering with myenteric neuromodulation mediated by the activation of AT1 /AT2 receptors. In this article, we aimed to explore which mediators and cells were involved in Ang II-mediated colonic contraction in the TNBS-induced rat model of colitis. The contractile responses to Ang II were evaluated in distinct regions of the colon of control animals or animals with colitis in the absence and presence of different antagonists/inhibitors. Endogenous levels of Ang II in the colon were assessed by ELISA and the number of AT1/AT2 receptors by qPCR. Ang II caused AT1 receptor-mediated colonic contraction that was markedly decreased along the colons of TNBS-induced rats, consistent with reduced AT1 mRNA expression. However, the effect mediated by Ang II is much more intricate, involving (in addition to smooth muscle cells and nerve terminals) ICC and EGC, which communicate by releasing ACh and NO in a complex mechanism that changes colitis, unveiling new therapeutic targets.

A Leishmania amastigote-specific hypothetical protein evaluated as recombinant protein plus Th1 adjuvant or DNA plasmid-based vaccine to protect against visceral leishmaniasis.

Most studies evaluating vaccine candidates against visceral leishmaniasis (VL) have used parasite promastigote-expressed antigens; however, Leishmania proteins expressed in the amastigote forms should be considered, since few hours after infection this stage comes into contact with the host immune system and is responsible for the development of the disease. In this context, in the present study, a Leishmania amastigote-specific hypothetical protein, called LiHyJ, was evaluated as a recombinant protein plus saponin as an adjuvant or DNA vaccine to protect against VL. The vaccine effect was evaluated by means of the evaluation of immunological and parasitological analyses performed in BALB/c mice against Leishmania infantum infection. Results showed that rLiHyJ/saponin and DNA LiHyJ induced significantly higher levels of anti-protein and anti-parasite IFN-γ, IL-12, GM-CSF, and IgG2a isotype antibodies, which were associated with a low presence of IL-4 and IL-10. DNA vaccination induced higher IFN-γ production, mainly by CD8+ T cells, while rLiHyJ/saponin stimulated the production of this cytokine, mainly by CD4+ T cells. The parasite load evaluated in distinct organs showed that both immunization schedules significantly reduced organic parasitism, when compared to the controls. Similar results were found in the immunological and parasitological assays when using the recombinant protein or DNA, although the vaccination with rLiHyJ plus saponin induced a slightly higher Th1 response and lower parasite load, when compared to the use of DNA plasmid. The protein also proved to be immunogenic when peripheral blood mononuclear cells of treated VL patients and healthy subjects were in vitro stimulated, since higher IFN-γ and lower IL-4 and IL-10 levels were found in the culture supernatants. In conclusion, LiHyJ should be considered in future studies as a vaccine candidate to protect against VL.

Parasitological and immunological evaluation of a novel chemotherapeutic agent against visceral leishmaniasis.

AIMS: Treatment for visceral leishmaniasis (VL) is hampered by the toxicity and/or high cost of drugs, as well as by emergence of parasite resistance. Therefore, there is an urgent need for new antileishmanial agents. METHODS AND RESULTS: In this study, the antileishmanial activity of a diprenylated flavonoid called 5,7,3,4'-tetrahydroxy-6,8-diprenylisoflavone (CMt) was tested against Leishmania infantum and L amazonensis species. Results showed that CMt presented selectivity index (SI) of 70.0 and 165.0 against L infantum and L amazonensis promastigotes, respectively, and of 181.9 and 397.8 against respective axenic amastigotes. Amphotericin B (AmpB) showed lower SI values of 9.1 and 11.1 against L infantum and L amazonensis promastigotes, respectively, and of 12.5 and 14.3 against amastigotes, respectively. CMt was effective in the treatment of infected macrophages and caused alterations in the parasite mitochondria. L infantum-infected mice treated with miltefosine, CMt alone or incorporated in polymeric micelles (CMt/Mic) presented significant reductions in the parasite load in distinct organs, when compared to the control groups. An antileishmanial Th1-type cellular and humoral immune response were developed one and 15 days after treatment, with CMt/Mic-treated mice presenting a better protective response. CONCLUSION: Our data suggest that CMt/Mic could be evaluated as a chemotherapeutic agent against VL.