A new standardization for the use of chicken embryo: selection of target from the phage display library and infection.

The filamentous bacteriophage M13KO7 (M13) is the most used in phage display (PD) technology and, like other phages, has been applied in several areas of medicine, agriculture, and in the food industry. One of the advantages is that they can modulate the immune response in the presence of pathogenic microorganisms, such as bacteria and viruses. This study evaluated the use of phage M13 in the chicken embryos model. We inoculated 13-day-old chicken embryos with Salmonella Pullorum (SP) and then evaluated survival for the presence of phage M13 or E. coli ER2738 (ECR) infected with M13. We found that the ECR bacterium inhibits SP multiplication in 0.32 (M13-infected ECR) or 0.44 log UFC/mL (M13-uninfected ECR) and that the ECR-free phage M13 from the PD library can be used in chicken embryo models. This work provides the use of the chicken embryo as a model to study systemic infection and can be employed as an analysis tool for various peptides that M13 can express from PD selection. KEY POINTS: • SP-infected chicken embryo can be a helpful model of systemic infection for different tests. • Phage M13 does not lead to embryonic mortality or cause serious injury to embryos. • Phage M13 from the PD library can be used in chicken embryo model tests.

Development of collagenous scaffolds for wound healing: characterization and in vivo analysis.

The development of wound dressings from biomaterials has been the subject of research due to their unique structural and functional characteristics. Proteins from animal origin, such as collagen and chitosan, act as promising materials for applications in injuries and chronic wounds, functioning as a repairing agent. This study aims to evaluate in vitro effects of scaffolds with different formulations containing bioactive compounds such as collagen, chitosan, N-acetylcysteine (NAC) and ε-poly-lysine (ε-PL). We manufactured a scaffold made of a collagen hydrogel bioconjugated with chitosan by crosslinking and addition of NAC and ε-PL. Cell viability was verified by resazurin and live/dead assays and the ultrastructure of biomaterials was evaluated by SEM. Antimicrobial sensitivity was assessed by antibiogram. The healing potential of the biomaterial was evaluated in vivo, in a model of healing of excisional wounds in mice. On the 7th day after the injury, the wounds and surrounding skin were processed for evaluation of biochemical and histological parameters associated with the inflammatory process. The results showed great cell viability and increase in porosity after crosslinking while antimicrobial action was observed in scaffolds containing NAC and ε-PL. Chitosan scaffolds bioconjugated with NAC/ε-PL showed improvement in tissue healing, with reduced lesion size and reduced inflammation. It is concluded that scaffolds crosslinked with chitosan-NAC-ε-PL have the desirable characteristics for tissue repair at low cost and could be considered promising biomaterials in the practice of regenerative medicine.

Exploring Inflammatory Asthma Phenotypes: Proteomic Signatures in Serum and Induced Sputum.

Asthma drug responses may differ due to inflammatory mechanisms triggered by the immune cells in the pulmonary microenvironment. Thus, asthma phenotyping based on the local inflammatory profile may aid in treatment definition and the identification of new therapeutic targets. Here, we investigated protein profiles of induced sputum and serum from asthma patients classified into eosinophilic, neutrophilic, mixed granulocytic, and paucigranulocytic asthma, according to inflammatory phenotypes. Proteomic analyses were performed using an ultra-performance liquid chromatography (ultra-HPLC) system coupled to the Q Exactive Hybrid Quadrupole Orbitrap Mass Spectrometer. Fifty-two (52) proteins showed significant differences in induced sputum among the groups, while only 12 were altered in patients' sera. Five proteins in the induced sputum were able to discriminate all phenotypic groups, while four proteins in the serum could differentiate all except the neutrophilic from the paucigranulocytic inflammatory pattern. This is the first report on comparative proteomics of inflammatory asthma phenotypes in both sputum and serum samples. We have identified a potential five-biomarker panel that may be able to discriminate all four inflammatory phenotypes in sputum. These findings not only provide insights into potential therapeutic targets but also emphasize the potential for personalized treatment approaches in asthma management.

N-Formyl-Methionyl-Leucyl-Phenylalanine Plays a Neuroprotective and Anticonvulsant Role in Status Epilepticus Model.

Status epilepticus (SE) is described as continuous and self-sustaining seizures, which triggers hippocampal neurodegeneration, inflammation, and gliosis. N-formyl peptide receptor (FPR) has been associated with inflammatory process. N-formyl-methionyl-leucyl-phenylalanine (fMLP) peptide plays an anti-inflammatory role, mediated by the activation of G-protein-coupled FPR. Here, we evaluated the influence of fMLP peptides on the behavior of limbic seizures, memory consolidation, and hippocampal neurodegeneration process. Male Wistar rats (Rattus norvegicus) received microinjections of pilocarpine in hippocampus (H-PILO, 1.2 mg/µL, 1 µL) followed by fMLP (1 mg/mL, 1 µL) or vehicle (VEH, saline 0.9%, 1 µL). During the 90 min of SE, epileptic seizures were analyzed according to the Racine's Scale. After 24 h of SE, memory impairment was assessed by the inhibitory avoidance test and the neurodegeneration process was evaluated in hippocampal areas. There was no change in latency and number of wet dog shake (WDS) after administration of fMLP. However, our results showed that the intrahippocampal infusion of fMLP reduced the severity of seizures, as well as the number of limbic seizures. In addition, fMLP infusion protected memory dysfunction followed by SE. Finally, the intrahippocampal administration of fMLP attenuated the process of neurodegeneration in both hippocampi. Taken together, our data suggest a new insight into the functional role of fMLP peptides, with important implications for their potential use as a therapeutic agent for the treatment of brain disorders, such as epilepsy. Schematic drawing on the neuroprotective and anticonvulsant role of fMLP during status epilepticus. Initially, a cannula was implanted in hippocampus and pilocarpine/saline was administered into the hippocampus followed by fMLP/saline (A-C). fMLP reduced seizure severity and neuronal death in the hippocampus, as well as protecting against memory deficit (D).

CdSe magic-sized quantum dots attenuate reactive oxygen species generated by neutrophils and macrophages with implications in experimental arthritis.

The biological applicability of nanomaterials has been limited due to cytotoxicity. Studies have described the effects of nanomaterials on different tissues and cell types, but their actions on immune cells are less elucidated. This study describes unprecedented in vitro and in vivo antioxidant activities of cadmium selenide magic-sized quantum dots (CdSe MSQDs) with implications on rheumatoid arthritis. While the generation of ROS induced by nanomaterials is linked to cytotoxicity, we found that CdSe MSQDs reduced ROS production by neutrophils and macrophages following opsonized-zymosan stimuli, and we did not find cytotoxic effects. Interestingly, inherent antioxidant properties of CdSe MSQDs were confirmed through DPPH, FRAP, and ORAC assays. Furthermore, CdSe MSQDs reduced ROS levels generated by infiltrating leukocytes into joints in experimental model of rheumatoid arthritis. Briefly, we describe a novel application of CdSe MSQDs in modulating the inflammatory response in experimental rheumatoid arthritis through an unexpected antioxidant activity.

Mitochondrial dysfunction on Leishmania (Leishmania) amazonensis induced by ketoconazole: insights into drug mode of action.

BACKGROUND: Leishmania parasites cause leishmaniasis that range from self-limiting cutaneous lesions to more serious forms of the disease. The search for potential drug targets focusing on biochemical and metabolic pathways revealed the sterol biosynthesis inhibitors (SBIs) as a promising approach. In this class of inhibitors is found ketoconazole, a classical inhibitor of 14α-methysterol 14-demethylase. OBJECTIVE: The present study aimed to better understand the biological response of Leishmania (Leishmania) amazonensis promastigotes at the cellular level after ketoconazole treatment. METHODS: Herein, techniques, such as fluorimetry, flow cytometry, fluorescence microscopy, electron and scanning microscopy were used to investigate the cellular structures and to identify organelles affected by ketoconazole treatment. FINDINGS: The study demonstrated, for the first time, the effect of ketoconazole on mitochondrion functioning and its probable relationship to cell cycle and death on L. (L.) amazonensis promastigotes (IFLA/BR/67/PH8 strain). MAIN CONCLUSIONS: Ketoconazole-induced mitochondrial damages led to hyperpolarisation of this single organelle and autophagic vacuoles formation, as a parasite survival strategy. These damages did not reflect directly on the parasite cell cycle, but drove the parasites to death, making them susceptible to ketoconazole treatment in in vitro models.

Development of a Molecular Aptamer Beacon Applied to Magnetic-Assisted RNA Extraction for Detection of Dengue and Zika Viruses Using Clinical Samples.

Limitations in the detection of cocirculating flaviviruses such as Dengue and Zika lead us to propose the use of aptameric capture of the viral RNA in combination with RT-PCR (APTA-RT-PCR). Aptamers were obtained via SELEX and next-generation sequencing, followed by colorimetric and fluorescent characterizations. An APTA-RT-PCR assay was developed, optimized, and tested against the viral RNAs in 108 serum samples. After selection, sequence APTAZC10 was designed as a bifunctional molecular beacon (APTAZC10-MB), exhibiting affinity for the viral targets. APTA-RT-PCR was able to detect Dengue and Zika RNA in 43% and 8% of samples, respectively. Our results indicate that APTAZC10-MB and APTA-RT-PCR will be useful to improve the detection of Dengue and Zika viruses in a fast molecular assay for the improvement of infectious disease surveillance.

Angiotensin type 2 receptor antagonism as a new target to manage gout.

BACKGROUND: There is a growing search for therapeutic targets in the treatment of gout. The present study aimed to evaluate the analgesic and anti-inflammatory potential of angiotensin type 2 receptor (AT2R) antagonism in an acute gout attack mouse model. METHODS: Male wild-type (WT) C57BL/6 mice either with the AT2R antagonist, PD123319 (10 pmol/joint), or with vehicle injections, or AT2R KO mice, received intra-articular (IA) injection of monosodium urate (MSU) crystals (100 µg/joint), that induce the acute gout attack, and were tested for mechanical allodynia, thermal hyperalgesia, spontaneous nociception and ankle edema development at several times after the injections. To test an involvement of AT2R in joint pain, mice received an IA administration of angiotensin II (0.05-5 nmol/joint) with or without PD123319, and were also evaluated for pain and edema development. Ankle joint tissue samples from mice undergoing the above treatments were assessed for myeloperoxidase activity, IL-1ß release, mRNA expression analyses and nitrite/nitrate levels, 4 h after injections. RESULTS: AT2R antagonism has robust antinociceptive effects on mechanical allodynia (44% reduction) and spontaneous nociception (56%), as well as anti-inflammatory effects preventing edema formation (45%), reducing myeloperoxidase activity (54%) and IL-1ß levels (32%). Additionally, Agtr2tm1a mutant mice have largely reduced painful signs of gout. Angiotensin II administration causes pain and inflammation, which was prevented by AT2R antagonism, as observed in mechanical allodynia 4 h (100%), spontaneous nociception (46%), cold nociceptive response (54%), edema formation (83%), myeloperoxidase activity (48%), and IL-1ß levels (89%). PD123319 treatment also reduces NO concentrations (74%) and AT2R mRNA levels in comparison with MSU untreated mice. CONCLUSION: Our findings show that AT2R activation contributes to acute pain in experimental mouse models of gout. Therefore, the antagonism of AT2R may be a potential therapeutic option to manage gout arthritis.

Low Annexin A1 level in HTLV-1 infected patients is a potential biomarker for the clinical progression and diagnosis of HAM/TSP.

BACKGROUND: Human T-lymphotropic virus 1 (HTLV-1) is etiologically associated with the chronic inflammatory neurodegenerative disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) Annexin A1 (AnxA1) is an anti-inflammatory protein with proposed neuroprotective and anti-neuroinflammatory functions. We hypothesized that ANXA1 gene expression may be dysregulated in HTLV-1-infected HAM/TSP patients. METHODS: This study involved 37 individuals infected with HTLV-1, including 21 asymptomatic (AS) carriers and 16 with HAM/TSP, and a control group of 30 individuals negative for HTLV-1 and HTLV-2. For AS HTLV-1-positive and HAM/TSP patients, ANXA1 and formyl peptide receptor (FPR1, FPR2 and FPR3) expression and HTLV-1 proviral load (PVL) in peripheral blood cells were evaluated by real-time quantitative PCR (qPCR), and plasma AnxA1 levels were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: ANXA1 gene expression was increased in the AS group compared with the HAM/TSP and control groups, but the differences were not statistically significant. FPR1 gene expression was higher in patients with HTLV-1 than in controls (AS, p = 0.0032; HAM/TSP, p < 0.0001). Plasma AnxA1 levels were higher in the AS group than in the HAM/TSP group (p = 0.0045), and PVL was higher in patients with HAM/TSP than in AS individuals (p = 0.0162). The use of a combined ROC curve using Annexin 1 levels and proviral load significantly increased the sensitivity and specificity to predict progression to HAM/TSP (AUC = 0.851 and AUC = 0.937, respectively, to AUC = 1000). CONCLUSIONS: Our results suggest that AnxA1 may be dysregulated in HAM/TSP patients. Serological detection of AnxA1 in association with proviral load may provide a prognostic biomarker for HTLV-1-associated neurodegenerative disease.

Diagnostic application of sensitive and specific phage-exposed epitopes for visceral leishmaniasis and human immunodeficiency virus coinfection.

The diagnosis of visceral leishmaniasis (VL) has improved with the search of novel antigens; however, their performance is limited when samples from VL/human immunodeficiency virus (HIV)-coinfected patients are tested. In this context, studies conducted to identify more suitable antigens to detect both VL and VL/HIC coinfection cases should be performed. In the current study, phage display was performed using serum samples from healthy subjects and VL, HIV-infected and VL/HIV-coinfected patients; aiming to identify novel phage-exposed epitopes to be evaluated with this diagnostic purpose. Nine non-repetitive and valid sequences were identified, synthetized and tested as peptides in enzyme-linked immunosorbent assay experiments. Results showed that three (Pep2, Pep3 and Pep4) peptides showed excellent performance to diagnose VL and VL/HIV coinfection, with 100% sensitivity and specificity values. The other peptides showed sensitivity varying from 50.9 to 80.0%, as well as specificity ranging from 60.0 to 95.6%. Pep2, Pep3 and Pep4 also showed a potential prognostic effect, since specific serological reactivity was significantly decreased after patient treatment. Bioinformatics assays indicated that Leishmania trypanothione reductase protein was predicted to contain these three conformational epitopes. In conclusion, data suggest that Pep2, Pep3 and Pep4 could be tested for the diagnosis of VL and VL/HIV coinfection.