Leishmania (Sauroleishmania) tarentolae versus pathogenic species: comparative evaluation of protease activity, glycoconjugates, resistance to complement and metabolome composition.

BACKGROUND: Leishmania tarentolae is a non-pathogenic species found in lizards representing an important model for Leishmania biology. However, several aspects of this Sauroleishmania remain unknown to explain its low level of virulence. OBJECTIVES: We reported several aspects of L. tarentolae biology including glycoconjugates, proteolytic activities and metabolome composition in comparison to pathogenic species (Leishmania amazonensis, Leishmania braziliensis, Leishmania infantum and Leishmania major). METHODS: Parasites were cultured for extraction and purification of lipophosphoglycan (LPG), immunofluorescence probing with anti-gp63 and resistance against complement. Parasite extracts were also tested for proteases activity and metabolome composition. FINDINGS: Leishmania tarentolae does not express LPG on its surface. It expresses gp63 at lower levels compared to pathogenic species and, is highly sensitive to complement-mediated lysis. This species also lacks intracellular/extracellular activities of proteolytic enzymes. It has metabolic differences with pathogenic species, exhibiting a lower abundance of metabolites including ABC transporters, biosynthesis of unsaturated fatty acids and steroids, TCA cycle, glycine/serine/threonine metabolism, glyoxylate/dicarboxylate metabolism and pentose-phosphate pathways. MAIN CONCLUSIONS: The non-pathogenic phenotype of L. tarentolae is associated with alterations in several biochemical and molecular features. This reinforces the need of comparative studies between pathogenic and non-pathogenic species to elucidate the molecular mechanisms of virulence during host-parasite interactions.

Complete assembly, annotation of virulence genes and CRISPR editing of the genome of Leishmania amazonensis PH8 strain.

We report the sequencing and assembly of the PH8 strain of Leishmania amazonensis one of the etiological agents of leishmaniasis. After combining data from long Pacbio reads, short Illumina reads and synteny with the Leishmania mexicana genome, the sequence of 34 chromosomes with 8317 annotated genes was generated. Multigene families encoding three virulence factors, A2, amastins and the GP63 metalloproteases, were identified and compared to their annotation in other Leishmania species. As they have been recently recognized as virulence factors essential for disease establishment and progression of the infection, we also identified 14 genes encoding proteins involved in parasite iron and heme metabolism and compared to genes from other Trypanosomatids. To follow these studies with a genetic approach to address the role of virulence factors, we tested two CRISPR-Cas9 protocols to generate L. amazonensis knockout cell lines, using the Miltefosine transporter gene as a proof of concept.

Genomic Surveillance of Monkeypox Virus, Minas Gerais, Brazil, 2022.

Phylogenetic analysis of 34 monkeypox virus genome sequences isolated from patients in Minas Gerais, Brazil, revealed initial importation events in early June 2022, then community transmission within the state. All generated genomes belonged to the B.1 lineage responsible for a global mpox outbreak. These findings can inform public health measures.

Survey of SARS-CoV-2 genetic diversity in two major Brazilian cities using a fast and affordable Sanger sequencing strategy.

Genetic variants of SARS-CoV-2 have been emerging and circulating in many places across the world. Rapid detection of these variants is essential since their dissemination can impact transmission rates, diagnostic procedures, disease severity, response to vaccines or patient management. Sanger sequencing has been used as the preferred approach for variant detection among circulating human immunodeficiency and measles virus genotypes. Using primers to amplify a fragment of the SARS-CoV-2 genome encoding part of the Spike protein, we showed that Sanger sequencing allowed us to rapidly detect the introduction and spread of three distinct SARS-CoV-2 variants in two major Brazilian cities. In both cities, after the predominance of variants closely related to the virus first identified in China, the emergence of the P.2 variant was quickly followed by the detection of the P1 variant, which became dominant in less than one month after it was first detected.

Early use of nitazoxanide in mild COVID-19 disease: randomised, placebo-controlled trial.

BACKGROUND: Nitazoxanide is widely available and exerts broad-spectrum antiviral activity in vitro. However, there is no evidence of its impact on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: In a multicentre, randomised, double-blind, placebo-controlled trial, adult patients presenting up to 3 days after onset of coronavirus disease 2019 (COVID-19) symptoms (dry cough, fever and/or fatigue) were enrolled. After confirmation of SARS-CoV-2 infection using reverse transcriptase PCR on a nasopharyngeal swab, patients were randomised 1:1 to receive either nitazoxanide (500 mg) or placebo, three times daily, for 5 days. The primary outcome was complete resolution of symptoms. Secondary outcomes were viral load, laboratory tests, serum biomarkers of inflammation and hospitalisation rate. Adverse events were also assessed. RESULTS: From June 8 to August 20, 2020, 1575 patients were screened. Of these, 392 (198 placebo, 194 nitazoxanide) were analysed. Median (interquartile range) time from symptom onset to first dose of study drug was 5 (4-5) days. At the 5-day study visit, symptom resolution did not differ between the nitazoxanide and placebo arms. Swabs collected were negative for SARS-CoV-2 in 29.9% of patients in the nitazoxanide arm versus 18.2% in the placebo arm (p=0.009). Viral load was reduced after nitazoxanide compared to placebo (p=0.006). The percentage viral load reduction from onset to end of therapy was higher with nitazoxanide (55%) than placebo (45%) (p=0.013). Other secondary outcomes were not significantly different. No serious adverse events were observed. CONCLUSIONS: In patients with mild COVID-19, symptom resolution did not differ between nitazoxanide and placebo groups after 5 days of therapy. However, early nitazoxanide therapy was safe and reduced viral load significantly.

Differential expression of Acanthamoeba castellanii proteins during amoebic keratitis in rats.

Amoebic keratitis (AK) is a sight-threatening infection characterized by a severe inflammation of the cornea, caused by the free-living protozoan of the genus Acanthamoeba. Identification of amoebic proteins involved in AK pathogenesis may help to elucidate molecular mechanisms of infection and contribute to indicate diagnostic and therapeutic targets. In this study, we evaluated changes in the expression profile of Acanthamoeba proteins triggered by the invasive process, using an approach involving two-dimensional polyacrylamide gel electrophoresis (2DE PAGE), followed by mass spectrometry identification (ESI-IT-TOF LC-MSn). AK was induced by intrastromal inoculation in Wistar rats, using trophozoites from a T4 genotype, human case-derived A. castellanii strain under prolonged axenic culture. Cultures re-isolated from the lesions after two successive passages in the animals were used as biological triplicate for proteomic experiments. Analysis of the protein profile comparing long-term and re-isolated cultures indicated 62 significant spots, from which 27 proteins could be identified in the Acanthamoeba proteome database. Five of them (Serpin, Carboxypeptidase A1, Hypothetical protein, Calponin domain-containing protein, aldo/keto reductase) were exclusively found in the re-isolated trophozoites. Our analysis also revealed that a concerted modulation of several biochemical pathways is triggered when A. castellanii switches from a free-living style to a parasitic mode, including energetic metabolism, proteolytic activity, control of gene expression, protein degradation and methylation of DNA, which may be also involved in gain of virulence in an animal model of AK.

Anti-inflammatory effects of C-peptide on kidney of type 1 diabetes mellitus animal model.

Type 1 diabetes mellitus (T1DM) is characterized by C-peptide deficiency and elevated levels of pro-inflammatory cytokines. The aim of this study was to investigate the role of C-peptide in renal and inflammatory complications in streptozotocin (STZ)-diabetic mice model of T1DM with kidney disease. The study was performed in 8-week old male C57BL/6 mice. Two streptozotocin-diabetic groups (a T1DM animal model), after 4 weeks of diabetes, were treated with subcutaneous infusion of either vehicle (n = 12) or C-peptide (n = 11). Two non-diabetic groups (vehicle, n = 10; C-peptide, n = 9) were treated using the same protocol as described for the diabetic mice. The treatment with C-peptide in the diabetic group reduced the urinary levels of IL17 and TNFα, as well as IL4 and IL10 (p < 0.05). Contrary, the diabetic + C-peptide group presented higher IL10 gene expression in kidney. Besides, it displayed a reduction of TNFα gene expression. The data suggest that C-peptide may modulate pro- and anti-inflammatory signalling pathways, resulting in attenuation of kidney inflammation in T1DM animal model.

An immunoproteomics approach to identify Leishmania infantum proteins to be applied for the diagnosis of visceral leishmaniasis and human immunodeficiency virus co-infection.

The co-infection between visceral leishmaniasis (VL) and human immunodeficiency virus (HIV) has increased in several countries in the world. The current serological tests are not suitable since they present low sensitivity to detect the most of VL/HIV cases, and a more precise diagnosis should be performed. In this context, in the present study, an immunoproteomics approach was performed using Leishmania infantum antigenic extracts and VL, HIV and VL/HIV patients sera, besides healthy subjects samples; aiming to identify antigenic markers for these clinical conditions. Results showed that 43 spots were recognized by antibodies in VL and VL/HIV sera, and 26 proteins were identified by mass spectrometry. Between them, ß-tubulin was expressed, purified and tested in ELISA experiments as a proof of concept for validation of our immunoproteomics findings and results showed high sensitivity and specificity values to detect VL and VL/HIV patients. In conclusion, the identified proteins in the present work could be considered as candidates for future studies aiming to improvement of the diagnosis of VL and VL/HIV co-infection.

Evaluation of different total Leishmania amazonensis antigens for the development of a first-generation vaccine formulated with a Toll-like receptor-3 agonist to prevent cutaneous leishmaniasis.

BACKGROUND Unfortunately, no any vaccine against leishmaniasis has been developed for human use. Therefore, a vaccine based on total Leishmania antigens could be a good and economic approach; and there are different methodologies to obtain these antigens. However, it is unknown whether the method to obtain the antigens affects the integrity and immune response caused by them. OBJECTIVES to compare the protein profile and immune response generated by total L. amazonensis antigens (TLA) produced by different methods, as well as to analyse the immune response and protection by a first-generation vaccine formulated with sonicated TLA (sTLA) and polyinosinic:polycytidylic acid [Poly (I:C)]. METHODS TLA were obtained by four different methodologies and their integrity and immune response were evaluated. Finally, sTLA was formulated with Poly (I:C) and their protective immune response was measured. FINDINGS sTLA presented a conserved protein profile and induced a strong immune response. In addition, Poly (I:C) improved the immune response generated by sTLA. Finally, sTLA + Poly (I:C) formulation provided partial protection against L. amazonensis infection. MAIN CONCLUSIONS The protein profile and immune response depend on the methodology used to obtain the antigens. Also, the formulation sTLA + Poly (I:C) provides partial protection against cutaneous leishmaniasis in mice.

The dual effect of C-peptide on cellular activation and atherosclerosis: Protective or not?

C-peptide is a cleavage product of proinsulin that acts on different type of cells, such as blood and endothelial cells. C-peptide biological effects may be different in type 1 and type 2 diabetes. Besides, there are further evidence for a functional interaction between C-peptide and insulin. In this way, C-peptide has ambiguous effects, acting as an antithrombotic or thrombotic molecule, depending on the physiological environment and disease conditions. Moreover, C-peptide regulates interaction of leucocytes, erythrocytes, and platelets with the endothelium. The beneficial effects include stimulation of nitric oxide production with its subsequent release by platelets and endothelium, the interaction with erythrocytes leading to the generation of adenosine triphosphate, and inhibition of atherogenic cytokine release. The undesirable action of C-peptide includes the chemotaxis of monocytes, lymphocytes, and smooth muscle cells. Also, C-peptide was related with increased lipid deposits and elevated smooth muscle cells proliferation in the vessel wall, contributing to atherosclerosis. Purpose of this review is to explore these dual roles of C-peptide on the blood, contributing at one side to haemostasis and the other to atherosclerotic process.

Annexin A1 Is Involved in the Resolution of Inflammatory Responses during Leishmania braziliensis Infection.

Leishmaniases are diseases caused by several Leishmania species. Leishmania (Viannia) braziliensis can cause localized cutaneous leishmaniasis (LCL), which heals spontaneously, or mucosal leishmaniasis (ML), characterized by chronic and intense inflammation and scanty parasitism. Annexin A1 (AnxA1) is a protein involved in modulation and resolution of inflammation through multiple mechanisms. In the present study, the role of AnxA1 was investigated in L. braziliensis-infected BALB/c mice. AnxA1 levels increased at the peak of tissue lesion and parasitism in infected mice. AnxA1 increased also after L. braziliensis infection of BALB/c (wild-type [WT]) bone marrow derived macrophages. Despite a lower parasite intake, parasite burden in bone marrow-derived macrophages from AnxA1-/- mice was similar to WT and associated with an early increase of TNF-α and, later, of IL-10. AnxA1-/- mice controlled tissue parasitism similarly to WT animals, but they developed significantly larger lesions at later stages of infection, with a more pronounced inflammatory infiltrate and increased specific production of IFN-γ, IL-4, and IL-10. AnxA1-/- mice also presented higher phosphorylation levels of ERK-1/2 and p65/RelA (NF-κB) and inducible NO synthase expression, suggesting that AnxA1 may be involved in modulation of inflammation in this model of experimental leishmaniasis. Finally, assessment of AnxA1 levels in sera from patients with LCL or ML revealed that ML patients had higher levels of serum AnxA1 than did LCL patients or control subjects. Collectively, these data indicate that AnxA1 is actively expressed during L. braziliensis infection. In the absence of AnxA1, mice are fully able to control parasite replication, but they present more intense inflammatory responses and delayed ability to resolve their lesion size.

Reticular and erosive oral lichen planus have a distinct metabolomic profile: A preliminary study using gas chromatography-mass spectrometry.

BACKGROUND: Oral Lichen Planus is a chronic inflammatory disorder that affects the oral mucosa, with the reticular and erosive forms representing the primary clinical variants of the disease. Previous studies have shown that metabolic alterations may well be involved in the pathogenesis of the disease; however, the molecular mechanisms related to the clinicopathological differences between erosive and reticular forms remain unknown. METHODS: A comparative metabolomic analysis was performed on formalin-fixed and paraffin-embedded tissue samples of erosive (n = 6) and reticular (n = 10) oral lichen planus using gas chromatography-mass spectrometry. RESULTS: The metabolomic analysis showed a distinct profile between the two clinical variants. Five metabolites (cyclohexanamine, glycine, mannitol/sorbitol, methyl palmitate and trehalose) were significantly diminished in erosive oral lichen planus as compared to the reticular form. CONCLUSIONS: Reticular and erosive forms of oral lichen planus have a distinct metabolic profile. However, further studies using a large number of fresh tissue samples are necessary to confirm this data.

The importance of BRAF-V600E mutation to ameloblastoma metabolism.

BACKGROUND: Ameloblastoma is a locally infiltrative, aggressive epithelial odontogenic neoplasm. BRAF-V600E mutation is frequently found in this tumor and has a pivotal role in its pathogenesis, but the consequences of this alteration need to be addressed. An untargeted metabolomics approach was applied to verify whether metabolic disturbances are related to tumor biology and whether BRAF-V600E mutation contributes to these alterations. METHODS: Formalin-fixed and paraffin-embedded tissue specimens from thirteen ameloblastoma and six dental follicles were included in this study. BRAF mutational status was determined by competitive allele-specific real-time PCR. Metabolite extracts were analyzed using gas chromatography coupled to mass spectrometry. Univariate and multivariate statistical methods were employed to compare the metabolic profiles of the samples. RESULTS: The abundance of eleven metabolites was significantly higher in ameloblastoma in relation to dental follicles, including amino acids, fatty acids, carbohydrates, inorganic acids, and organoheterocyclic compounds. The presence of BRAF-V600E mutations in ameloblastoma was related to decreased levels of glycerol in comparison with tumors carrying only wild-type alleles of this gene. No metabolic differences were observed between recurrent and primary manifestations of ameloblastoma. CONCLUSIONS: Ameloblastoma exhibits a distinct metabolic profile from normal odontogenic epithelium. BRAF-V600E may contribute to metabolic alterations in ameloblastoma. Collectively, our findings suggest that metabolic alterations might play a role in tumor pathogenesis.

Molecular epidemiology of Hepatitis delta virus infection in Minas Gerais state from Brazil, an area outside the hyperendemic region of the Amazon Basin.

BACKGROUND: Hepatitis delta virus (HDV) infections in hepatitis B virus (HBV) carriers are the most severe form of viral hepatitis. HDV prevalence is high in the Brazilian Amazon, but studies in other regions of the country are still scarce and often underestimated its prevalence by including a small numbers of individuals. OBJECTIVE: This study aimed to determine the serological prevalence of hepatitis D, the genotypes circulating and to evaluate the associated risk factors for acquisition of HDV in Minas Gerais state, Brazil. METHODS: We screened plasma samples (n = 498) from HBV chronic carriers for anti-HD antibodies using a commercial enzyme-linked immunosorbent assay (ELISA) kit. For those samples that were positive for anti-HD antibodies, we performed a reverse transcriptase (RT) nested-polymerase chain reaction (nested-PCR) in order to detect the viral genome and identify the viral genotypes circulating in the state. FINDINGS: The prevalence was 6.22% (31/498). Blood transfusion was the only risk factor associated with HDV infection [risk ratio: 3.73; 95% confidence interval (CI): 1.44 to 9.65]. For 26 anti-HD positive patients, HDAg gene sequences were determined and in all patients HDV genotype 1 was found. CONCLUSIONS: This study confirmed the circulation of HDV in Minas Gerais, an area previously considered non-endemic for hepatitis D in Brazil. The prevalence found in this study is much higher when compared to other studies performed in Brazil, probably because the population in our study was selected with minimal bias. Furthermore, in 26 anti-HD positive plasma samples, we were also able to detect the viral genome, indicating that these patients were experienced an active infection at the time of sample collection. These findings emphasise the importance of anti-HD testing in HBV infected individuals, which may contribute to this disease control in Brazil.

Evaluation of three recombinant proteins for the development of ELISA and immunochromatographic tests for visceral leishmaniasis serodiagnosis.

BACKGROUND: Visceral Leishmaniasis (VL) is an infectious disease that is a significant cause of death among infants aged under 1 year and the elderly in Brazil. Serodiagnosis is a mainstay of VL elimination programs; however, it has significant limitations due to low accuracy. OBJECTIVE: This study aimed to evaluate three recombinant Leishmania infantum proteins (rFc, rC9, and rA2) selected from previous proteomics and genomics analyses to develop enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of human VL (HVL) and canine VL (CVL). METHODS: A total of 186 human (70 L. infantum-infected symptomatic, 20 other disease-infected, and 96 healthy) and 185 canine (82 L. infantum-infected symptomatic, 27 L. infantum-infected asymptomatic, and 76 healthy) sera samples were used for antibody detection. FINDINGS: Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9 (95.7% sensitivity and 87.5% specificity) displayed the best performance in ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and had high concordance with immunofluorescence antibody tests (IFAT), ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9, and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with specificity values of 84.0%, 92.0%, and 100%, respectively for CVL diagnosis. MAIN CONCLUSIONS: The three antigens selected by us are promising candidates for VL diagnosis regardless of the test format, although the antigen combinations and test parameters may warrant further optimisation.

Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes.

Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole.

Evidence of interventions for the risk of dry eye in critically ill patients: An integrative review.

AIMS: Identify the best scientific evidence available to eye care in order to prevent dry eye. METHOD: Review study conducted according to the three steps of the evidence-based practice, guided by the following question, grounded in the Patient, Intervention, Comparison, and Outcome strategy: "What is the best scientific evidence available to eye care related to preventing dry eye?" Two databases were used, the web portal Medical Literature Analysis and Retrieval System Online and two digital libraries. Data were organized by using three structured forms. RESULTS: Ten studies made up the final sample, in English, with evidence levels between I and III. The results pointed out differences regarding the best or most appropriate occlusion and ocular lubrication methods to prevent dry eye. CONCLUSION: Several care methods showed strong scientific evidence to prevent dry eye, related to occlusion and ocular lubrication. There is a need for further studies to determine the strength of this evidence.

A2 and other visceralizing proteins of Leishmania: role in pathogenesis and application for vaccine development.

Visceral leishmaniasis is a re-emergent disease and a significant cause of morbidity worldwide. Amongst the more than 20 Leishmania species, Leishmania donovani, Leishmania infantum and more rarely Leishmania amazonensis are associated with visceral leishmaniasis. A major question in leishmaniasis research is how these species migrate to and infect visceral organs whereas other species such as Leishmania major and Leishmania braziliensis remain in the skin, causing tegumentary leishmaniasis. Here we present the more recent advances and approaches towards the identification of species-specific visceralizing factors of Leishmania, such as the A2 protein, leading to a better understanding of parasite biology. We also discuss their potential use for the development of a vaccine for visceral leishmaniasis.

Low-level laser therapy as an alternative for pulpotomy in human primary teeth.

This study aimed to evaluate the effects of low-level laser therapy (LLLT) on pulpal response of primary teeth. Twenty mandibular primary molars were randomly divided into the following groups: group I Buckley's formocresol (diluted at 1:5), group II calcium hydroxide, group III LLLT + zinc oxide/eugenol, and group IV LLLT + calcium hydroxide. LLLT parameters were set at 660-nm wavelength, 10-mW power output, and 2.5 J/cm(2) energy density for 10 s in continuous mode (InGaAlP laser, Twin Laser®, MMOptics, Sao Carlos, Sao Paulo, Brazil). The teeth were extracted at the regular exfoliation period. The dentin-pulp complex was graded by an established histopathological score system. Statistical analysis was performed by Kruskal-Wallis and chi-square test. The histopathological assessment revealed statistically significant differences among groups (P < 0.05). The lowest degree of pulpal inflammation was present in LLLT + calcium hydroxide (P = 0.0296). Calcium hydroxide showed the highest rate of hard tissue barrier (P = 0.0033), odontoblastic layer (P = 0.0033), and dense collagen fibers (P = 0.0095). On the other hand, formocresol showed the highest incidence of internal resorption (P = 0.0142). Based on this study, low-level laser therapy preceding the use of calcium hydroxide exhibited satisfactory results on pulp tissue healing. However, further clinical studies on human teeth with long-term follow-up are needed to test the low-level laser therapy efficacy.