Factors related to the development of high antibody titres against SARS-CoV-2 in convalescent plasma donors from the ConPlas-19 trial.

BACKGROUND AND OBJECTIVES: The efficacy of COVID-19 convalescent plasma (CP) associates with high titres of antibodies. ConPlas-19 clinical trial showed that CP reduces the risk of progression to severe COVID-19 at 28 days. Here, we aim to study ConPlas-19 donors and characteristics that associate with high anti-SARS-CoV-2 antibody levels. MATERIALS AND METHODS: Four-hundred donors were enrolled in ConPlas-19. The presence and titres of anti-SARS-CoV-2 antibodies were evaluated by EUROIMMUN anti-SARS-CoV-2 S1 IgG ELISA. RESULTS: A majority of 80.3% of ConPlas-19 donor candidates had positive EUROIMMUN test results (ratio ≥1.1), and of these, 51.4% had high antibody titres (ratio ≥3.5). Antibody levels decline over time, but nevertheless, out of 37 donors tested for an intended second CP donation, over 90% were still EUROIMMUN positive, and nearly 75% of those with high titres maintained high titres in the second sample. Donors with a greater probability of developing high titres of anti-SARS-CoV-2 antibodies include those older than 40 years of age (RR 2.06; 95% CI 1.24-3.42), with more than 7 days of COVID-19 symptoms (RR 1.89; 95% CI 1.05-3.43) and collected within 4 months from infection (RR 2.61; 95% CI 1.16-5.90). Male donors had a trend towards higher titres compared with women (RR 1.67; 95% CI 0.91-3.06). CONCLUSION: SARS-CoV-2 CP candidate donors' age, duration of COVID-19 symptoms and time from infection to donation associate with the collection of CP with high antibody levels. Beyond COVID-19, these data are relevant to inform decisions to optimize the CP donor selection process in potential future outbreaks.

Transcriptomics reveals a distinct metabolic profile in T cells from severe allergic asthmatic patients.

The reasons behind the onset and continuation of chronic inflammation in individuals with severe allergies are still not understood. Earlier findings indicated that there is a connection between severe allergic inflammation, systemic metabolic alterations and impairment of regulatory functions. Here, we aimed to identify transcriptomic alterations in T cells associated with the degree of severity in allergic asthmatic patients. T cells were isolated from severe (n = 7) and mild (n = 9) allergic asthmatic patients, and control (non-allergic, non-asthmatic healthy) subjects (n = 8) to perform RNA analysis by Affymetrix gene expression. Compromised biological pathways in the severe phenotype were identified using significant transcripts. T cells' transcriptome of severe allergic asthmatic patients was distinct from that of mild and control subjects. A higher count of differentially expressed genes (DEGs) was observed in the group of individuals with severe allergic asthma vs. control (4,924 genes) and vs. mild (4,232 genes) groups. Mild group also had 1,102 DEGs vs. controls. Pathway analysis revealed alterations in metabolism and immune response in the severe phenotype. Severe allergic asthmatic patients presented downregulation in genes related to oxidative phosphorylation, fatty acid oxidation and glycolysis together with increased expression of genes coding inflammatory cytokines (e.g. IL-19, IL-23A and IL-31). Moreover, the downregulation of genes involved in TGFß pathway together with a decreased tendency on the percentage of T regulatory cell (CD4 + CD25+), suggest a compromised regulatory function in severe allergic asthmatic patients. This study demonstrates a transcriptional downregulation of metabolic and cell signalling pathways in T cells of severe allergic asthmatic patients associated with diminished regulatory T cell function. These findings support a link between energy metabolism of T cells and allergic asthmatic inflammation.

Pathogen reduction with methylene blue does not have an impact on the clinical effectiveness of COVID-19 convalescent plasma.

BACKGROUND AND OBJECTIVES: There is a concern about a possible deleterious effect of pathogen reduction (PR) with methylene blue (MB) on the function of immunoglobulins of COVID-19 convalescent plasma (CCP). We have evaluated whether MB-treated CCP is associated with a poorer clinical response compared to other inactivation systems at the ConPlas-19 clinical trial. MATERIALS AND METHODS: This was an ad hoc sub-study of the ConPlas-19 clinical trial comparing the proportion of patients transfused with MB-treated CCP who had a worsening of respiration versus those treated with amotosalen (AM) or riboflavin (RB). RESULTS: One-hundred and seventy-five inpatients with SARS-CoV-2 pneumonia were transfused with a single CCP unit. The inactivation system of the CCP units transfused was MB in 90 patients (51.4%), RB in 60 (34.3%) and AM in 25 (14.3%). Five out of 90 patients (5.6%) transfused with MB-treated CCP had worsening respiration compared to 9 out of 85 patients (10.6%) treated with alternative PR methods (p = 0.220). Of note, MB showed a trend towards a lower rate of respiratory progressions at 28 days (risk ratio, 0.52; 95% confidence interval, 0.18-1.50). CONCLUSION: Our data suggest that MB-treated CCP does not provide a worse clinical outcome compared to the other PR methods for the treatment of COVID-19.

Deciphering the role of platelets in severe allergy by an integrative omics approach.

BACKGROUND: Mechanisms causing the onset and perpetuation of inflammation in severe allergic patients remain unknown. Our previous studies suggested that severe allergic inflammation is linked to platelet dysfunction. METHODS: Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) samples were obtained by platelet-apheresis from severe (n = 7) and mild (n = 10) allergic patients and nonallergic subjects (n = 9) to perform platelet lipidomics by liquid chromatography coupled to mass spectrometry (LC-MS) and RNA-seq analysis. Significant metabolites and transcripts were used to identify compromised biological pathways in the severe phenotype. Platelet and inflammation-related proteins were quantified by Luminex. RESULTS: Platelets from severe allergic patients were characterized by high levels of ceramides, phosphoinositols, phosphocholines, and sphingomyelins. In contrast, they showed a decrease in eicosanoid precursor levels. Biological pathway analysis performed with the significant lipids revealed the alteration of phospholipases, calcium-dependent events, and linolenic metabolism. RNAseq confirmed mRNA overexpression of genes related to platelet activation and arachidonic acid metabolism in the severe phenotypes. Pathway analysis indicated the alteration of NOD, MAPK, TLR, TNF, and IL-17 pathways in the severe phenotype. P-Selectin and IL-17AF proteins were increased in the severe phenotype. CONCLUSIONS: This study demonstrates that platelet lipid, mRNA, and protein content is different according to allergy severity. These findings suggest that platelet load is a potential source of biomarkers and a new chance for therapeutic targets in severe inflammatory pathologies.

Mutant PRPF8 Causes Widespread Splicing Changes in Spliceosome Components in Retinitis Pigmentosa Patient iPSC-Derived RPE Cells.

Retinitis pigmentosa (RP) is a rare, progressive disease that affects photoreceptors and retinal pigment epithelial (RPE) cells with blindness as a final outcome. Despite high medical and social impact, there is currently no therapeutic options to slow down the progression of or cure the disease. The development of effective therapies was largely hindered by high genetic heterogeneity, inaccessible disease tissue, and unfaithful model organisms. The fact that components of ubiquitously expressed splicing factors lead to the retina-specific disease is an additional intriguing question. Herein, we sought to correlate the retinal cell-type-specific disease phenotype with the splicing profile shown by a patient with autosomal recessive RP, caused by a mutation in pre-mRNA splicing factor 8 (PRPF8). In order to get insight into the role of PRPF8 in homeostasis and disease, we capitalize on the ability to generate patient-specific RPE cells and reveal differentially expressed genes unique to RPE cells. We found that spliceosomal complex and ribosomal functions are crucial in determining cell-type specificity through differential expression and alternative splicing (AS) and that PRPF8 mutation causes global changes in splice site selection and exon inclusion that particularly affect genes involved in these cellular functions. This finding corroborates the hypothesis that retinal tissue identity is conferred by a specific splicing program and identifies retinal AS events as a framework toward the design of novel therapeutic opportunities.

Blood Donations and Transfusions during the COVID-19 Pandemic in Spain: Impact According to Autonomous Communities and Hospitals.

Worldwide, the COVID-19 pandemic has caused a decline in blood donations, between 30% and 70% in some of the most affected countries. In Spain, during the initial eight weeks after the State of Emergency was decreed on 14 March 2020, in the weekly reports of the Health Ministry, an average decrease of 20% was observed between 11 and week 25 compared with the 2018 donation. We aimed to investigate the impact of the COVID-19 pandemic on blood donations and blood distribution in four autonomous communities, and to explore the evolution of the consumption of blood components (BCs) in ten hospitals of six autonomous communities. We performed a prospective study of grouped cohorts on the donation and distribution of blood in four regional transfusion centers in four autonomous communities in Spain, and a retrospective study of the consumption of blood components in ten hospitals in six autonomous communities. Regarding donations, there was no significant decrease in donations, with differences between autonomous communities, which started between 1 and 15 March 2020 (-11%). The increase in donations in phase II (from 26 May 2020) stands out. Regarding consumption, there was a significant reduction in the consumption of packed red blood cells (RBCs) (24.5%), plasma (45.3%), and platelets (25.3%) in the central period (16 March-10 May). The reduction in the consumption of RBCs was significant in the period from 1-15 March. Conclusions: The COVID-19 pandemic has affected the donation and consumption of BCs.

Natural photosensitizers in photodynamic therapy: In vitro activity against monolayers and spheroids of human colorectal adenocarcinoma SW480 cells.

Photodynamic Therapy (PDT), is a treatment option for cancer.It involves the photochemical interaction of light, photosensitizer (PS) and molecular oxygen to produce radical species as well as singlet oxygen which induce cell death. Anthraquinones (AQs) have been extensively studied with respect to their UV/Vis absorption characteristics and their photosensitizing properties in photodynamic reactions. We study the photoactivity of different natural AQs (Parietin, Soranjidiol and Rubiadin) in treating monolayers and multicellular tumor spheroids (MCTSs). Rubiadin and soranjidiol were isolated and purified from the stem and leaves of Heterophyllae pustulata, and PTN was from the liquen Teloschistes flavicans by using repeated combination of several chromatographic techniques. Monolayer and spheroids of human colorectal adenocarcinoma SW480 cells were incubated with different concentrations of the AQs and then irradiated at room temperature. 24 h post-PDT cell viability, nuclear morphology and type of cell death were analyzed. We observed that Soranjidiol and Rubiadin showed no significant difference in the photosensitizing ability on monoculture of colon cancer cells (LD80 at 50 µM and 10 J/cm2, for both AQs). Nevertheless, for Parietin (PTN) LD80 was achieved at (20 µM using the same light dose (10 J/cm2). The death mechanism induced post-PDT was necrosis by use of Soranjidol and Rubiadin and apoptosis by use of PTN. Furthermore, in MCTSs of 300 and 900 µm, the treatment PTN- PDT produces the greatest cytotoxic effect. The three AQs analyzed could be promising chemotherapeutic candidates as anticancer PDT agents.

Cellular and Molecular Biomarkers Indicate Premature Aging in Pseudoxanthoma Elasticum Patients.

The molecular processes of aging are very heterogenic and not fully understood. Studies on rare progeria syndromes, which display an accelerated progression of physiological aging, can help to get a better understanding. Pseudoxanthoma elasticum (PXE) caused by mutations in the ATP-binding cassette sub-family C member 6 (ABCC6) gene shares some molecular characteristics with such premature aging diseases. Thus, this is the first study trying to broaden the knowledge of aging processes in PXE patients. In this study, we investigated aging associated biomarkers in primary human dermal fibroblasts and sera from PXE patients compared to healthy controls. Determination of serum concentrations of the aging biomarkers eotaxin-1 (CCL11), growth differentiation factor 11 (GDF11) and insulin-like growth factor 1 (IGF1) showed no significant differences between PXE patients and healthy controls. Insulin-like growth factor binding protein 3 (IGFBP3) showed a significant increase in serum concentrations of PXE patients older than 45 years compared to the appropriate control group. Tissue specific gene expression of GDF11 and IGFBP3 were significantly decreased in fibroblasts from PXE patients compared to normal human dermal fibroblasts (NHDF). IGFBP3 protein concentration in supernatants of fibroblasts from PXE patients were decreased compared to NHDF but did not reach statistical significance due to potential gender specific variations. The minor changes in concentration of circulating aging biomarkers in sera of PXE patients and the significant aberrant tissue specific expression seen for selected factors in PXE fibroblasts, suggests a link between ABCC6 deficiency and accelerated aging processes in affected peripheral tissues of PXE patients.

Melanogenic inhibitory effects of Triangularin in B16F0 melanoma cells, in vitro and molecular docking studies.

The lack of secure therapies for hyperpigmentation disorders, without serious adverse effects, and the latest reports relating melanogenic disorders with development of neurodegenerative diseases, encourage the continuing search for new drugs for the treatment of such disorders. In this sense, the plant kingdom is an important source of bioactive natural products with great potential for the research and development of new therapeutics. The present study evaluated the anti-melanogenic activity of the natural methoxylated chalcone, 2',6'-dihydroxy-4'-methoxy-3'-methylchalcone (Triangularin, T), on diphenolase activity from mushroom tyrosinase and on murine B16F0 melanoma cell model. In addition, molecular modelling studies were carried out in order to understand the inhibitory activity observed. T showed a potent anti-melanogenic activity being more active than kojic acid (KA) on tyrosinase isolated of both sources and on intracellular tyrosinase. Molecular docking studies displayed important interactions between T and the active site of tyrosinase. Our results suggest that T may be useful for the treatment of hyperpigmentary disorders.

La sesión de educación física en la escuela primaria: prácticas y obstáculos / The session of physical education in primary school: practices and obstacles / A sessão de educação física no ensino fundamental: práticas e obstáculos

Problema: a pesar de las reformas frecuentes al curriculum de Educación Física, este campo es de los menos investigados en México. Objetivo: indagar cómo se desarrolla la sesión de Educación Física en la escuela primaria, a través del Programa de Estudios de la Secretaria de Educación Pública 2011. Se buscó saber cómo los educadores físicos llevan a cabo la sesión, y qué tanto incorporan del programa de Educación Física, así como los obstáculos que tienen para desarrollar la sesión. Método: en el estudio participaron 10 educadores físicos. Los instrumentos empleados fueron la observación directa y la entrevista en profundidad. Conclusión: los docentes utilizan el programa de manera limitada. Durante la sesión, los educadores físicos promueven la actividad física de intensidad moderada a vigorosa, pero enfrentan diversos obstáculos para desarrollarla

Problem: Despite frequent reforms to the physical education curriculum, this field is one of the least researched in Mexico. Objective: To investigate how the sessions of physical education take place in primary school by means of the program of the Secretary of Public Education 2011. It was sought to know how physical educators carry out the session ,and how much they incorporate from the hysical education program, as well the obstacles present in the session. . Method: Ten physical educators participated in the study. The instruments used were direct observation and in-depth interview. Conclusions: t Teachers use the program in a limited way . During the session, physical educators promote physical activity of moderate to vigorous intensity, but they face various obstacles to develop it.

Problema: apesar de frequentes reformas no currículo de Educação Física, esse campo é um dos menos pesquisados em México. Objetivo: investigar como se desenvolve a sessão de Educação Física no ensino fundamental, através do Programa de Estudos da Secretaria de Educação Pública em 2011. Buscamos saber como os educadores físicos conduzem a sessão e quanto integram o programa de Educação Física. Além disso, assinalam-se os obstáculos que eles têm para desenvolver a sessão. Método: realizou-se um estudo onde participaram dez educadores físicos, os instrumentos utilizados foram observação direta e entrevista em profundidade. Conclusão : os professores utilizam o programa de maneira limitada: durante a sessão, os educadores físicos promovem atividade física de intensidade moderada a vigorosa, porém enfrentam vários obstáculos para desenvolvê-la

Natural anthraquinones as novel photosentizers for antiparasitic photodynamic inactivation.

BACKGROUND: Cutaneous leishmaniasis (CL) is a vector-borne disease caused by obligate protist parasites from the genus Leishmania. The potential toxicity as well as the increased resistance of standard treatments has encouraged the development of new therapeutical strategies. Photodynamic inactivation (PDI) combines the use of a photosensitizer and light to generate reactive oxygen species and kill cells, including microorganisms. Vegetal kingdom constitutes an important source of bioactive compounds that deserve to be investigated in the search of naturally occurring drugs with leishmanicidal activity. PURPOSE: The purpose of this study was to test the antiparasitic activity of PDI (ApPDI) of five natural anthraquinones (AQs) obtained from Heterophyllaea lycioides (Rusby) Sandwith (Rubiacae). To support our results, effect of AQ mediated-PDI on parasite´s morphology and AQ uptake were studied. Cytotoxicity on fibroblasts was also evaluated. STUDY DESIGN/METHODS: Two monomers, soranjidiol (Sor) and 5-chlorosoranjidiol (5-ClSor) plus three bi-anthraquinones (bi-AQs), bisoranjidiol (Bisor), 7-chlorobisoranjidiol (7-ClBisor) and Lycionine (Lyc) were selected for this study. Recombinant L. amazonensis promastigote strain expressing luciferase was subjected to AQs and LED treatment. Following irradiation with variable light parameters, cell viability was quantified by bioluminescence. Alteration on parasite's morphology was analyzed by scanning electron microscopy (SEM). In addition, we verified the AQ uptake in Leishmania cells by fluorescence and their toxicity on fibroblasts by using MTT assay. RESULTS: Bisor, Sor and 5-ClSor exhibited photodynamic effect on L. amazonensis. SEM showed that promastigotes treated with Bisor-mediated PDI exhibited a significant alteration in shape and size. Sor and 5-ClSor presented higher uptake levels than bi-AQs (Bisor, Lyc and 7-ClBisor). Finally, Sor and Bisor presented the lowest toxic activity against fibroblasts. CONCLUSION: Taking together, our results indicate that Sor presents the highest specificity towards Leishmania cells with no toxicity on fibroblasts.

Experimental poisoning by Heterophyllaea pustulata Hook. f. (Rubiaceae) in goats. Clinical, biochemical and toxicological aspects.

The aim of this study was to investigate the clinical, biochemical and toxicological findings of the experimentally poisoning induced by Heterophyllaea pustulata in goats. Ten healthy adult female Saanen breed goats were used in the experiment. The goats were randomly assigned to two groups of five individuals: control and experimental group (CG and EG). Both groups were kept in the same enclosure devoid of shade for 8 h daily. The EG received only H. pustulata samples (leaves and thin steam) and water ad libitum. The CG received lucerne hay. Blood samples were taken at different times after oral administration of vegetal samples, and level of hepatic enzymes, total bilirubin, conjugated and non-conjugated bilirubin was measured, together with the detection of anthraquinones (AQs) and phylloerythrin by High Performance Liquid Chromatography with Diode-Array Detector and Mass Spectrometry with Electron Spray Ionization and Quadrupole Time Of Fly analysis. At the same time, skin biopsy samples were collected for AQs determinations. For histopathological examination, hepatic biopsy samples were collected on day 8. Clinically, all goats of the EG revealed photophobia, dermatitis and photosensitization. None of these goats developed jaundice or died during the experiment (15 days). In addition, affected goats exhibited a significant elevation in the serum levels of glutamic oxaloacetic transaminase, direct bilirubin, and total bilirubin. Microscopic examination of the liver samples revealed slight degenerative lesions. Although phylloerythrin was not detected in sera, a high level of two predominant AQs in H. pustulata (rubiadin/soranjidiol) were noted between 24 and 72 h after plant consumption, which coincided with the period in which the clinical signs were more obvious. Since those AQs were not identified in skin samples, the clinical findings were supported by the presence of AQs in sera. Finally, toxicological studies of the AQs are important, since many current works suggest their potential use in the photodynamic therapy.

Flavonoids as protective agents against oxidative stress induced by gentamicin in systemic circulation. Potent protective activity and microbial synergism of luteolin.

The flavonoids effect on gentamicin (GEN)-induced oxidative stress (OS) in systemic circulation was evaluated in terms of reactive oxygen species (ROS) production, enzymatic antioxidant defenses superoxide dismutase (SOD) and catalase (CAT), and lipid peroxidation (LP) in vitro on human leukocytes and in vivo on rat whole blood. The inhibitory activity of ROS was ATS < QTS < isovitexin < vitexin < luteolin. Luteolin, the most active, showed more inhibition in ROS production than vitamin C (reference inhibitor) in mononuclear cells and a slightly lower protective behavior compared to this inhibitor in polymorphonuclear cells. In both cellular systems, luteolin tends to level SOD and CAT activities modified by GEN, reaching basal values and preventing LP. In Wistar rats, GEN plus luteolin can suppress ROS generation, collaborate with SOD and CAT and diminish LP produced by GEN at therapeutic doses. Finally, luteolin and antibiotic association was evaluated on the antimicrobial activity in S. aureus and E. coli showing a synergism between GEN and luteolin on S. aureus ATCC and an additive effect on E. coli ATCC. Therefore, simultaneous administration of luteolin and GEN could represent a potential therapeutic option capable of protecting the host against OS induced by GEN in the systemic circulation while enhancing the antibacterial activity of GEN.

The antioxidant activity of a prenyl flavonoid alters its antifungal toxicity on Candida albicans biofilms.

The antioxidant effect of 8PP, a prenylflavonoid from Dalea elegans on Candida albicans biofilms, was investigated. We previously reported that sensitive (SCa) and resistant C. albicans (RCa) biofilms were strongly inhibited by this compound, in a dose-depending manner (50 µM-100 µM), with a prooxidant effect leading to accumulation of endogenous oxidative metabolites and increased antioxidant defenses. In this work, the antifungal activity of high concentrations of 8PP (200-1000 µM), the cellular stress imbalance and the architecture of biofilms were evaluated. Biofilms were studied by crystal violet and confocal scanning laser microscopy (CSLM) with COMSTAT analysis. Superoxide anion radical, the activity of the superoxide dismutase and the total antioxidant capacity were measured. Intracellular ROS were detected by a DCFH-DA and visualized by CSLM; reactive nitrogen intermediates by Griess. An antioxidant effect was detected at 1000 µM and levels of oxidant metabolites remained low, with major changes in the SCa. COMSTAT analysis showed that biofilms treated with higher concentrations exhibited different diffusion distances with altered topographic surface architectures, voids, channels and pores that could change the flow inside the matrix of biofilms. We demonstrate for first time, a concentration-dependent antioxidant action of 8PP, which can alter its antifungal activity on biofilms.

Histological Examination in Obtaining a Diagnosis in Patients with Lymphadenopathy in Lima, Peru.

The differential diagnosis for lymphadenopathy is wide and clinical presentations overlap, making obtaining an accurate diagnosis challenging. We sought to characterize the clinical and radiological characteristics, histological findings, and diagnoses for a cohort of patients with lymphadenopathy of unknown etiology. 121 Peruvian adults with lymphadenopathy underwent lymph node biopsy for microbiological and histopathological evaluation. Mean patient age was 41 years (Interquartile Range 26-52), 56% were males, and 39% were HIV positive. Patients reported fever (31%), weight loss (23%), and headache (22%); HIV infection was associated with fever (P < 0.05) and gastrointestinal symptoms (P < 0.05). Abnormalities were reported in 40% of chest X-rays (N = 101). Physicians suspected TB in 92 patients (76%), lymphoma in 19 patients (16%), and other malignancy in seven patients (5.8%). Histological diagnoses (N = 117) included tuberculosis (34%), hyperplasia (27%), lymphoma (13%), and nonlymphoma malignancy (14%). Hyperplasia was more common (P < 0.001) and lymphoma less common (P = 0.005) among HIV-positive than HIV-negative patients. There was a trend toward reduced frequency of caseous necrosis in samples from HIV-positive than HIV-negative TB patients (67 versus 93%, P = 0.055). The spectrum of diagnoses was broad, and clinical and radiological features correlated poorly with diagnosis. On the basis of clinical features, physicians over-diagnosed TB, and under-diagnosed malignancy. Although this may not be inappropriate in resource-limited settings where TB is the most frequent easily treatable cause of lymphadenopathy, diagnostic delays can be detrimental to patients with malignancy. It is important that patients with lymphadenopathy undergo a full diagnostic work-up including sampling for histological evaluation to obtain an accurate diagnosis.

Interactions of a prenylated flavonoid from Dalea elegans with fluconazole against azole- resistant Candida albicans.

BACKGROUND: The prenylated flavonoid 2', 4'-dihydroxy-5'-(1'″, 1'″-dimethylallyl)-8-prenylpinocembrin (8PP, formerly 6PP) shows antifungal activity, inhibits rhodamine 6G efflux and reverses fluconazole (FCZ) resistance in azole-resistant Candida albicans overexpressing cdr1, cdr2 and mdr1 transporters. PURPOSE AND DESIGN: In this paper, we tried to characterize 8PP in vitro interactions on the cell growth and lethality of C. albicans. We also initiated preliminary in vivo toxicological studies on mice. METHODS: The effects of 8PP and FCZ on cell growth and viability of C. albicans were evaluated by CLSI guidelines. The checkerboard assay was used to search for interactions on cell growth. The time-kill assay was used to study fungicidal effects. Acute toxicity was evaluated at a single dose schedules. RESULTS: From the checkerboard design, and using a starting inoculum of 103CFU/ml, the fractional inhibitory concentration (FIC) of FCZ and 8PP could be determined as 0.11 and 0.50, respectively, with a FIC index value (FICI) of 0.61. This FICI and the isobologram showing a concave shape suggests an additive interaction between them. At a higher starting inoculum (105CFU/ml), C. albicans growth and viability were decreased by FCZ, 8PP and their combination in a concentration-dependent way. For FCZ, minimum fungicidal concentration (MFC) and FC50 (the concentration that kills 50% of the fungal cells) were 4-fold reduced (280-70µM) in combination with 125µM 8PP. A decrease of 3 log units in viable counts with respect to control was reached (3.65 ± 1.05 ‰, p< 0.0001). Thus, both fungistatic compounds when combined achieved an almost complete fungicidal effect at lower concentrations respecting of each of them alone. In preliminary toxicological assessment, lethal dose 50% (LD50) for 8PP by the i.p. route was 357 and 245mg/kg, for female and male adult albino mice, respectively. FCZ LD50 was 785 and 650mg/kg for female and male animals, respectively CONCLUSIONS: In vitro results suggest additive interactions between 8PP and FCZ with respect to C. albicans cell growth. Besides killing per se, 8PP helps FCZ to achieve an almost complete fungicidal effect, which would be crucial to eradicate fungal infections.

Chemical and bioactivity of flavanones obtained from roots of Dalea pazensis Rusby.

Two new prenylated flavanones, pazentin A (3',4'-dihydroxy-6,2'-diprenylpinocembrin, 1) and pazentin B [4'-hydroxy-2'-methoxy-5'-(1‴, 1‴-dimethylallyl)-6-prenylpinocembrin, 2] together with two known ones (3 and 4) previously isolated from other Dalea species were obtained from the benzene extract of Dalea pazensis Rusby roots. The compounds were evaluated in vitro for their inhibition on mushroom tyrosinase enzyme and in relation to their effect on melanogenesis in B16 murine melanoma cells, by using a spectrophotometric method. The information obtained could be relevant to the knowledge of the structure-activity relationship for these flavonoids with the aim to explore the rational design for skin-whitening agents.

Usnic Acid Activity on Oxidative and Nitrosative Stress of Azole-Resistant Candida albicans Biofilm.

Several studies report that (+)-usnic acid, a lichen secondary metabolite, inhibits growth of different bacteria and fungi; however, the mechanism of its antimicrobial activity remains unknown. In this study, we explored the ability of usnic acid, obtained from Usnea amblyoclada, as an antibiofilm agent against azole-resistant and azole-sensitive Candida albicans strains by studying the cellular stress and antioxidant response in biofilms. The biofilm inhibitory concentration of usnic acid (4 µg/mL) exhibited a significant biofilm inhibition, 71.08 % for azole-resistant and 87.84 % for azole-sensitive C. albicans strains. Confocal scanning laser microscopy showed that the morphology of mature biofilm was altered (reduced the biomass and thickness) in the presence of usnic acid. The antifungal effect was mediated by an oxidative and nitrosative stress, with a significant accumulation of intracellular and extracellular reactive oxygen species detected by confocal scanning laser microscopy and by nitro blue tetrazolium, respectively. In fact, azole-resistant and azole-sensitive C. albicans biofilms treated at the biofilm inhibitory concentration of usnic acid presented 30-fold and 10-fold increased reactive oxygen species measurements compared to basal levels, respectively, and important nitric oxide generation, showing 25-fold and 60-fold increased reactive nitrogen intermediates levels with respect to the controls, respectively. Nonenzymatic and enzymatic antioxidant defenses were increased in both strains compared to biofilm basal levels as response to the increase of oxidant metabolites. The present study shows for the first time that usnic acid can alter the prooxidant-antioxidant balance, which may be the cause of the irreversible cell damage and lead to cell death. Our results suggest that usnic acid could be an alternative for the treatment of Candida infections, which deserves further investigation.

Protective effect of quercetin in gentamicin-induced oxidative stress in vitro and in vivo in blood cells. Effect on gentamicin antimicrobial activity.

We have evaluated the effect of gentamicin and gentamicin plus quercetin on ROS production, endogenous antioxidant defenses (SOD and CAT) and lipid peroxidation in vitro on human leukocytes and in vivo on whole rat blood. Gentamicin generated ROS production in human leukocytes, produced a dual effect on both enzymes dosage-dependent and generated an increase in lipid peroxidation. Quercetin, in leukocytes stimulated by gentamicin, showed more inhibitory capacity in ROS production than the reference inhibitor (vitaminC) in mononuclear cells and a similar protective behavior at this inhibitor in polymorphonuclear cells. Quercetin, in both cellular systems, tend to level SOD and CAT activities, reaching basal values and could prevent lipidic peroxidation induced by gentamicin. The results in Wistar rats confirmed that therapeutic doses of gentamicin can induce oxidative stress in whole blood and that the gentamicin treatment plus quercetin can suppress ROS generation, collaborate with SOD and CAT and diminish lipid peroxidation. Finally, flavonoid and antibiotic association was evaluated on the antimicrobial activity in S. aureus and E. coli, showing that changes were not generated in the antibacterial activity of gentamicin against E. coli strains, while for strains of S. aureus a beneficial effect observes. Therefore, we have demonstrated that gentamicin could induce oxidative stress in human leukocytes and in whole blood of Wistar rats at therapeutic doses and that quercetin may to produce a protective effect on this oxidative stress generated without substantially modifying the antibacterial activity of gentamicin against E. coli strains, and it contributes to this activity against S. aureus strains.

The anthraquinones rubiadin and its 1-methyl ether isolated from Heterophyllaea pustulata reduces Candida tropicalis biofilms formation.

BACKGROUND: Candida tropicalis is increasingly becoming among the most commonly isolated pathogens causing fungal infections with an important biofilm-forming capacity. PURPOSE: This study addresses the antifungal effect of rubiadin (AQ1) and rubiadin 1-methyl ether (AQ2), two photosensitizing anthraquinones (AQs) isolated from Heterophyllaea pustulata, against C. tropicalis biofilms, by studying the cellular stress and antioxidant response in two experimental conditions: darkness and irradiation. The combination with Amphotericin B (AmB) was assayed to evaluate the synergic effect. STUDY DESIGN/METHODS: Biofilms of clinical isolates and reference strain of Candida tropicalis were treated with AQs (AQ1 or AQ2) and/or AmB, and the biofilms depletion was studied by crystal violet and confocal scanning laser microscopy (CSLM). The oxidant metabolites production and the response of antioxidant defense system were also evaluated under dark and irradiation conditions, being the light a trigger for photo-activation of the AQs. The Reactive Oxygen Species (ROS) were detected by the reduction of Nitro Blue Tetrazolium test, and Reactive Nitrogen Intermediates (RNI) by the Griess assay. ROS accumulation was also detected inside biofilms by using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe, which was visualized by CSLM. Superoxide dismutase (SOD) activity and the total antioxidant capacity of biofilms were measured by spectrophotometric methods. The minimun inhibitory concentration for sessile cells (SMIC) was determined for each AQs and AmB. The fractional inhibitory concentration index (FICI) was calculated for the combinations of each AQ with AmB by the checkerboard microdilution method. RESULTS: Biofilm reduction of both strains was more effective with AQ1 than with AQ2. The antifungal effect was mediated by an oxidative and nitrosative stress under irradiation, with a significant accumulation of endogenous ROS detected by CSLM and an increase in the SOD activity. Thus, the prooxidant-antioxidant balance was altered especially by AQ1. The best synergic combination with AmB was also obtained with AQ1 (80.5%) (FICI=0.74). CONCLUSION: Under irradiation, the oxidative stress was the predominant effect, altering the prooxidant-antioxidant balance, which may be the cause of the irreversible cell injury in the biofilm. Our results showed synergism of these natural AQs with AmB. Therefore, the photosensitizing AQ1 could be an alternative for the Candida infections treatment, which deserves further investigation.