Cupuassu juice fermentation by Lactiplantibacillus plantarum Lp62 produces an antioxidant enriched probiotic beverage.

The present work aimed to produce a cupuassu juice (Theobroma grandiflorum) fermented by the probiotic bacterium Lactiplantibacillus plantarum Lp62 and to analyze its antioxidant potential, antimicrobial activity, and resistance to biological barriers. The fermented beverage showed an increase in the content of phenolics, flavonoids, and antioxidant potential. The culture showed antagonistic activity against pathogens, but this result was not observed when the juice was tested. The probiotic strain remained viable under refrigeration, even in an acidified environment, and survived simulated gastrointestinal transit in vitro. L. plantarum Lp62 showed 30% adherence to HT-29 intestinal cells and proved to be safe in terms of antibiotic resistance and production of virulence factors. Fermentation increased the functional characteristics of cupuassu juice. This drink proved to be a good vehicle for the delivery of the probiotic bacteria L. plantarum Lp62.

Ex-vivo mucolytic and anti-inflammatory activity of BromAc in tracheal aspirates from COVID-19.

COVID-19 is a lethal disease caused by the pandemic SARS-CoV-2, which continues to be a public health threat. COVID-19 is principally a respiratory disease and is often associated with sputum retention and cytokine storm, for which there are limited therapeutic options. In this regard, we evaluated the use of BromAc®, a combination of Bromelain and Acetylcysteine (NAC). Both drugs present mucolytic effect and have been studied to treat COVID-19. Therefore, we sought to examine the mucolytic and anti-inflammatory effect of BromAc® in tracheal aspirate samples from critically ill COVID-19 patients requiring mechanical ventilation. METHOD: Tracheal aspirate samples from COVID-19 patients were collected following next of kin consent and mucolysis, rheometry and cytokine analysis using Luminex kit was performed. RESULTS: BromAc® displayed a robust mucolytic effect in a dose dependent manner on COVID-19 sputum ex vivo. BromAc® showed anti-inflammatory activity, reducing the action of cytokine storm, chemokines including MIP-1alpha, CXCL8, MIP-1b, MCP-1 and IP-10, and regulatory cytokines IL-5, IL-10, IL-13 IL-1Ra and total reduction for IL-9 compared to NAC alone and control. BromAc® acted on IL-6, demonstrating a reduction in G-CSF and VEGF-D at concentrations of 125 and 250 µg. CONCLUSION: These results indicate robust mucolytic and anti-inflammatory effect of BromAc® ex vivo in tracheal aspirates from critically ill COVID-19 patients, indicating its potential to be further assessed as pharmacological treatment for COVID-19.

Differentially expressed proteins in the interaction of Paracoccidioides lutzii with human monocytes.

BACKGROUND: Fungi of the genus Paracoccidioides are the etiological agents of paracoccidioidomycosis, a highly prevalent mycosis in Latin America. Infection in humans occurs by the inhalation of conidia, which later revert to the form of yeast. In this context, macrophages are positioned as an important line of defense, assisting in the recognition and presentation of antigens, as well as producing reactive oxygen species that inhibit fungal spreading. AIMS: The objective of this study was to identify differentially expressed proteins during the interaction between Paracoccidioides lutzii Pb01 strain and human U937 monocytes. METHODS: Two-dimensional electrophoresis, combined with mass spectrometry, was used to evaluate the differential proteomic profiles of the fungus P. lutzii (Pb01) interacting with U937 monocytes. RESULTS: It was possible to identify 25 proteins differentially expressed by Pb01 alone and after interacting with U937 monocytes. Most of these proteins are directly associated with fungal metabolism for energy generation, such as glyceraldehyde-3-phosphate dehydrogenase, and intracellular adaptation to monocytes. Antioxidant proteins involved in the response to oxidative stress, such as peroxiredoxin, cytochrome, and peroxidase, were expressed in greater quantity in the interaction with monocytes, suggesting their association with survival mechanisms inside phagocytic cells. We also identified 12 proteins differentially expressed in monocytes before and after the interaction with the fungus; proteins involved in the reorganization of the cytoskeleton, such as vimentin, and proteins involved in the response to oxidative stress, such as glioxalase 1, were identified. CONCLUSIONS: The results of this proteomic study of a P. lutzii isolate are novel, mimicking in vitro what occurs in human infections. In addition, the proteins identified may aid to understand fungal-monocyte interactions and the pathogenesis of paracoccidioidomycosis.

Immobilization of PR4A3 enzyme in pluronic F127 polymeric micelles against colorectal adenocarcinoma cells and increase of in vitro bioavailability.

Traditional therapy for malignant neoplasms involving surgical procedures, radiotherapy and chemotherapy aims to kill neoplastic cells, but also affects normal cells. Therefore, exogenous proteases are the target of studies in cancer therapy, as they have been shown to be effective in suppressing tumors and reducing metastases. Pluronic F127 (F127) is a copolymer of amphiphilic blocks that has shown significant potential for drug administration, as it is capable of incorporating hydrophobic drugs and self-assembling in micrometers of nanometric size. This study investigated the effects of immobilization of the alkaline protease PR4A3 with pluronic F127 micelles on the enzyme-induced cytotoxicity. Protease immobilization was demonstrated through UV-visible and circular dichroism (CD) spectroscopies, as the enzyme interacts with the polymeric micelle of Pluronic F127 without changing its secondary structure. In addition, the immobilized form of the enzyme showed greater bioavailability after passing through the simulated gastrointestinal transit. Cell viability was assessed using the tetrazoic methylthiazole (MTT) assay. The results open perspectives for new research and development for PR4A3 in the treatment of colorectal carcinoma.

Biotechnological potential of mangrove sediments: Identification and functional attributes of thermostable and salinity-tolerant ß-glucanase.

Microorganisms native to mangroves are expected to contain enzymes capable of hydrolyzing different carbon sources. However, most of these microorganisms aren't cultivable; hence, alternative techniques as metagenomics are tools for studying and obtaining some of the natural genomes, genes and enzymes of biotechnological interest. The ß-glucanase was produced using a metagenomic clone of mangrove sediments and detected by functional screening on carboxymethylcellulose substrate. The enzyme was purified by cation exchange chromatography. The peptides detected by mass spectrometry showed 20% identity with the polypeptide deduced from the genomic fragment sequenced. The ORF identified as BglfosD9 possessed 729 bp and the encoded protein showed predicted MW and pI of 28kD and 6.8, respectively. The enzyme was active in a wide range of pH (5-10) with optimum pH at 8, had relative activity greater than 50% at all temperatures tested (5-90 °C), was stable at temperatures of 5, 50 and 90 °C and showed excellent relative activity at high NaCl concentrations. This ß-glucanase also showed high relative activity in the presence of SDS and it could hydrolyze ß-glucan, CMC and Avicel as substrates. These findings support the idea of a new thermostable and active enzyme at basic pH from metagenomic library of mangrove sediment.

Potential of Maintaining a Healthy Vaginal Environment by Two Lactobacillus Strains Isolated from Cocoa Fermentation.

Bacteria in the genera Mycoplasma and Ureaplasma do not have cell walls and therefore interact with host cells through lipid-associated membrane proteins (LAMP). These lipoproteins are important for both surface adhesion and modulation of host immune responses. Mycoplasma and Ureaplasma have been implicated in cases of bacterial vaginosis (BV), which can cause infertility, abortion, and premature delivery. In contrast, bacteria of the genus Lactobacillus, which are present in the vaginal microbiota of healthy women, are thought to inhibit local colonization by pathogenic microorganisms. The aim of the present study was to evaluate the in vitro interactions between lipoproteins of Mycoplasma and Ureaplasma species and vaginal lineage (HMVII) cells and to study the effect of Lactobacillus isolates from cocoa fermentation on these interactions. The tested Lactobacillus strains showed some important probiotic characteristics, with autoaggregation percentages of 28.55% and 31.82% for L. fermentum FA4 and L. plantarum PA3 strains, respectively, and percent adhesion values of 31.66 and 41.65%, respectively. The two strains were hydrophobic, with moderate to high hydrophobicity values, 65.33% and 71.12% for L. fermentum FA4 and L. plantarum PA3 in toluene. Both strains secreted acids into the culture medium with pH=4.32 and pH=4.33, respectively, and showed antibiotics susceptibility profiles similar to those of other lactobacilli. The strains were also able to inhibit the death of vaginal epithelial cells after incubation with U. parvum LAMP from 41.03% to 2.43% (L. fermentum FA4) and 0.43% (L. plantarum PA3) and also managed to significantly decrease the rate of cell death caused by the interaction with LAMP of M. hominis from 34.29% to 14.06% (L. fermentum FA4) and 14.61% (L. plantarum PA3), thus demonstrating their potential for maintaining a healthy vaginal environment.

Co-infection of sexually transmitted pathogens and Human Papillomavirus in cervical samples of women of Brazil.

BACKGROUND: Some sexually transmitted infectious agents, such as Chlamydia trachomatis and Herpes simplex, cause local inflammation, and could contribute to Human Papillomavirus (HPV) and cervical lesion progression. Thus, the aim of this study was to determine any association between the presence of microorganisms of gynecological importance, sexual behavior, clinical and demographical variables to the development and progress of cervical lesions. METHODS: One hundred and thirty-two women between 14 and 78 years and living at Vitória da Conquista, Bahia, Brazil, were included (62 individuals with cervical lesions and 70 without lesions). They answered a questionnaire to provide data for a socioeconomic and sexual activity profile. Samples of cervical swabs were collected and analyzed by PCR to detect genital microorganisms and HPV. Quantitative PCR was used to detect and quantify Ureaplasma urealyticum and Ureaplasma parvum. Univariate and multiple logistic regression were performed to measure the association with the cervical lesions, and an odds ratio (OR) with 95% confidence intervals (95%CI) were calculated. The Mann-Whitney U test was also used to compare the microorganism load in the case and control groups. The significance level was 5% in all hypotheses tested. RESULTS: Cervical lesions were associated with: women in a stable sexual relationship (OR = 14.21, 95%CI = 3.67-55.018), positive PCR for HPV (OR = 16.81, 95%CI = 4.19-67.42), Trichomonas vaginalis (OR = 8.566, 95%CI = 2.04-35.94) and Gardnerella vaginalis (OR = 6.13, 95%CI = 1.53-24.61), adjusted by age and qPCR for U. parvum. U. parvum load showed a statistical difference between the case and control groups (p-value = 0.002). CONCLUSION: Variables such as stable relationship, HPV, T. vaginalis, G. vaginalis were associated with cervical lesions in epidemiological studies. U. parvum load was higher in woman with cervical lesions compared with women without lesions. Additional studies are needed to better understand the role of these factors in cervical lesion development.

The role of cytokines in immune regulation of female reproductive physiology

Cytokines act as protein mediators of the immune system and exert pleiotropic effects on the source cells and/or on target cells. Cytokines are formed in a cascade, bind to specific receptors, and influence the activity, differentiation, proliferation, and survival of immune cells of both T helper 1 (Th1) type (which has proinflammatory properties) and Th2 type (with an anti-inflammatory function). The female reproductive system is regulated by the immune system via cytokines at various physiological stages: during the ovarian cycle, maternal recognition, embryo implantation, gestation, and birth, participating in homeostasis and protection from pathogens. These processes interact under the hormonal influence of the hypothalamic­pituitary­gonadal axis. This review is aimed at addressing the involvement of some cytokines in female reproductive physiology, highlighting the maternal recognition of the embryo and implantation as immunologically important steps for fetal survival. The scientific knowledge on the role of cytokines in female reproduction processes, such as the Th1/Th2 balance and immune tolerance should advance the research in various fields of assisted reproduction in humans and animals, such as artificial insemination, embryo transfer, and in vitro fertilization. The same is true for the development of contraceptive methods and understanding of pathological processes such as uterine infections and autoimmune diseases.

Immunomodulatory Effects of Lactobacillus plantarum Lp62 on Intestinal Epithelial and Mononuclear Cells.

Probiotic lactic acid bacteria are known for their ability to modulate the immune system. They have been shown to inhibit inflammation in experiments with animal models, cell culture, and clinical trials. The objective of this study was to elucidate the anti-inflammatory potential of Lactobacillus plantarum Lp62, isolated from cocoa fermentation, in a cell culture model. Lp62 inhibited IL-8 production by Salmonella Typhi-stimulated HT-29 cells and prevented the adhesion of pathogens to these epithelial cells. The probiotic strain was able to modulate TNF-α, IL1-ß, and IL-17 secretion by J774 macrophages. J774 activation was reduced by coincubation with Lp62. PBMC culture showed significantly higher levels of CD4(+)CD25(+) T lymphocytes following treatment with Lp62. Probiotics also induced increased IL-10 secretion by mononuclear cells. L. plantarum Lp62 was able to inhibit inflammatory stimulation in epithelial cells and macrophages and activated a tolerogenic profile in mononuclear cells of healthy donors. These results indicate this strain for a possible application in the treatment or prevention of inflammatory diseases.

Analysis of Protein Composition and Bioactivity of Neoponera villosa Venom (Hymenoptera: Formicidae).

Ants cause a series of accidents involving humans. Such accidents generate different reactions in the body, ranging from a mild irritation at the bite site to anaphylactic shock, and these reactions depend on the mechanism of action of the venom. The study of animal venom is a science known as venomics. Through venomics, the composition of the venom of several ant species has already been characterized and their biological activities described. Thus, the aim of this study was to evaluate the protein composition and biological activities (hemolytic and immunostimulatory) of the venom of Neoponera villosa (N. villosa), an ant widely distributed in South America. The protein composition was evaluated by proteomic techniques, such as two-dimensional electrophoresis. To assess the biological activity, hemolysis assay was carried out and cytokines were quantified after exposure of macrophages to the venom. The venom of N. villosa has a profile composed of 145 proteins, including structural and metabolic components (e.g., tubulin and ATPase), allergenic and immunomodulatory proteins (arginine kinase and heat shock proteins (HSPs)), protective proteins of venom (superoxide dismutase (SOD) and catalase) and tissue degradation proteins (hyaluronidase and phospholipase A2). The venom was able to induce hemolysis in human erythrocytes and also induced release of both pro-inflammatory cytokines, as the anti-inflammatory cytokine release by murine macrophages. These results allow better understanding of the composition and complexity of N. villosa venom in the human body, as well as the possible mechanisms of action after the bite.

Infection cycle of Salmonella enterica serovar Enteritidis in latent carrier mice.

This work reports the distribution of an oral dose of Salmonella enterica serovar Enteritidis (SE) in C57Bl/6-Bcgr mice, to study its pathogenesis in a latent carrier animal. Mice orally inoculated with a high dose of SE developed a latent infection characterized by the absence of clinical symptoms in which the cecum is functioning as a "strategic site" of SE proliferation, releasing bacteria into feces intermittently over the 4-week study. A sequence of disruptions occurred in the small intestine at 1 day postinculation (PI). The microvilli exhibited different degrees of degeneration, which were reversible as the cells became vacuolated. From 2 days PI, SE was detected in the mononuclear phagocytic system, and an exponential growth of the remaining bacteria in tissues was observed until 4 days PI. The production of interferon gamma from 3 days PI is restricting the SE growth, and a plateau phase was observed from 4 to 15 days PI. A recurrence of the bacterial growth in tissue occurred from 15 to 28 days PI, especially in the cecum. Increasing our knowledge about the host-pathogen interaction of adapted pathogens with the ability to develop latency is essential for the development of an efficient strategy for Salmonella control.

Primeiro escore de risco inflamatório das endopróteses de aorta / First inflammatory risk score for aortic endoprostheses

OBJETIVO: Propor um escore de risco inflamatório para tratamento endovascular dos aneurismas da aorta. MÉTODOS: Vinte e cinco pacientes foram seguidos do período pré-operatório até 3º mês de pós-operatório (1 hora, 6 horas, 24 horas, 48 horas, 7 dias, 1 mês, 2 meses e 3 meses). Variáveis inflamatórias avaliadas foram proteína C reativa, velocidade de hemossedimentação, interleucinas (IL-6, IL8), fator de necrose tumoral alfa, L-selectina, molécula de adesão intercelular (ICAM-1), transfusão de hemáceas, volume de cristalóide, volume de contraste, material da prótese, número de próteses, contagem total de leucócitos e linfócitos. O teste de Spearman apontou as variáveis candidatas ao maior risco inflamatório, segundo P < 20 por cento. A regressão logística apontou variáveis selecionáveis para escore final segundo P < 10 por cento. A análise da curva ROC revelou valores de corte para variáveis selecionadas pela regressão logística. RESULTADOS: Variáveis apresentadas pelo teste de Spearman foram: volume de cristalóide (P = 0,04), material da prótese (P = 0,04), volume de contraste (P=0,02), IL-8 préoperatória (P= 0,10), ICAM-1 1 mês (P=0,03) e L-selectina 1 mês (P = 0,06). A regressão logística revelou que os valores do volume de cristalóide e IL-8 pré-operatória são primordiais para constituição do escore de risco inflamatório para tratamento endovascular dos aneurismas da aorta. O escore de risco seria dividido em três categorias (leve, moderado e grave), com base em intervalos numéricos das duas variáveis selecionadas e as categorias seriam correlacionadas com achados clínicos CONCLUSÃ: Volume de cristalóide e IL-8 pré-operatória são variáveis que poderiam contribuir para categorizar risco inflamatório e, desse modo, ter um papel prognóstico no tratamento endovascular dos aneurismas da aorta.

OBJECTIVE: To purpose an inflammatory risk score for aortic aneurysm endovascular treatment. METHODS: Twenty-five patients were followed-up from preoperative period to third month postoperatively (1-hour, 6-hour, 24-hour, 48-hour, 7-day, 1-month, 2- month and 3month). Inflammatory variables were C-reactive protein, hemosedimentation velocity, interleukins (IL-6, IL-8), tumor necrosis factor-Alpha, L-selectin, intercellular adhesion molecule (ICAM-1), red blood cells transfusion, volume of crystalloid, volume of contrast, type of endoprosthesis, number of endoprostheses, total count of leukocytes and lymphocytes. Spearman test defined the variables considered as candidates to higher inflammatory risk based on P < 20 percent. Logistic regression defined the variables considered as selected for final score based on P < 10 percent. ROC curve analysis revealed the cut-off values for variables selected by logistic regression. RESULTS: Variables defined by Spearman test were: volume of crystalloid (P=0.04), type of endoprosthesis (P=0.04), volume of contrast (P=0.02), preoperative IL-8 (P = 0.10), 1 - month ICAM-1 (P=0.03) and 1-month L-selectin (P=0.06). Logistic regression revealed that volume of crystalloid and preoperative IL-8 values are relevant for composition of inflammatory risk score for aortic aneurysm endovascular treatment. Risk score would be divided into three categories (mild, moderate and severe) based on numeric intervals of these two variables and the categories would be correlated to clinical findings. CONCLUSION: Volume of crystalloid and preoperative IL-8 are variables that might contribute to categorize inflammatory risk and thereby might play a prognostic role for aortic aneurysm endovascular treatment.

First inflammatory risk score for aortic endoprostheses.

OBJECTIVE: To purpose an inflammatory risk score for aortic aneurysm endovascular treatment. METHODS: Twenty-five patients were followed-up from preoperative period to third month postoperatively (1-hour, 6-hour, 24-hour, 48-hour, 7-day, 1-month, 2- month and 3month). Inflammatory variables were C-reactive protein, hemosedimentation velocity, interleukins (IL-6, IL-8), tumor necrosis factor-Alpha, L-selectin, intercellular adhesion molecule (ICAM-1), red blood cells transfusion, volume of crystalloid, volume of contrast, type of endoprosthesis, number of endoprostheses, total count of leukocytes and lymphocytes. Spearman test defined the variables considered as candidates to higher inflammatory risk based on P < 20%. Logistic regression defined the variables considered as selected for final score based on P < 10%. ROC curve analysis revealed the cut-off values for variables selected by logistic regression. RESULTS: Variables defined by Spearman test were: volume of crystalloid (P=0.04), type of endoprosthesis (P=0.04), volume of contrast (P=0.02), preoperative IL-8 (P = 0.10), 1 - month ICAM-1 (P=0.03) and 1-month L-selectin (P=0.06). Logistic regression revealed that volume of crystalloid and preoperative IL-8 values are relevant for composition of inflammatory risk score for aortic aneurysm endovascular treatment. Risk score would be divided into three categories (mild, moderate and severe) based on numeric intervals of these two variables and the categories would be correlated to clinical findings. CONCLUSION: Volume of crystalloid and preoperative IL-8 are variables that might contribute to categorize inflammatory risk and thereby might play a prognostic role for aortic aneurysm endovascular treatment.

Analysis of the inflammatory response in endovascular treatment of aortic aneurysms.

OBJECTIVE: The objective of this study is to evaluate the inflammatory response caused by endovascular stents in the treatment of aortic aneurysms. METHODS: Twenty-five patients underwent endovascular stent treatment from March through December 2005. The evolution of mediators (sedimentation velocity, C reactive protein, interleukin-6, interleukin-8, tumor necrosis factor-alpha, intercellular adhesion molecule-1, l-selectin), inflammatory cells (leukocytes, lymphocytes, platelets), serum creatinine and body temperature within preoperative period and in the following postoperative periods--1, 6, 24 and 48 h, 7 days, 1-3 months, was analyzed. In order to achieve statistic significance, Friedman test and Wilcoxon test were used, with index of significance of 5% (p<0.05). RESULTS: Peak values of sedimentation velocity, C reactive protein and interleukin-6 were observed at 7 days (p<0.0001), 48 h (p<0.0001) and 24h (p<0.0001), respectively. Tumor necrosis factor-alpha and interleukin-8 did not show statistically significant variability during the entire follow-up. In terms of intercellular adhesion molecule-1 and l-selectin, their expressive values were found in late phase of follow-up, although without statistical significance. Elevation of leukocytes count occurred in premature phase of follow-up (p<0.0001), while lymphocyte and platelet count occurred in a late phase of follow-up (p<0.0001). Serum levels of creatinine did not show significant variability during follow-up. The period between 24 and 48 h corresponded to major frequency for fever (p<0.0001). CONCLUSION: Individual mediators analysis and inflammatory cells demonstrated variability of their values during postoperative follow-up. This could help in the analysis of the inflammatory response evolution caused by endovascular stent treatment for aortic aneurysms in premature and late phases after implantation of the vascular prosthesis.