Comparison of the extracellular polymeric substances of Candida albicans and Candida dubliniensis biofilms

Candida albicans and Candida dubliniensis live as benign commensal organisms in the oral cavity of both healthy and unhealthy individuals behaving, under certain conditions, as opportunistic pathogens, causing candidiasis. These two Candida species have been mismatched for years, but recently Candida dubliniensis was recovered from the mouth of imunnosupressed patients and identified as a different species. Candidiasis is usually related with the Candida capacity of forming biofilms on inert or biological surfaces, being this phenotype associated with infections. Biofilms are complex structures of microbial communities attached to a surface, in which microorganisms are embedded in a matrix of extracellular polymeric substances (EPS), composed mainly by proteins and polysaccharides. The biofilm matrix holds the potential of determining possible mechanisms of resistance of Candida biofilms. Several factors are known as affecting the production of EPS, namely, growth medium, growth phase and substratum. This study focused the influence of artificial saliva growth medium in the composition of EPS of biofilms formed by both Candida albicans and Candida dubliniensis strains. Biofilms of one strain of Candida albicans and two strains of Candida dubliniensis were formed in an artificial saliva growth medium (ASGM) and compared with those formed in Sabouraud Dextrose Broth (SDB) and analysed after 48h.The differences between the EPS of biofilms were evaluated (after sonication) in terms of proteins (quantified using the BCA protein assay kit) and polysaccharides (quantified using the phenol-sulphuric method). Proteins were also analysed by SDS-PAGE. In SDB the amount of proteins and polysaccharides in the EPS of biofilms formed by Candida albicans was lower than in the EPS of biofilms formed by Candida dubliniensis strains. In the presence of ASGM the amount of proteins and polysaccharides was similar among the EPS of biofilms of Candida albicans and one of the Candida dubliniensis strains and was lower in biofilms of Candida albicans than in biofilms of the other Candida dubliniensis. Analysis of protein profiles obtained by SDS-PAGE showed that all strains present similar patterns independently of the medium of biofilm formation. Biofilms formed in ASGM originated different amounts of EPS, either in terms of polysaccharides or proteins, compared to the ones formed in SDB. Differences were also found in the profile of extracellular proteins of each strain, depending on the medium

Influence of saliva and mucin on the adhesion of Candida oral clinical isolates

Objectives: This research work intends to clarify the role of artificial saliva, in particularly the role of mucin, a salivary protein, on the surface properties and adhesion ability of Candida spp. oral clinical isolates to abiotic surfaces. Methods: Four oral clinical isolates of Candida spp. were used: two Candida albicans strains (AC; AM) and two Candida parapsilosis strains (AD; AM2). The strains were isolated from patients using oral prosthesis. The microorganisms were cultured in the absence or presence of mucin and artificial saliva, and their adhesion to an abiotic surface (coated with mucin and artificial saliva) was evaluated. Results: The presence of mucin per se onto the abiotic surface decreased the adhesion of all strains, although the combination of mucin with artificial saliva had reduced this effect. No direct correlation between adhesion and the surface free energies of adhesion of the microorganisms was found. Significance: Candida spp. were human commensal microorganisms that became pathogenic when the host immune defenses were compromised. Medical devices were colonized by Candida spp. particularly, oral prostheses, which might lead to the degradation of the prostheses and systemic infections. The salivary secretions that constantly cover the oral cavity influenced Candida spp. adhesion process. Therefore, it was important to understand the interactions between Candida spp., salivary proteins and the characteristic of oral prosthesis when developing materials for oral prostheses.The authors thank the Project “BioHealth-Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER. The would also like to thank the Fundação para a Ciência e Tecnologia for the Strategic Project Pest-OE/EQB/LA0023/2013 and Fundação para a Ciência e Tecnologia (FCT) for Ana Oliveira PhD Grant (SFRH/BD/68588/2010) and Catarina L. Seabra PhD Grant (SFRH/BD/89001/2012). The authors would also like to acknowledge Professora Rosário Oliveira, which is no longer with us, for her exceptional contribution and dedication to this work

Monoclonal antibodies production in wave and stirred tank bioreactors

The rapid increase in the number of monoclonal antibodies (mAbs) which are being regularly approved for therapeutic use results in the need of their large scale production. However, this requires the development of bioreactors and processes simple to operate and easily scaledup, that allow cultivation of mammalian cells. The Stirred Tank bioreactor is the traditional and most widely used reactor type, mainly due to the know-how acquired with microbial fermentation, its flexibility and suitability for different cell types, operation modes, products and working volumes.[1] However, recently, disposable bioreactors, such as the new Wave reactor, are becoming increasingly popular due to their low initial and lifetime costs, simplified scale-up, reduced turn-around time between runs and low risk of crosscontamination.[2] Due to the current lack of comparative studies about these different technologies, we are optimizing and comparing mAb production in both Stirred Tank and Wave bioreactors. Different modes of operation are being tested (batch and fed-batch), and the use of microcarrier technology for culture of anchorage-dependent cells will also be assayed. Furthermore, it is important to evaluate the product in terms of biological activity, assuring that it maintains full functionality, by assessing how the glycosylation pattern is affected during the process of production in both reactors

Vascular Pathways of Testosterone: Clinical Implications

Cardiovascular diseases (CVD) are one of the leading causes of death worldwide. Testosterone (T) is an important sex hormone that triggers several genomic and non-genomic pathways, leading to improvements of several cardiovascular risk factors and quality of life in men. At the vascular level, the key effect of T is the vasorelaxation. This review discusses the molecular pathways and clinical implications of T in the vascular system. Firstly, the mechanisms involved in the T vasodilator effect will be presented. Then, it will be discussed the association of T with the main risks for CVD, namely metabolic syndrome, type 2 diabetes mellitus, obesity, atherosclerosis, dyslipidaemia and hypertension. Several studies have shown a correlation between low T levels and an increased prevalence of several CVD. These observations suggest that T has beneficial effects on the cardiovascular system and that testosterone replacement therapy may become a therapeutic reality for some of these disorders.UBI-Santander Totta (BID/FCS/2018)info:eu-repo/semantics/publishedVersio

Adhesion of non-Candida albicans Candida spp to urinary epithelial cells

Non-Candida albicans Candida (NCAC) spp, are nowadays responsible for more than 50% of Candida infections and it has been shown that the most prevalent NCAC spp are C.tropicalis, C. parapsilosis and C. glabrata. An important step on NCAC spp infection is their ability to adhere to the host tissues. Therefore, the aim of this study was to evaluate the ability of C.tropicalis, C. parapsilosis and C. glabrata to adhere to a urinary epithelial cell line (TCC-SUP). The ability of C.tropicalis ATCC 750, C. parapsilosis ATCC 2201, C. glabrata ATCC 2001 and C. albicans SC5413 to adhere to TCC-SUP cells was evaluated after 2 and 24h. For that, yeast cells (1x107 cell/ml) were incubated with a confluent layer of epithelial cells at 37 ºC and 5 % of CO2. The number of yeast cells adhered to the epithelial cells was determined using an adaptation of the Crystal Violet (CV) staining method. Additionally, the epithelial cell activity was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium (MTS) viability assay. After 24h of incubation the number of yeasts adhered to epithelial cells was higher than after 2h, unexpectedly except for C. glabrata. Nevertheless, after 24 h, C. glabrata extent of adhesion was still higher than C. tropicalis and C. parapsilosis. This particular behavior of C. glabrata was also observed after 2 h of adhesion, showing a higher number of adhered cells in comparison with the other NCAC spp, which behaved very similarly. The trend observed by CV staining does not seem to be reflected on the metabolic activity of the epithelial cells.The main conclusion of the present work is that NCAC spp are biofilm producers on silicone, and that the adhesion and biofilm formation ability appear to be species and strain dependent

Candida tropicalis biofilms : formation and virulence factors

Significance and objectives: A substantial proportion of Candida tropicalis infections is associated with biofilm formation, especially on catheters. Thus, the aim of this study was to investigate C. tropicalis biofilm formation on silicone and its effect on epithelial cells and enzyme production (hemolysins and proteinases). Methods and results: This study was performed with C. tropicalis (clinical isolate and reference strain ATCC 750). Biofilms formed on silicone coupons immersed in artificial urine, were quantified by crystal violet (CV) staining and by enumeration of colony forming units (CFU) and the matrix content in proteins and polysaccharides was also determined. Biofilm cells and matrix were assessed in terms of hemolysins and proteinases production and their effect on TCC-SUP urinary epithelial cells was evaluated as well. Biofilms of C. tropicalis ATCC 750 presented a higher number of cells than the clinical isolate although less biofilm biomass and less polysaccharides. Moreover C. tropicalis biofilm was able to express total hemolytic activity and higher proteinase but these factors were not detectable within the matrix. Additionally, C. tropicalis biofilm adhered in higher extent to epithelial cells than their planktonic counterparts. Moreover, epithelial cells showed low metabolic activity when in contact with biofilms. Conclusions: Therefore, it is possible to conclude that enzyme production was detected in C. tropicalis biofilm cells, but not in its matrix and that biofilm cells can cause more damage to epithelial cells than their placktonic counterparts. This highlights the importance of biofilm formation, associated to the use of urinary catheters, on C. tropicalis virulence

IL-1R and inflammasomes mediate early pulmonary protective mechanisms in respiratory brucella abortus infection

Brucella spp. infection is frequently acquired through contaminated aerosols. The role of interleukin-1 beta (IL-1β) in the early pulmonary response to respiratory Brucella infection is unknown. As shown here, IL-1β levels in lung homogenates and bronchoalveolar lavage fluid (BALF) of mice intratracheally inoculated with B. abortus were increased at 3 and 7 days p.i. At 7 days p.i., pulmonary CFU numbers were higher in IL-1 receptor (IL-1R) knockout (KO) mice than in wild type (WT) mice. At different times p.i. CFU in lungs and BALF were higher in mice lacking some inflammasome components (caspase-1, AIM2, NLRP3) than in WT mice. At 2 days p.i. pulmonary levels of IL-1b and CXCL1 (neutrophils chemoattractant) were lower in caspase-1/11 KO mice. At day 3 p.i., neutrophils counts in BALF were lower in caspase-1/11 KO mice than in WT mice. During in vitro infections, IL-1β secretion was lower in alveolar macrophages from caspase-1/11, NLRP3 or AIM2 KO mice than in WT controls. Similarly, IL-1β production by B. abortus-infected alveolar epithelial cells was reduced by pretreatment with a specific caspase-1 inhibitor. This study shows that IL-1R, probably through IL-1β action, and the NLRP3 and AIM2 inflammasomes are involved in pulmonary innate immune protective mechanisms against respiratory B. abortus infection.Fil: Hielpos, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Fernandez, Andrea Giselle. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Falivene, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Alonso Paiva, Iván Mathias. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Muñoz González, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Ferrero, Mariana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Campos, Priscila C.. Universidade Federal de Minas Gerais; BrasilFil: Vieira, Angelica T.. Universidade Federal de Minas Gerais; BrasilFil: Oliveira, Sergio Costa. Universidade Federal de Minas Gerais; BrasilFil: Baldi, Pablo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentin