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ABSTRACT: Microcarriers are widely used for the large-scale culture of attachment-dependent cells with increased cell densities and, ultimately, higher product yield. In these processes, the specific culture conditions can affect the quality of the product, which is closely related to its glycosylation pattern. Furthermore, the lack of studies in the area reinforces the need to better understand the effects of microcarrier culture in product glycosylation. Consequently, in this work, the glycosylation profile of a monoclonal antibody (mAb) produced by adherent CHO-K1 cells grown in Cytodex 3 was evaluated under different conditions, and compared to that obtained of typical adherent cultures. It was found that microcarrier cultures result in a glycosylation profile with different characteristics from T-flask cultures, with a general increase in galactosylation and decrease in fucosylation levels, both with a potentially positive impact on mAb activity. Sialylation also varied but without a general tendency. This study then showed that the specific culture conditions used in microcarrier culture influence the mAb glycan profile, and each functional element (galactose, core fucose, sialic acid) is independently affected by these conditions. In particular, great reductions of fucosylation (from 79 to 55%) were obtained when using half volume at inoculation, and notable decreases in sialylation (from 23 to 2%) and glycoform heterogeneity (from 20 to 11 glycoforms) were observed for shake flask culture, potentially associated with the improved cell densities achieved in these culture vessels.
SpringerPlus. 12/2013; 2(1):25.
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ABSTRACT: Currently, mammalian cell technology has become the focus of biopharmaceutical production, with strict regulatory scrutiny of the techniques employed. Major concerns about the presence of animal-derived components in the culture media led to the development of serum-free (SF) culture processes. However, cell adaptation to SF conditions is still a major challenge and limiting step of process development. Thus, this study aims to assess the impact of SF adaptation on monoclonal antibody (mAb) production, identify the most critical steps of cell adaptation to the SF EX-CELL medium, and create basic process guidelines. The success of SF adaptation was dependent on critical steps that included accentuated cell sensitivity to common culture procedures (centrifugation, trypsinization), initial cell concentration, time given at each step of serum reduction, and, most importantly, medium supplements used to support adaptation. Indeed, only one of the five supplement combinations assessed (rhinsulin, ammonium metavanadate, nickel chloride, and stannous chloride) succeeded for the Chinese hamster ovary-K1 cell line used. This work also revealed that the chemically defined EX-CELL medium benefits mAb production in comparison with the general purpose Dulbecco's Modified Eagle's Medium, but the complete removal of serum attenuates these positive effects.
Applied biochemistry and biotechnology 01/2013; · 1.94 Impact Factor
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ABSTRACT: N-glycosylation is one of the most critical parameters affecting the biological activity of therapeutic monoclonal antibodies (mAbs), and should therefore be closely monitored and controlled to guarantee a consistent and high-quality product in biopharmaceutical processes. In the present work, the effect of the time-consuming step of gradual cell adaptation to serum-free conditions on the glycosylation profile of a mAb produced by CHO-K1 cells was evaluated. High-performance liquid chromatography analysis revealed important changes in mAb glycosylation patterns in all steps of serum reduction. These changes could be grouped in two distinct phases of the process of adaptation: middle (2.5 to 0.15% serum) and final (0.075 and 0% serum). For intermediate levels of serum, a desirable increase of galactosylation and decrease of fucosylation, but an undesirable increase in sialylation were observed; while the inverse was obtained at the final stages of adaptation. These divergences may be related to the reduction of serum supplementation, to variations in the levels of cell density and viability achieved at these stages, and to the natural shift of the cell growth mode during adaptation from adherent to suspended. The divergent glycan profiles obtained in this study demonstrate a strong influence of the adaptation process on mAb glycosylation, suggesting that control and monitoring of product quality should be implemented at the early stages of process development.
New Biotechnology 12/2012; · 2.76 Impact Factor
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ABSTRACT: AIM: The aim of this study was to assess the effect of different silver nanoparticles (SN) concentrations on the matrix composition and the structure of Candida albicans and Candida glabrata biofilms. METHODS AND RESULTS: Candida biofilms were developed in 6-well microtiter plates during 48 h. After, these biofilms were exposed to 13.5 or 54 μg SN ml(-1) for 24 h. Then, extracellular matrices were extracted from biofilms and analyzed chemically in terms of proteins, carbohydrates and DNA. To investigate the biofilm structure, scanning electron microscopy (SEM) and epifluorescence microscopy were used. SN interfered with the matrix composition of Candida biofilms tested in terms of protein, carbohydrate and DNA, except for the protein content of C. albicans biofilm. By SEM, Candida biofilms treated with SN revealed structural differences, when compared to the control groups. Further, SN showed a trend of agglomeration within the biofilms. Epifluorescence microscopy images suggest that SN induced damage to cell walls of Candida isolates tested. CONCLUSIONS: In general, irrespective of concentration, SN affected the matrix composition and structure of Candida biofilms and these findings may be related to the mechanisms of biocide action of SN. © 2012The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.
Journal of Applied Microbiology 12/2012; · 2.34 Impact Factor
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ABSTRACT: The selection of a serum-free medium for a particular process of production using mammalian cells is a critical step for its success. In this study, seven commercially available serum-free media (EX-CELL, ISF-I, CD CHO, CDM4CHO, CHO-III-A, Octomed and HybridoMed) were evaluated and compared for cell growth and monoclonal antibody (mAb) production of a transfected CHO-K1 cell line. In the conditions assayed, EX-CELL and particularly CDM4CHO are the most recommended media for extended biopharmaceutical processes, on account of inducing superior levels of cell proliferation and mAb production, accentuated by a tendency to improve over time. Furthermore, the less positive results obtained with some media emphasize the importance and the impact of the correct medium selection.
International journal of pharmaceutics 08/2012; 437(1-2):303-5. · 2.96 Impact Factor
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Sónia Silva,
Priscila Pires,
Douglas R Monteiro,
Melyssa Negri,
Luiz F Gorup,
Emerson R Camargo,
Débora B Barbosa,
Rosário Oliveira,
David W Williams, Mariana Henriques,
Joana Azeredo
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ABSTRACT: The aim of this study was to compare biofilm formation by Candida glabrata and Candida albicans on acrylic, either individually or when combined (single and dual species) and then examine the antimicrobial effects of silver nanoparticles and nystatin on these biofilms. Candidal adhesion and biofilm assays were performed on acrylic surface in the presence of artificial saliva (AS) for 2 h and 48 h, respectively. Candida glabrata and C. albicans adherence was determined by the number of colony forming units (CFUs) recovered from the biofilms on CHROMagar(®) Candida. In addition, crystal violet (CV) staining was used as an indicator of biofilm biomass and to quantify biofilm formation ability. Pre-formed biofilms were treated either with silver nanoparticles or nystatin and the effect of these agents on the biofilms was evaluated after 24 h. Results showed that both species adhered to and formed biofilms on acrylic surfaces. A significantly (P < 0.05) higher number of CFUs was evident in C. glabrata biofilms compared with those formed by C. albicans. Comparing single and dual species biofilms, equivalent CFU numbers were evident for the individual species. Both silver nanoparticles and nystatin reduced biofilm biomass and the CFUs of single and dual species biofilms (P < 0.05). Silver nanoparticles had a significantly (P < 0.05) greater effect on reducing C. glabrata biofilm biomass compared with C. albicans. Similarly, nystatin was more effective in reducing the number of CFUs of dual species biofilms compared with those of single species (P < 0.05). In summary, C. glabrata and C. albicans can co-exist in biofilms without apparent antagonism, and both silver nanoparticles and nystatin exhibit inhibitory effects on biofilms of these species.
Medical mycology: official publication of the International Society for Human and Animal Mycology 07/2012; · 2.13 Impact Factor
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ABSTRACT: Candida tropicalis infection is strongly associated with the presence of biofilms in urinary catheters. Thus, the aim of this work was to study the behaviour of C. tropicalis in biofilms of different ages (24-120 h) formed in artificial urine (AU) and their effect in human urinary bladder cells (TCC-SUP). Reference strain ATCC 750 and two isolates from patients with candiduria (U69 and U75) were used in this study. The adhesion to human cells was evaluated after 2 h of contact with Candida biofilms, using the Crystal violet staining method, and the human cells response was evaluated in terms of activity inhibition and cell damage. Candida tropicalis aspartyl proteinase (SAPT) gene expression was determined by real-time PCR. Candida tropicalis biofilm cells were able to adhere to TCC-SUP cells. The highest extent of yeast attachment was obtained for the 72 h old biofilm cells. Yeasts affected TCC-SUP cells, with 120 h-biofilm cells causing the highest levels of cell injury. Generally, SAPT3 was highly expressed and SAPT4 was only detected in the reference strain. Overall, it is important to highlight that C. tropicalis cells detached from biofilms are able to colonize human cells and cause some injury and reduction of metabolic activity.
Microbial Pathogenesis 05/2012; 53(2):95-9. · 1.94 Impact Factor
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ABSTRACT: Antimicrobial fabrics are increasingly important in a great variety of applications and thus several standard methods to evaluate
their efficiency have been developed. However, there is no consensus on the most adequate method to be used. Therefore, aim
of this work was to compare the practical applicability of the best known standards: AATCC 147, ISO 20645:2004, AATCC:100
and JIS L 1902. Four samples, with different amounts of antimicrobial agents, were used. It was tested 3 qualitative methods
(AATCC 147, ISO 20645 and JIS L 1902–Halo method) and 2 quantitative (AATCC 100 and JIS L 1902–Absorption method). For each
method, both Gram-positive (Staphylococcus aureus) and Gram-negative (Klebsiella pneumoniae) bacteria were used. Textiles samples assayed did not present diffusible activity, thus only the qualitative results from
the AATCC 147 and the Halo method could be analyzed and no differences were observed between them. Therefore, the AATCC 147
or the JIS L 1902–Halo method can be used for a simple and expedite screening of a large amount of samples with or without
diffusible antimicrobial activity. In contrast, the ISO 20645 can only be used when diffusible antimicrobial agents are present.
Concerning the two quantitative methods, the results showed that the JIS L 1902 method is more sensitive to the amount of
antimicrobial agent than the AATCC 100 test. An additional assay also showed that the JIS L 1902 is sensitive enough to distinguish
serial dilutions of the antimicrobial agent.
KeywordsAntimicrobial textiles–Standard methods–
Staphylococcus aureus
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Klebsiella pneumoniae
Annals of Microbiology 05/2012; 61(3):493-498. · 0.69 Impact Factor
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ABSTRACT: In the last decade, the number and diversity of nosocomial Candida infections has increased significantly, resulting in an emergent need for rapid and accurate methods for Candida identification. Therefore, the aim of this study was to evaluate the performance of three biochemical systems (Auxacolor,
ID32C, and Vitek 2 YST) for the identification of Candida species, comparing them with molecular identification (polymerase chain reaction and gel agarose electrophoresis). These
methods were used to assess Candida spp. (229 clinical isolates) prevalence and distribution among clinical specimens. The biochemical methods with higher percentages
of correct identification were Vitek 2 YST (79.6%) and Auxacolor (78.6%). However, overall the biochemical methods assayed
differed from the molecular identification. Thus, due to their rapid and precise identification, molecular methods are promising
techniques for Candida species identification in clinical laboratories. Candida albicans and Non Candida albicans Candida species had a similar prevalence (50.4 and 49.6%, respectively), corroborating the epidemiological shift observed for these
pathogens in the recent years.
Keywords
Candida species-Discriminating potential-PCR-Vitek 2 YST-Auxacolor-ID32C
Annals of Microbiology 04/2012; 60(1):105-112. · 0.69 Impact Factor
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ABSTRACT: The goal of this work was to create a new generation of greener fabrics made of natural materials. For that, resveratrol (Res),
obtained from Polygonum cuspidatum extract and known to have antibacterial, antifungal, and anti-inflammatory activity, was applied by an exhaustion method
to cotton, bamboo, and silk knit fabrics. The fabrics adsorption behavior was tested and the amount of Res adsorbed was determined
by its decrease on the immersion solutions with time and measured by spectrophotometry at 350 nm. The maximum adsorption capacity
was observed for silk and it was independent of pH conditions used (50.5 % at pH=7 and 58.3 % at pH=5 of the initial Res concentration).
At acidic pH conditions, cotton adsorbed 51.2 % of Res and Bamboo adsorbed only 28.1 % in 15 min. However, neither cotton
nor bamboo adsorbed Res at pH=7. The release behavior was also analyzed and the highest Res release was observed for cotton
in alkaline sweat and urine mimic solutions. The lowest release was achieved by cotton in water (1.0 ng/ml). Moreover, no relation was found between the amounts of Res adsorbed or released and cell viability. In conclusion, this
work shows that it is possible to obtain cotton, bamboo, and silk functionalized with resveratrol. The incorporating process
here described is simple and silk-Res can be presented as a good combination.
KeywordsBiofunctional fabrics-Resveratrol-Antinflammatory-Antioxidant-Exhaustion process
Fibers and Polymers 04/2012; 11(2):271-276. · 0.84 Impact Factor
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ABSTRACT: Biopharmaceutical production of complex recombinant protein therapeutics currently relies on mammalian cells. The development of high-yielding stable cell lines requires processes of transfection, selection and adaptation. With several technologies available, selection has been most frequently based on dihydrofolate reductase or glutamine synthetase systems, which can be very time-consuming. Due to the pressure to reduce development costs and speed up time to market, new technologies are emerging, as the promising OSCAR expression system that could provide more rapid development of high-yielding stable cell lines than the traditional systems. However, further evaluation of its application in a wider range of cell types and media is still necessary. In this study, application of OSCAR for the transfection of a CHO-K1 cell line with a monoclonal antibody was evaluated. OSCAR was reasonably fast and simple, without negative impact on cell growth characteristics. However, minigene selection was critical, with only pDWM128 working for the cell line assessed. Initial relatively high levels of production decreased significantly in the first few weeks of passing, remaining relatively stable although with low yield thereafter. The results suggest that more work is required to develop methodologies and prove that OSCAR has significant value to the bioproduction industry.
International journal of pharmaceutics 03/2012; 430(1-2):42-6. · 2.96 Impact Factor
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ABSTRACT: The high dose requirements of biopharmaceutical products led to the development of mammalian cell culture technologies that increase biomanufacturing capacity. The disposable Wave bioreactor is one of the most promising technologies, providing ease of operation and no cross-contamination, and using an innovative undulation movement that ensures good mixing and oxygen transfer without cell damage. However, its recentness demands further characterization. This study evaluated the residence time distribution (RTD) in Wave, allowing the characterization of mixing and flow and the comparison with ideal models and a Stirred tank reactor (STR) used for mammalian cell culture. RTD was determined using methylene blue with pulse input methodology, at three flow rates common in mammalian cell culture (3.3×10(-5)m(3)/h, 7.9×10(-5)m(3)/h, and 1.25×10(-4)m(3)/h) and one typical of microbial culture (5×10(-3)m(3)/h). Samples were taken periodically and the absorbance read at 660nm. It was observed that Wave behavior diverted from ideal models, but was similar to STR. Therefore, the deviations are not related to the particular Wave rocking mechanism, but could be associated with the inadequacy of these reactors to operate in continuous mode or to a possible inability of the theoretical models to properly describe the behavior of reactors designed for mammalian cell culture. Thus, the development of new theoretical models could better characterize the performance of these reactors.
New Biotechnology 02/2012; 29(3):402-8. · 2.76 Impact Factor
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ABSTRACT: The purpose of this work was to evaluate the size-dependent antifungal activity of different silver nanoparticles (SN) colloidal suspensions against Candida albicans and Candida glabrata mature biofilms.
The research presented herein used SN of three different average sizes (5, 10 and 60 nm), which were synthesized by the reduction of silver nitrate through sodium citrate and which were stabilized with ammonia or polyvinylpyrrolidone. Minimal inhibitory concentration (MIC) assays were performed using the microdilution methodology. The antibiofilm activity of SN was determined by total biomass quantification (by crystal violet staining) and colony forming units enumeration. MIC results showed that all SN colloidal suspensions were fungicidal against the tested strains at very low concentrations (0·4-3·3 μg ml(-1) ). With regard to biomass quantification, SN colloidal suspensions were very effective only against C. glabrata biofilms, achieving biomass reductions around 90% at a silver concentration of 108 μg ml(-1) . In general, all SN suspensions promoted significant log(10) reduction of the mean number of cultivable biofilm cells after exposure to silver concentrations at or higher than 108 μg ml(-1) . Moreover, the results showed that the particle size and the type of stabilizing agent used did not interfere in the antifungal activity of SN against Candida biofilms.
This study suggests that SN have antifungal therapeutic potential, but further studies are still required namely regarding formulation and delivery means.
SN may contribute to the development of new strategies for the improvement of oral health and quality of life particularly of the complete denture wearers.
Letters in Applied Microbiology 02/2012; 54(5):383-91. · 1.62 Impact Factor
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BMC proceedings 11/2011; 5 Suppl 8:P112.
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BMC proceedings 11/2011; 5 Suppl 8:P111.
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ABSTRACT: Candida albicans supernatants contain a mixture of autoregulatory alcohols. In vitro, when added individually or in combination, these alcohols inhibit the yeast to filamentous form conversion. Here we evaluate the in vivo effect of the exogenous administration of a Cocktail solution simulating the composition of alcohols present in a C. albicans culture supernatant (1 ml; 94 μmol l(-1) isoamyl alcohol, 70 μmol l(-1) 2-phenylethanol, 3.2 n mol l(-1) E -nerolidol, and 18 n mol l(-1) E,E -farnesol) using the well established murine model of hematogenously disseminated candidiasis. Mice injected intraperitoneally with the Cocktail solution demonstrated increased survival and decreased organ fungal burden compared to control mice. Histological observations suggest that the Cocktail, to some extent, has an inhibitory effect on cell filamentation within the kidney. These findings suggest that the exogenous administration of C. albicans autoregulatory alcohols displays a protective effect during disseminated candidiasis.
Journal of Basic Microbiology 11/2011; 52(4):487-91. · 1.27 Impact Factor
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ABSTRACT: Central venous catheters (CVCs) nowadays constitute critical devices used in medical care, namely in intensive care units. However, CVCs also represent one of the indwelling medical devices with enhanced risk of nosocomial device-related infection. Catheter-related infections (CRIs) are a major cause of patient morbidity and mortality, often justifying premature catheter removal and an increase in costs and use of resources. Adhesion and subsequent biofilm formation on the surfaces of indwelling catheters is elemental to the onset of pathogenesis. Seeking the prevention of CVC colonisation and CRI, a variety of approaches have been studied, tested and, in some cases, already applied in clinical practice. This review looks at the current preventive strategies often used to decrease the risk of CRIs due to colonization and biofilm formation on catheter surfaces, as well as at the more recent approaches under investigation.
Biofouling 07/2011; 27(6):609-20. · 4.43 Impact Factor
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ABSTRACT: Cells within Candida albicans biofilms display decreased susceptibility to most clinically used antifungal agents. We recently demonstrated that extracellular DNA (eDNA) plays an important role in biofilm integrity, as a component of the biofilm matrix. This study aimed at gaining insights into the contributions of eDNA to C. albicans biofilms antifungal susceptibility by the investigation of the impact of the combined use of deoxyribonuclease I (DNase) and antifungals to treat biofilms. Candida albicans biofilms were formed using a simple and reproducible 96-well plate-based method. The activity of the combined use of 0.13 mg l(-1) DNase and antifungals was estimated using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay and total viable counts. Herein, we report the improved efficacy of amphotericin B when in combination with DNase against C. albicans biofilms, as assessed using XTT readings and viable counts. Furthermore, although DNase increased the efficacy of caspofungin in the reduction of mitochondrial activity, no changes were observed in terms of culturable cells. Deoxyribonuclease I did not affect biofilm cells susceptibility to fluconazole. This work suggests that agents that target processes affecting the biofilm structural integrity may have potential use as adjuvants of a catheter-lock therapy.
Mycoses 06/2011; 55(1):80-5. · 2.25 Impact Factor
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ABSTRACT: The incidence of infections caused by Candida species (candidosis) has increased considerably over the past three decades, mainly due to the rise of the AIDS epidemic, an increasingly aged population, higher numbers of immunocompromised patients and the more widespread use of indwelling medical devices. Candida albicans is the main cause of candidosis; however, non-C. albicans Candida (NCAC) species such as Candida glabrata, Candida tropicalis and Candida parapsilosis are now frequently identified as human pathogens. The apparent increased emergence of these species as human pathogens can be attributed to improved identification methods and also associated with the degree of diseases of the patients, the interventions that they were subjected and the drugs used. Candida pathogenicity is facilitated by a number of virulence factors, most importantly adherence to host surfaces including medical devices, biofilm formation and secretion of hydrolytic enzymes (e.g. proteases, phospholipases and haemolysins). Furthermore, despite extensive research to identify pathogenic factors in fungi, particularly in C. albicans, relatively little is known about NCAC species. This review provides information on the current state of knowledge on the biology, identification, epidemiology, pathogenicity and antifungal resistance of C. glabrata, C. parapsilosis and C. tropicalis.
FEMS microbiology reviews 05/2011; 36(2):288-305. · 10.96 Impact Factor
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ABSTRACT: The aim of this study was to investigate the interaction of Candida tropicalis with three different human cell lines: TCC-SUP (epithelial cells from urinary bladder), HeLa (epithelial cells from cervical carcinoma) and Caco-2 (epithelial cells from colorectal adenocarcinoma). In particular we sought to assess the degree of cell damage and activity reduction induced by C. tropicalis adhesion and the role of secreted aspartyl proteinase (SAP) gene expression in this process. Two C. tropicalis strains were used: the reference strain ATCC 750 and a clinical isolate from urine (U69). The ability of C. tropicalis to adhere to a confluent layer of human cells was determined using an adaptation of the crystal violet staining method; cell damage and cell activity inhibition induced by the adhesion of C. tropicalis were assessed by measuring lactate dehydrogenase and tetrazolium salt (MTS) reduction, respectively. C. tropicalis SAP gene expression was determined by real-time PCR. Both C. tropicalis strains were able to adhere to the different human cells, although in a strain- and cell-line-dependent manner. Concerning the cellular response to C. tropicalis, the highest inhibition of cell activity was obtained for Caco-2, followed by TCC-SUP and HeLa cells. The highest percentage of cell damage (around 14 %) was observed for TCC-SUP cells in contact with the U69 isolate and for Caco-2 in contact with the reference strain. Real-time PCR analysis revealed a wide range of expression profiles of SAP genes for both C. tropicalis strains in contact with the different types of epithelial cells. SAPT3 was the gene expressed at the highest level for both C. tropicalis strains in contact with the three human epithelial cell lines. The results highlight that the response of human cells to C. tropicalis adhesion, as well as production of SAPs, is dependent on both the strain and the epithelial cell line.
Journal of Medical Microbiology 05/2011; 60(Pt 9):1270-5. · 2.50 Impact Factor
