Cecilia Tapia

The optimized capsid gene of porcine circovirus type 2 expressed in yeast forms virus-like particles and elicits antibody responses in mice fed with recombinant yeast extracts

Porcine circovirus type 2 (PCV2)-associated diseases are considered to be the biggest problem for the worldwide swine industry. The PCV2 capsid protein (Cap) is an important antigen for development of vaccines. At present, most anti-PCV2 vaccines are produced as injectable formulations. Although effective, these vaccines have certain drawbacks, including stress with concomitant immunosuppresion, and involve laborious and time-consuming procedures. In this study, Saccharomyces cerevisiae was used as a vehicle to deliver PCV2 antigen in a preliminary attempt to develop an oral vaccine, and its immunogenic potential in mice was tested after oral gavage-mediated delivery. The cap gene with a yeast-optimized codon usage sequence (opt-cap) was chemically synthesized and cloned into Escherichia coli/Saccharomyces cerevisiae shuttle vector, pYES2, under the control of the Gal1 promoter. Intracellular expression of the Cap protein was confirmed by Western blot analysis and its antigenic properties were compared with those of baculovirus/insect cell-produced Cap protein derived from the native PCV2 cap gene. It was further demonstrated by electron micrography that the yeast-derived PCV2 Cap protein self-assembles into virus-like particles (VLPs) that are morphologically and antigenically similar to insect cell-derived VLPs. Feeding raw yeast extract containing Cap protein to mice elicited both serum- and fecal-specific antibodies against the antigen. These results show that it is feasible to use S. cerevisiae as a safe and simple system to produce PCV2 virus-like particles, and that oral yeast-mediated antigen delivery is an alternative strategy to efficiently induce anti-PCV2 antibodies in a mouse model, which is worthy of further investigation in swine.

Antifungal activity of low molecular weight chitosan against clinical isolates of Candida spp.

Chitosan is a natural polymer derived from chitin, a structural component of fungi, insects and shrimp, which exerts antimicrobial effects against bacteria and fungi. The aim of this study was to investigate the in vitro antifungal activity of low molecular weight chitosan (LMWC), and the potential synergy between chitosan and a currently used antifungal drug, fluconazole. The in vitro minimal inhibitory concentrations (MICs) of chitosan and fluconazole against 105 clinical Candida isolates were measured by the broth microdilution method. LMWC exhibited a significant antifungal activity, inhibiting over 89.9% of the clinical isolates examined (68.6% of which was completely inhibited). The species included several fluconazole-resistant strains and less susceptible species such as C. glabrata, which was inhibited at a concentration of 4.8 mg/l LMWC. Although some strains were susceptible at pH 7.0, a greater antifungal activity of LMWC was observed at pH 4.0. There was no evidence of a synergistic effect of the combination of LMWC and fluconazole at pH 7.0. This is the first report in which the antifungal activity of LMWC was investigated with clinical Candida strains. The use of LMWC as an antifungal compound opens new therapeutic perspectives, as the low toxicity of LMWC in humans supports its use in new applications in an environment of pH 4.0-4.5, such as a topical agent for vulvovaginal candidiasis.

Melanocytes and melanin represent a first line of innate immunity against Candida albicans

Melanocytes are dendritic cells located in the skin and mucosae that synthesize melanin. Some infections induce hypo- or hyperpigmentation, which is associated with the activation of Toll-like receptors (TLRs), especially TLR4. Candida albicans is an opportunist pathogen that can switch between blastoconidia and hyphae forms; the latter is associated with invasion. Our objectives in this study were to ascertain whether C. albicans induces pigmentation in melanocytes and whether this process is dependent on TLR activation, as well as relating this with the antifungal activity of melanin as a first line of innate immunity against fungal infections. Normal human melanocytes were stimulated with C. albicans supernatants or with crude extracts of the blastoconidia or hyphae forms, and pigmentation and TLR2/TLR4 expression were measured. Expression of the melanosomal antigens Melan-A and gp100 was examined for any correlation with increased melanin levels or antifungal activity in melanocyte lysates. Melanosomal antigens were induced earlier than cell pigmentation, and hyphae induced stronger melanization than blastoconidia. Notably, when melanocytes were stimulated with crude extracts of C. albicans, the cell surface expression of TLR2/TLR4 began at 48 h post-stimulation and peaked at 72 h. At this time, blastoconidia induced both TLR2 and TLR4 expression, whereas hyphae only induced TLR4 expression. Taken together, these results suggest that melanocytes play a key role in innate immune responses against C. albicans infections by recognizing pathogenic forms of C. albicans via TLR4, resulting in increased melanin content and inhibition of infection.

Behavioral effects of intracerebral administration of luteinizing hormone releasing hormone (LHRH) in rats

The effects of LHRH intracerebrally infused on acquisition of conditioned avoidance responses (CARs) and spontaneous motility were studied in adult male rats. The results were the following: 1) LHRH (1 and 2.5 micrograms/rat) administered through a cannula stereotaxically implanted into the lateral ventricle induced an impairment in the acquisition of CARs along with an increase in global motility, rearing, head shaking and grooming behavior; 2) LHRH 1 microgram/rat injected into the hippocampus or nucleus accumbens induced also an impairment in acquisition which is evident 15 min after treatment. In contrast, intrastriatal injection induced an immediate disruption of this behavior; and 3) there is a good dose-response relationship for intrastriatal LHRH between 7.8 and 62.5 ng/rat. The results suggest that the estriatum could be the locus of the LHRH-induced inhibition of CARs. Then the possibility of an involvement of the dopamine nigrostriatal system is discussed.

Actividad antifúngica de melanina en cepas clínicas de Candida spp

Introduccion: Los melanocitos son celulas presentes en piel y en mucosas que sintetizan melanina, ademas de citoquinas. Es sabido que melanina presenta actividad antimicrobiana y que los melanocitos se melanizan al ser expuestos a moleculas microbianas. Objetivo: Estudiar la actividad antifungica de melanina en cepas clinicas de Candida spp. Metodologia: Se midio la concentracion inhibitoria minima (CIM) a melanina, de 4 cepas de Candida ATCC (C. albicans SC5314, C. parapsilosis 22019, C. glabrata 2001 y C. krusei 6258) y 56 aislados clinicos de Candida spp. (33 C. albicans, 12 C. glabrata, 3 C. famata, 3 C. krusei, 3 C. parapsilosis, 2 C. tropicalis) mediante un metodo de microdilucion en caldo. Ademas se estudio el efecto antifungico de lisados de melanocitos y celulas de melanoma de raton en C. albicans. Resultados: Melanina inhibio las cepas analizadas, incluso cepas susceptibles dosis-dependiente y resistentes a fluconazol, siendo los rangos de CIM y CIM50 de 0,09-50 μg/mL y 6,25 μg/ mL, respectivamente. Los lisados de celulas pigmentadas inhibieron C. albicans. Conclusiones: Melanina es capaz de inhibir cepas clinicas de Candida spp. La melanizacion podria ser un importante mecanismo protector de los melanocitos.BACKGROUND Melanocytes are cells located in epidermis and mucous membranes that synthesize melanin and cytokines. It is known that melanin has antimicrobial activity and that melanocytes are melanized in presence of microbial molecules. OBJECTIVE To study the antifungal activity of melanin on Candida spp. METHODOLOGY The minimum inhibitory concentration (MIC) to melanin was determined in 4 Candida ATCC strains (C. albicans SC5314, C. parapsilosis 22019, C. glabrata 2001, C. krusei 6258) and 56 clinical isolates of Candida spp. (33 C. albicans, 12 C. glabrata, 3 C. famata, 3 C. krusei, 3 C. parapsilosis, 2 C. tropicalis) using a broth microdilution method. In addition, the antifungal activity of melanocytes and mice melanoma cells was tested against C. albicans. RESULTS Melanin inhibited the tested isolates, including the susceptible dose-dependent and fluconazole-resistant strains; MIC range and MIC50 were 0.09-50 μg/mL and 6.25 μg/mL, respectively. Pigmented cells lysates inhibited C. albicans. CONCLUSIONS Melanin is able to inhibit clinical isolates of Candida spp. Melanization could be an important protective mechanism of melanocytes.

Differential antifungal activity of human and cryptococcal melanins with structural discrepancies

Melanin is a pigment found in all biological kingdoms, and plays a key role in protection against ultraviolet radiation, oxidizing agents, and ionizing radiation damage. Melanin exerts an antimicrobial activity against bacteria, fungi, and parasites. We demonstrated an antifungal activity of synthetic and human melanin against Candida sp. The members of the Cryptococcus neoformans and C. gattii species complexes are capsulated yeasts, which cause cryptococcosis. For both species melanin is an important virulence factor. To evaluate if cryptococcal and human melanins have antifungal activity against Cryptococcus species they both were assayed for their antifungal properties and physico-chemical characters. Melanin extracts from human hair and different strains of C. neoformans (n = 4) and C. gattii (n = 4) were investigated. The following minimum inhibitory concentrations were found for different melanins against C. neoformans and C. gattii were (average/range): 13.7/(7.8–15.6) and 19.5/(15.6–31.2) μg/mL, respectively, for human melanin; 273.4/(125–>500) and 367.2/(125.5–>500) μg/mL for C. neoformans melanin and 125/(62.5–250) and 156.2/(62–250) μg/mL for C. gattii melanin. Using Scanning Electron Microscopy we observed that human melanin showed a compact conformation and cryptococcal melanins exposed an amorphous conformation. Infrared spectroscopy (FTIR) showed some differences in the signals related to C-C bonds of the aromatic ring of the melanin monomers. High Performance Liquid Chromatography established differences in the chromatograms of fungal melanins extracts in comparison with human and synthetic melanin, particularly in the retention time of the main compound of fungal melanin extracts and also in the presence of minor unknown compounds. On the other hand, MALDI-TOF-MS analysis showed slight differences in the spectra, specifically the presence of a minor intensity ion in synthetic and human melanin, as well as in some fungal melanin extracts. We conclude that human melanin is more active than the two fungal melanins against Cryptococcus. Although some physico-chemical differences were found, they do not explain the differences in the antifungal activity against Cryptococcus of human and cryptococcal melanins. More detailed studies on the structure should be considered to associate structure and antifungal activity.

Distribución de especies y perfil de susceptibilidad de aislados de Candida spp: la importancia de vigilar también cepas de la comunidad

BACKGROUND The most of the surveillance studies has been conducted in hospitalized patients with invasive infections. Recently, new clinical breakpoints (CBPs) have been proposed for antifungal susceptibility testing and epidemiological cutoffs (ECVs). AIM To evaluate species distribution and susceptibility pattern of Candida spp. obtained from in and outpatients in a period of 6 months. MATERIAL AND METHODS The isolates (n=223) came from vaginal discharge (51.6%), lower respiratory tract (24.7%), urine (20.2%), wounds (1.8%), blood (0.9%), peritoneal fluid (0.4%) and nails (0.4%). RESULTS The species distribution was C. albicans 84.8% (n: 189), C. glabrata 7.6% (n: 17), C. tropicalis 2.7% (n: 6), C. parapsilosis 2.2% (n: 5), C. kefyr 0.9% (n: 2) and others 1.8% (C. krusei, C. lusitanie, C. guilliermondii, C. intermedia) (n: 4). The susceptibility dose dependence (SDD) and resistance were 3.2% for fluconazole and 2.2% for voriconazole. The most of SDD and resistant strains were isolated from ambulatory patients. Also, a higher percentage of MICs over the new CBPs and ECVs were found in strains from ambulatory patients and especially in C. glabrata isolates to caspofungin. CONCLUSION Taking into consideration that most of the invasive infections are caused by strains from the endogenous microbiota, and that there is a resistant population of Candida spp. in the community, should be important to include in surveillance studies strains isolated from ambulatory patients.

Modified profile of matrix metalloproteinase-2 and -9 production by human Fallopian tube epithelial cells following infection in vitro with Neisseria gonorrhoeae.

Epithelial shedding and scarring of Fallopian tube mucosa are the main consequences of sexually transmitted Neisseria gonorrhoeae infection and likely involves an imbalance of host extracellular matrix components (ECM) and their regulators such as matrix metalloproteinases (MMPs). In this brief report, primary human Fallopian tube epithelial cells were infected with N. gonorrhoeae and MMP patterns examined. Gonococcal infection induced a significant increase in secreted MMP-9 and an accumulation of cytoplasmic MMP-2 over time, but no significant MMP-3 or MMP-8 production was observed. Thus, MMP-9 in particular could play a role in tubal scarring in response to gonococcal infection.Epithelial shedding and scarring of fallopian tube mucosa are the main consequences of sexually transmitted Neisseria gonorrhoeae infection and probably involve an imbalance of host extracellular matrix components and their regulators such as matrix metalloproteinases (MMPs). In the current study, primary human fallopian tube epithelial cells were infected with N. gonorrhoeae, and MMP patterns were examined. Gonococcal infection induced a significant increase in secreted MMP-9 and an accumulation of cytoplasmic MMP-2 over time, but no significant MMP-3 or MMP-8 production was observed. Thus, MMP-9 in particular could play a role in tubal scarring in response to gonococcal infection.

Caracterización de los mecanismos de resistencia a azoles en aislados clínicos chilenos de Candida albicans

INTRODUCTION The commensal yeast Candida albicans, can cause superficial or systemic candidiasis in susceptible hosts. In Chile, azole antifungals are the most widely used drugs in the treatment of candidiasis. In a previous study performed at our center, 2.1 and 1.6% of clinical isolates of C. albicans were found to be resistant to fluconazole and voriconazole, respectively. OBJECTIVE To characterize the resistance mechanisms involved in azoles resistance in Chilean clinical isolates. METHODOLOGY Eight resistant, nine susceptible-dose dependent (SDD) and 10 susceptible strains (n: 27) were selected according to the Clinical Laboratory Standards Institute (CLSI) M27-S3 criteria, from vaginal and urine samples. Mutations in the 408-488 region of the ERG11 gene were studied by sequencing, and the relative expression of ERG11 gene and efflux pump genes CDR1, CDR2 and MDR1, was evaluated by quantitative real-time PCR (q-PCR). RESULTS No mutations were detected in the ERG11 gene and its overexpression was found only in 12.5% of the resistant strains (1/8). The most prevalent mechanism of resistance was the over-expression of efflux pumps (62.5%; 5/8). CONCLUSION The study of the expression of efflux pumps by q-PCR could be a useful diagnostic tool for early detection of azole resistance in C. albicans.

Protective effect of inactivated blastoconidia in keratinocytes and human reconstituted epithelium against C. albicans infection

Candida albicans is commensal yeast that colonizes skin and mucosa; however, it can become an opportunist pathogen by changing from blastoconidia (commensal form) into hypha (pathogenic form). Each form activates a different cytokines response in epithelial cells. Little is known about the commensal role of C. albicans in the innate immunity. This work studied whether stimulation with C. albicans blastoconidia induces protection in keratinocytes and/or in a reconstituted human epithelium (RHE) infected with C. albicans. For this, inactivated C. albicans blastoconidia was used to stimulate keratinocytes and RHE prior to infection with C. albicans. Blastoconidia induced different cytokine expression profiles; in the case of RHE it decreased interleukin (IL)-1β and IL-10 and increased IL-8, tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ). A significant increase in the expression of human β-defensins (HBD) 2 and HBD3 was observed in blastoconidia stimulated keratinocytes and RHE, associated with impaired growth and viability of C. albicans. Additionally, blastoconidia stimulation decreased the expression of virulence factors in C. albicans that are associated with filamentation (EFG1, CPH1 and NRG1), adhesion (ALS5), and invasion (SAP2). Blastoconidia stimulated RHE was significantly less damaged by C. albicans invasion. These results show that the commensal form of C. albicans would exert a protective effect against self-infection.