In vitro evidence for immune evasion activity by human plasmin associated to pathogenic Leptospira interrogans

Leptospirosis is a widespread re-emerging zoonosis of human and veterinary concern. It has been shown that virulent leptospires protect themselves against the host`s innate immune system, a strategy that allows the bacteria to reach immunologically safe environments. Although extensive studies on host pathogen interactions have been performed, little is known on how leptospires deal with host immune attack. In a previous work, we demonstrated the ability of leptospires to bind human plasminogen (PLC), that after treatment with activators, conferred plasmin (PLA) activity on the bacteria surface. In this study, we show that the PLA activity associated to the outer surface of Leptospira could interfere with the host immune attack by conferring some evasion advantage during infection. We demonstrate that PLA-coated leptospires interfere with complement Ob and IgG depositions on the bacterial surface, probably through the degradation of these components, thus diminishing opsonization process. Similar decrease on the deposition was observed when normal and immune sera from patients diagnosed with leptospirosis were employed as a source of IgG. We believe that decreasing opsonization by PLA generation might be an important aspect of the leptospiral immune escape strategy and survival. To our knowledge, this is the first proteolytic activity of plasmin associated-Leptospira related to anti-opsonic properties reported to date. (C) 2011 Elsevier Ltd. All rights reserved.

Oviductal Fluid Proteins Associated with the Bovine Zona Pellucida and the Effect on In Vitro Sperm-Egg Binding, Fertilization and Embryo Development

Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus-associated protein, osteopontin (OPN), lipocalin-type prostaglandin D synthase (L-PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg-associated OPN and L-PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre-incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L-PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 x 10(5) frozen-thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre-treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.

Unexpected clobetasol propionate profile in human stratum corneum after topical application in vitro

Purpose. The validity of using drug amount-depth profiles in stratum corneum to predict uptake of clobetasol propionate into stratum corneum and its transport into deeper skin layers was investigated. Methods. In vitro diffusion experiments through human epidermis were carried out using Franz-type glass diffusion cells. A saturated solution of clobetasol propionate in 20% (V/V) aqueous propylene glycol was topically applied for 48 h. Steady state flux was calculated from the cumulative amount of drug permeated vs. time profile. Epidermal partitioning was conducted by applying a saturated drug solution to both sides of the epidermis and allowing time to equilibrate. The tape stripping technique was used to define drug concentration-depth profiles in stratum corneum for both the diffusion and equilibrium experiments. Results. The concentration-depth profile of clobetasol propionate in stratum corneum for the diffusion experiment is biphasic. A logarithmic decline of the drug concentration over the first four to five tape strips flattens to a relatively constant low concentration level in deeper layers. The drug concentration-depth profile for the equilibrium studies displays a similar shape. Conclusions. The shape of the concentration-depth profile of clobetasol propionate is mainly because of the variable partitioning coefficient in different stratum corneum layers.

Cultivo in vitro de Crambe abyssinica Hochst. : germinação, micropropagação, estabilidade genética e anatomia foliar

Crambe (Crambe abyssinica) pertence à família Brassicaceae, originário da Etiópia e principalmente destinado à produção de forragem (30 a 32% de proteína bruta). Atualmente, tem sido bastante cultivado visando à extração de óleo vegetal. Com os atuais incentivos à busca de fontes de energias renováveis, o cultivo de crambe vem ganhando papel de destaque na produção de biodiesel por suas diversas vantagens, como: (a) rápido ciclo de vida (colhida em torno de 90 dias); (b) alta produção de biomassa; (c) alta produtividade de sementes (1000 e 1500 kg ha-1); (d) menor custo de produção em relação a outras fontes oleaginosas, como, canola, girassol e soja; (e) um percentual de óleo total na semente entre 32 e 38%, superando, por exemplo, a soja; (f) potencial de fitorremediação, eficiente na descontaminação de arsênio, cromo e outros metais pesados; e (g) elevado percentual de ácido erúcico (50 a 60%) sendo útil na indústria de plástico e lubrificante. Devido aos poucos trabalhos realizados com crambe, abre-se um vasto campo de investigações científicas que tenham como objetivo desenvolver as potencialidades dessa cultura e, consequentemente, melhorar os aspectos agronômicos e tecnológicos para seu emprego na indústria de biodiesel. Nesse contexto, as técnicas de cultivo in vitro foram importantes tanto para a propagação massal, quanto como ferramenta para uma possível aplicação de outras técnicas biotecnológicas, contribuindo para uma produção homogênea, fiel e em larga escala. Portanto, este trabalho teve como objetivo geral avaliar as condições mais favoráveis à germinação, estabelecimento in vitro e micropropagação de Crambe abyssinica Hochst., além de verificar possíveis alterações genéticas e anatômicas, possibilitando a regeneração e produção de plântulas viáveis. Para a germinação e estabecimento in vitro de crambe, as condições mais favoráveis foram em meio B5 ou WPM, na presença ou ausência de pericarpo e na presença de luz. Na micropropagação dessa espécie, uma frequência satisfatória de regeneração de brotos foi obtida a partir de segmentos apicais utilizados como explante em meio contendo 5 μM de BAP (6- benzilaminopurina), e o alongamento foi satisfatório com 1 μM de GA3 (ácido giberélico). Os marcadores moleculares ISSR (Inter-Simples Sequence Repeats) utilizados para a análise da estabilidade genética indicaram que o segmento apical de crambe é um explante confiável para a micropropagação de plantas geneticamente verdadeiras (true-to-tipe), ou seja, mantém a estabilidade genética. As diversas fontes de citocininas e concentrações utilizadas neste trabalho não promoveram mudanças, no sentido de alterar a organização e/ou a espessura em relação ao controle, e as alterações observadas na estrutura e espessura das folhas dos tratamentos de aclimatização prejudicaram o processo de estabelecimento da plântula ex vitro. Contudo, existe a necessidade de um enraizamento e aclimatização eficiente para completa propagação in vitro de crambe. Portanto, este protocolo de regeneração de plantas in vitro de crambe pode ser útil no processo de criação e desenvolvimento de novas cultivares em um tempo mais curto e no melhoramento genético usando explantes apicais.

Synthesis, characterization, electrochemical behavior and in vitro protein tyrosine kinase inhibitory activity of the cymene-halogenobenzohydroxamato [Ru(eta(6)-cymene)(bha)Cl] complexes

The ruthenium(II)-cymene complexes [Ru(eta(6)-cymene)(bha)Cl] with substituted halogenobenzohydroxamato (bha) ligands (substituents = 4-F, 4-Cl, 4-Br, 2,4-F-2, 3,4-F-2, 2,5-F-2, 2,6-F-2) have been synthesized and characterized by elemental analysis, IR, H-1 NMR, C-13 NMR, cyclic voltammetry and controlled-potential electrolysis, and density functional theory (DFT) studies. The compositions of their frontier molecular orbitals (MOs) were established by DFT calculations, and the oxidation and reduction potentials are shown to follow the orders of the estimated vertical ionization potential and electron affinity, respectively. The electrochemical E-L Lever parameter is estimated for the first time for the various bha ligands, which can thus be ordered according to their electron-donor character. All complexes exhibit very strong protein tyrosine kinase (PTK) inhibitory activity, even much higher than that of genistein, the clinically used PTK inhibitory drug. The complex containing the 2,4-difluorobenzohydroxamato ligand is the most active one, and the dependences of the PTK activity of the complexes and of their redox potentials on the ring substituents are discussed. (C) 2012 Elsevier B.V. All rights reserved.

Avaliação do potencial de cultivo in vitro de Halopteris scoparia (Linnaeus), Sauvageau (1903), Heterokontophyta (Phaeophyceae)

Dissertação de Mestrado em Biodiversidade e Biotecnologia Vegetal.

In vitro and in situ activation of the complement system by the fungus Lacazia loboi

Since there are no studies evaluating the participation of the complement system (CS) in Jorge Lobo's disease and its activity on the fungus Lacazia loboi, we carried out the present investigation. Fungal cells with a viability index of 48% were obtained from the footpads of BALB/c mice and incubated with a pool of inactivated serum from patients with the mycosis or with sterile saline for 30 min at 37 ºC. Next, the tubes were incubated for 2 h with a pool of noninactivated AB+ serum, inactivated serum, serum diluted in EGTA-MgCl2, and serum diluted in EDTA. The viability of L. loboi was evaluated and the fungal suspension was cytocentrifuged. The slides were submitted to immunofluorescence staining using human anti-C3 antibody. The results revealed that 98% of the fungi activated the CS by the alternative pathway and no significant difference in L. loboi viability was observed after CS activation. In parallel, frozen histological sections from 11 patients were analyzed regarding the presence of C3 and IgG by immunofluorescence staining. C3 and IgG deposits were observed in the fungal wall of 100% and 91% of the lesions evaluated, respectively. The results suggest that the CS and immunoglobulins may contribute to the defense mechanisms of the host against L. loboi.

Resultado do Obstétrico de Gravidezes Gemelares Resultantes de Fecundação in Vitro, Microinjeção e Estimulação da Ovulação em Comparação com Espontâneas

Introduction: Uncontrolled studies suggest that twins conceived by in vitro fertilization have increased rates of preterm birth and low birth weight and would warrant increased antenatal monitoring. The objective of this study was to compare the obstetric outcome of twin pregnancies conceived by in vitro fertilization or intracytoplasmic sperm injection (IVF/ICSI) and ovulation induction with those conceived spontaneously. Methods: All twin deliveries achieved by IVF/ICSI (n=235) and ovulation induction (n=68) from September 1994 through December 2010 were evaluated. Both groups and an additional control group who conceived spontaneously (n=997) and was delivered during the same time period were compared with each other. Results: In univariate analysis, patients who conceived with the assistance of IVF/ICSI had a significantly higher risk of being older (p=0.01), nulliparous (p=0.01), having hypertensive disorders (p=0.012), gestational diabetes mellitus (p=0.031), cesarean section (p=0.008) and lower gestational age at birth, compared with the control group. Newborns had similar birthweights in all groups (2229±544g; 2102±619g; 2251±553g). Spontaneous pregnancies had a higher risk of being monochorionic 38.4% versus 16.2% and 10.2% (p=0.01). Multivariate analysis however showed that patients who conceived with the assistance of IVF/ICSI only had a higher risk of gestational diabetes (OR=1.91,95%CI 1.168-3.120; p=0.01). Conclusions: Our study shows that twin pregnancies conceived with the assistance of IVF/ICSI had a higher risk of gestacional diabetes and a lower gestacional age at birth. Birthweights were similar, as was the incidence of perinatal death, low birth weight infants, and congenital malformations.

In vitro ANTIGIARDIAL ACTIVITY OF THE CYSTEINE PROTEASE INHIBITOR E-64

The quest for new antiparasitic alternatives has led researchers to base their studies on insights into biology, host-parasite interactions and pathogenesis. In this context, proteases and their inhibitors are focused, respectively, as druggable targets and new therapy alternatives. Herein, we proposed to evaluate the in vitro effect of the cysteine protease inhibitor E-64 on Giardia trophozoites growth, adherence and viability. Trophozoites (105) were exposed to E-64 at different final concentrations, for 24, 48 and 72 h at 37 °C. In the growth and adherence assays, the number of trophozoites was estimated microscopically in a haemocytometer, whereas cell viability was evaluated by a dye-reduction assay using MTT. The E-64 inhibitor showed effect on growth, adherence and viability of trophozoites, however, its better performance was detected in the 100 µM-treated cultures. Although metronidazole was more effective, the E-64 was shown to be able to inhibit growth, adherence and viability rates by ≥ 50%. These results reveal that E-64 can interfere in some crucial processes to the parasite survival and they open perspectives for future investigations in order to confirm the real antigiardial potential of the protease inhibitors.

Evaluation of pathogenic potential of Aeromonas spp. strains using in vitro methodologies

Dissertation to obtain a Master Degree in Molecular Genetics and Biomedicine

In vitro antifungal activities of leaf extracts of Lippia alba (Verbenaceae) against clinically important yeast species

Introduction There are few studies reporting the antifungal activities of Lippia alba extracts. Methods A broth microdilution assay was used to evaluate the antifungal effects of Lippia alba extracts against seven yeast species of Candida and Cryptococcus. The butanol fraction was investigated by gas chromatography-mass spectrometry. Results The butanol fraction showed the highest activity against Candida glabrata. The fraction also acted synergistically with itraconazole and fluconazole against C. glabrata. The dominant compounds in the butanol fraction were 2,2,5-trimethyl-3,4-hexanedione, 3,5-dimethyl-4-octanone and hexadecane. Conclusions The butanol fraction may be a good candidate in the search for new drugs from natural products with antifungal activity.

Development of human central nervous system 3D in vitro models for preclinical research

Neurological disorders are a major concern in modern societies, with increasing prevalence mainly related with the higher life expectancy. Most of the current available therapeutic options can only control and ameliorate the patients’ symptoms, often be-coming refractory over time. Therapeutic breakthroughs and advances have been hampered by the lack of accurate central nervous system (CNS) models. The develop-ment of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of novel therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmentally, anatomically and physiologically) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity). The in vitro recapitulation of CNS phenotypic and functional features requires the implementation of advanced culture strategies that enable to mimic the in vivo struc-tural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. This thesis aimed at the development of novel human 3D in vitro CNS models by integrat-ing agitation-based culture systems and a wide array of characterization tools. Neural differentiation of hNSC as 3D neurospheres was explored in Chapter 2. Here, it was demonstrated that human midbrain-derived neural progenitor cells from fetal origin (hmNPC) can generate complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Chapter 3 focused on the development of cellular characterization assays for cell aggregates based on light-sheet fluorescence imaging systems, which resulted in increased spatial resolu-tion both for fixed samples or live imaging. The applicability of the developed human 3D cell model for preclinical research was explored in Chapter 4, evaluating the poten-tial of a viral vector candidate for gene therapy. The efficacy and safety of helper-dependent CAV-2 (hd-CAV-2) for gene delivery in human neurons was evaluated, demonstrating increased neuronal tropism, efficient transgene expression and minimal toxicity. The potential of human 3D in vitro CNS models to mimic brain functions was further addressed in Chapter 5. Exploring the use of 13C-labeled substrates and Nucle-ar Magnetic Resonance (NMR) spectroscopy tools, neural metabolic signatures were evaluated showing lineage-specific metabolic specialization and establishment of neu-ron-astrocytic shuttles upon differentiation. Chapter 6 focused on transferring the knowledge and strategies described in the previous chapters for the implementation of a scalable and robust process for the 3D differentiation of hNSC derived from human induced pluripotent stem cells (hiPSC). Here, software-controlled perfusion stirred-tank bioreactors were used as technological system to sustain cell aggregation and dif-ferentiation. The work developed in this thesis provides practical and versatile new in vitro ap-proaches to model the human brain. Furthermore, the culture strategies described herein can be further extended to other sources of neural phenotypes, including pa-tient-derived hiPSC. The combination of this 3D culture strategy with the implemented characterization methods represents a powerful complementary tool applicable in the drug discovery, toxicology and disease modeling.

In vitro mechanical fatigue behavior of poly-ε-caprolactone macroporous scaffolds for cartilage tissue engineering: Influence of pore filling by a poly(vinyl alcohol) gel

Polymeric scaffolds used in regenerative therapies are implanted in the damaged tissue and subjected to repeated loading cycles. In the case of articular cartilage engineering, an implanted scaffold is typically subjected to long term dynamic compression. The evolution of the mechanical properties of the scaffold during bioresorption has been deeply studied in the past, but the possibility of failure due to mechanical fatigue has not been properly addressed. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. In this work fatigue studies of polycaprolactone scaffolds were carried by subjecting the scaffold to repeated compression cycles in conditions simulating the scaffold implanted in the articular cartilage. The behaviour of the polycaprolactone sponge with the pores filled with a poly(vinyl alcohol) gel simulating the new formed tissue within the pores was compared with that of the material immersed in water. Results were analyzed with Morrow’s criteria for failure and accurate fittings are obtained just up to 200 loading cycles. It is also shown that the presence of poly(vinyl alcohol) increases the elastic modulus of the scaffolds, the effect being more pronounced with increasing the number of freeze/thawing cycles.

In vitro anti-Candida activity of Glycyrrhiza glabra L.

The severity and frequency of opportunistic fungal infections still growing, concomitantly to the increasing rates of antimicrobial drugs resistance. Natural matrices have been used over years due to its multitude of health benefits, including antifungal potential. Thus, the present work aims to evaluate the anti-Candida potential of the phenolic extract and individual phenolic compounds of Glycyrrhiza glabra L. (licorice), by disc diffusion assay, followed by determination of the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for both planktonic cells and biofilms. Licorice extract evidenced inhibitory potential against the nineteen tested Candida strains, but no pronounced effect was observed by testing the most abundant individual phenolic compounds. Candida tropicalis strains were the most sensible, followed by Candida glabrata, Candida parapsilosis and, then, Candida albicans. Lower MIC and MFC values were achieved to C. glabrata and C. tropicalis, which confirms its susceptibility to licorice extract; however, for C. tropicalis strains a higher variability was observed. Anti-biofilm potential was also achieved, being most evident in some C. glabrata and C. tropicalis strains. In general, a twice concentration of the MIC was necessary for planktonic cells to obtain a similar potential to that one observed for biofilms. Thus, an upcoming approach for new antifungal agents, more effective and safer than the current ones, is stablished; notwithstanding, further studies are necessary in order to understand its mechanism of action, as also to assess kinetic parameters.

Paroxetine and bupropion have no in vitro effects on lynphocyte proliferation and viability

OBJECTIVE: Initial studies with tricyclic antidepressants demonstrated that they jeopardize the immune system activity. Recent studies suggested that selective serotonin reuptake inhibitors would have stimulating immunological effects. Here, we explored the in vitro immunological effects of two antidepressants used in clinical practice, paroxetine (selective serotonin reuptake inhibitor) and bupropion (norepinephrine and dopamine reuptake inhibitor). METHOD: Peripheral blood samples were obtained from 16 healthy volunteers and the peripheral blood mononuclear cells were isolated and cultured in vitro. We evaluated the effects of bupropion and paroxetine on cell viability as well as the ability to suppress phytohemagglutinin-induced lymphocyte proliferation. RESULTS: Both antidepressants produced neither significant effect on cell viability nor on T-cell proliferation. CONCLUSIONS: This could be of valuable information for the clinical practice when these drugs are administered. These results indicate a more favorable effect of such psychopharmacological drugs when compared to reported immunological effects associated with tryciclic antidepressants.