Zebrafish embryos as in vivo model for toxicity evaluation: screening of nanoparticle formulations DODAB:MO and DODAC:MO

Dissertação de mestrado em Molecular Genetics

Correlation of wear in vivo and six laboratory wear methods.

OBJECTIVE: We examined the correlation between clinical wear rates of restorative materials and enamel (TRAC Research Foundation, Provo, USA) and the results of six laboratory test methods (ACTA, Alabama (generalized, localized), Ivoclar (vertical, volumetric), Munich, OHSU (abrasion, attrition), Zurich). METHODS: Individual clinical wear data were available from clinical trials that were conducted by TRAC Research Foundation (formerly CRA) together with general practitioners. For each of the n=28 materials (21 composite resins for intra-coronal restorations [20 direct and 1 indirect], 5 resin materials for crowns, 1 amalgam, enamel) a minimum of 30 restorations had been placed in posterior teeth, mainly molars. The recall intervals were up to 5 years with the majority of materials (n=27) being monitored, however, only for up to 2 years. For the laboratory data, the databases MEDLINE and IADR abstracts were searched for wear data on materials which were also clinically tested by TRAC Research Foundation. Only those data for which the same test parameters (e.g. number of cycles, loading force, type of antagonist) had been published were included in the study. A different quantity of data was available for each laboratory method: Ivoclar (n=22), Zurich (n=20), Alabama (n=17), OHSU and ACTA (n=12), Munich (n=7). The clinical results were summed up in an index and a linear mixed model was fitted to the log wear measurements including the following factors: material, time (0.5, 1, 2 and 3 years), tooth (premolar/molar) and gender (male/female) as fixed effects, and patient as random effect. Relative ranks were created for each material and method; the same was performed with the clinical results. RESULTS: The mean age of the subjects was 40 (±12) years. The materials had been mostly applied in molars (81%) and 95% of the intracoronal restorations were Class II restorations. The mean number of individual wear data per material was 25 (range 14-42). The mean coefficient of variation of clinical wear data was 53%. The only significant correlation was reached by OHSU (abrasion) with a Spearman r of 0.86 (p=0.001). Zurich, ACTA, Alabama generalized wear and Ivoclar (volume) had correlation coefficients between 0.3 and 0.4. For Zurich, Alabama generalized wear and Munich, the correlation coefficient improved if only composites for direct use were taken into consideration. The combination of different laboratory methods did not significantly improve the correlation. SIGNIFICANCE: The clinical wear of composite resins is mainly dependent on differences between patients and less on the differences between materials. Laboratory methods to test conventional resins for wear are therefore less important, especially since most of them do not reflect the clinical wear.

Present development concerning antimalarial activity of phospholipid metabolism inhibitors with special reference to in vivo activity

The systematic screening of more than 250 molecules against Plasmodium falciparum in vitro has previously shown that interfering with phospholipid metabolism is lethal to the malaria parasite. These compounds act by impairing choline transport in infected erythrocytes, resulting in phosphatidylcholine de novo biosynthesis inhibition. A thorough study was carried out with the leader compound G25, whose in vitro IC50 is 0.6 nM. It was very specific to mature parasites (trophozoïtes) as determined in vitro with P. falciparum and in vivo with P. chabaudi -infected mice. This specificity corresponds to the most intense phase of phospholipid biosynthesis activity during the parasite cycle, thus corroborating the mechanism of action. The in vivo antimalarial activity (ED50) against P. chabaudi was 0.03 mg/kg, and a similar sensitivity was obtained with P. vinckei petteri, when the drug was intraperitoneally administered in a 4 day suppressive test. In contrast, P. berghei was revealed as less sensitive (3- to 20-fold, depending on the P. berghei-strain). This difference in activity could result either from the degree of synchronism of every strain, their invasion preference for mature or immature red blood cells or from an intrinsically lower sensitivity of the P. berghei strain to G25. Irrespective of the mode of administration, G25 had the same therapeutic index (lethal dose 50 (LD50)/ED50) but the dose to obtain antimalarial activity after oral treatment was 100-fold higher than after intraperitoneal (or subcutaneous) administration. This must be related to the low intestinal absorption of these kind of compounds. G25 succeeded to completely inhibiting parasitemia as high as 11.2% without any decrease in its therapeutic index when administered subcutaneously twice a day for at least 8 consecutive days to P. chabaudi -infected-rodent model. Transition to human preclinical investigations now requires a synthesis of molecules which would permit oral absorption.

Chromoblastomycosis murine model and in vitro test to evaluate the sensitivity of Fonsecaea pedrosoi to ketoconazole, itraconazole and saperconazole

An experimental model of murine chromoblastomycosis and in vitro tests with Fonsecaea pedrosoi were used to test the sensitivity of this fungus to three different antimycotics. The experimental model was standardized in BALB/c mice inoculated intraperitoneally with a 10(6) CFU/ml suspension of a F. pedrosoi isolate. Clinical infection was evident after 5 days of inoculation. Three groups of 27 mice each were used in the experiment. One group was treated with ketoconazole (KTZ), another with itraconazole (ITZ) and the other with saperconazole (SPZ). Antimycotic therapy was continued for 21 days. The control group consisted of 40 mice which were inoculated, but not treated. Infection was documented by macroscopic and microscopic examination of affected tissue in addition to culture of tissue macerates. Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) for the F. pedrosoi strain used were done. The in vitro results showed that SPZ was the most active with MIC 0.01 mg/ml and MFC 0.1 mg/ml, followed by ITZ. SPZ was also the most effective in vivo since 63% of the treated animals (p=0.01) showed a curative effect after the observation period. We concluded that SPZ had the best in vitro and in vivo activity against F. pedrosoi.

Y-688, a new quinolone active against quinolone-resistant Staphylococcus aureus: lack of in vivo efficacy in experimental endocarditis.

Y-688 is a new fluoroquinolone with increased activity against ciprofloxacin-resistant staphylococci. The MICs of Y-688 and other quinolones were determined for 58 isolates of ciprofloxacin-resistant and methicillin-resistant Staphylococcus aureus (MRSA). The MICs at which 50% and 90% of bacteria were inhibited were >/=128 and >/=128 mg/liter, respectively, for ciprofloxacin, 16 and 32 mg/liter, respectively, for sparfloxacin, and 0.25 and 1 mg/liter, respectively, for Y-688. This new quinolone was further tested in rats with experimental endocarditis due to either of two isolates of ciprofloxacin-resistant MRSA (namely, P8/128 and CR1). Infected animals were treated for 3 days with ciprofloxacin, vancomycin, or Y-688. Antibiotics were administered through a computerized pump to simulate human-like pharmacokinetics in the serum of rats. The anticipated peak and trough levels of Y-688 were 4 and 1 mg/liter at 0.5 and 12 h, respectively. Treatment with ciprofloxacin was ineffective. Vancomycin significantly decreased vegetation bacterial counts for both organisms (P less, similar 0.05). In contrast, Y-688 only marginally decreased vegetation bacterial counts (P greater, similar 0.05). Moreover, several vegetation that failed Y-688 treatment grew staphylococci for which the MICs of the test antibiotic were increased two to eight times. Y-688 also selected for resistance in vitro, and isolates for which the MICs were increased eight times emerged at a frequency of ca. 10(-8). Thus, in spite of its low MIC for ciprofloxacin-resistant MRSA, Y-688 failed in vivo and its use carried the risk of resistance selection. The fact that ciprofloxacin-resistant staphylococci became rapidly resistant to this potent new drug suggests that the treatment of ciprofloxacin-resistant MRSA with new quinolones might be more problematic than expected.

Single-stranded oligonucleotide-mediated in vivo gene repair in the rd1 retina.

PURPOSE: The aim of this study was to test whether oligonucleotide-targeted gene repair can correct the point mutation in genomic DNA of PDE6b(rd1) (rd1) mouse retinas in vivo. METHODS: Oligonucleotides (ODNs) of 25 nucleotide length and complementary to genomic sequence subsuming the rd1 point mutation in the gene encoding the beta-subunit of rod photoreceptor cGMP-phosphodiesterase (beta-PDE), were synthesized with a wild type nucleotide base at the rd1 point mutation position. Control ODNs contained the same nucleotide bases as the wild type ODNs but with varying degrees of sequence mismatch. We previously developed a repeatable and relatively non-invasive technique to enhance ODN delivery to photoreceptor nuclei using transpalpebral iontophoresis prior to intravitreal ODN injection. Three such treatments were performed on C3H/henJ (rd1) mouse pups before postnatal day (PN) 9. Treatment outcomes were evaluated at PN28 or PN33, when retinal degeneration was nearly complete in the untreated rd1 mice. The effect of treatment on photoreceptor survival was evaluated by counting the number of nuclei of photoreceptor cells and by assessing rhodopsin immunohistochemistry on flat-mount retinas and sections. Gene repair in the retina was quantified by allele-specific real time PCR and by detection of beta-PDE-immunoreactive photoreceptors. Confirmatory experiments were conducted using independent rd1 colonies in separate laboratories. These experiments had an additional negative control ODN that contained the rd1 mutant nucleotide base at the rd1 point mutation site such that the sole difference between treatment with wild type and control ODN was the single base at the rd1 point mutation site. RESULTS: Iontophoresis enhanced the penetration of intravitreally injected ODNs in all retinal layers. Using this delivery technique, significant survival of photoreceptors was observed in retinas from eyes treated with wild type ODNs but not control ODNs as demonstrated by cell counting and rhodopsin immunoreactivity at PN28. Beta-PDE immunoreactivity was present in retinas from eyes treated with wild type ODN but not from those treated with control ODNs. Gene correction demonstrated by allele-specific real time PCR and by counts of beta-PDE-immunoreactive cells was estimated at 0.2%. Independent confirmatory experiments showed that retinas from eyes treated with wild type ODN contained many more rhodopsin immunoreactive cells compared to retinas treated with control (rd1 sequence) ODN, even when harvested at PN33. CONCLUSIONS: Short ODNs can be delivered with repeatable efficiency to mouse photoreceptor cells in vivo using a combination of intravitreal injection and iontophoresis. Delivery of therapeutic ODNs to rd1 mouse eyes resulted in genomic DNA conversion from mutant to wild type sequence, low but observable beta-PDE immunoreactivity, and preservation of rhodopsin immunopositive cells in the outer nuclear layer, suggesting that ODN-directed gene repair occurred and preserved rod photoreceptor cells. Effects were not seen in eyes treated with buffer or with ODNs having the rd1 mutant sequence, a definitive control for this therapeutic approach. Importantly, critical experiments were confirmed in two laboratories by several different researchers using independent mouse colonies and ODN preparations from separate sources. These findings suggest that targeted gene repair can be achieved in the retina following enhanced ODN delivery.

The in vivo antimalarial activity of methylene blue combined with pyrimethamine, chloroquine and quinine

The effectiveness of methylene blue (MB) combined with pyrimethamine (PYR), chloroquine (CQ) or quinine (Q) was examined in a classical four-day suppressive test against a causative agent of rodent malaria, Plasmodium berghei. A marked potentiation was observed when MB was administered at a non-curative dose of 15 mg/kg/day in combination with PYR (0.19 mg/kg/day) or Q (25 mg/kg/day). No synergy was found between MB (15 mg/Kg) and CQ (0.75 mg/Kg). Our results suggest that the combination of MB with PYR or Q may improve the efficacy of these currently used antimalarial drugs.

In vitro and in vivo antimalarial activity and cytotoxicity of extracts, fractions and a substance isolated from the Amazonian plant Tachia grandiflora (Gentianaceae)

Tachia sp. are used as antimalarials in the Amazon Region and in vivo antimalarial activity of a Tachia sp. has been previously reported. Tachia grandiflora Maguire and Weaver is an Amazonian antimalarial plant and herein its cytotoxicity and antimalarial activity were investigated. Spectral analysis of the tetraoxygenated xanthone decussatin and the iridoid aglyone amplexine isolated, respectively, from the chloroform fractions of root methanol and leaf ethanol extracts was performed. In vitro inhibition of the growth of Plasmodium falciparum Welch was evaluated using optical microscopy on blood smears. Crude extracts of leaves and roots were inactive in vitro. However, chloroform fractions of the root and leaf extracts [half-maximal inhibitory concentration (IC50) = 10.5 and 35.8 µg/mL, respectively] and amplexine (IC50= 7.1 µg/mL) were active in vitro. Extracts and fractions were not toxic to type MRC-5 human fibroblasts (IC50> 50 µg/mL). Water extracts of the roots of T. grandiflora administered by mouth were the most active extracts in the Peters 4-day suppression test in Plasmodium berghei-infected mice. At 500 mg/kg/day, these extracts exhibited 45-59% inhibition five to seven days after infection. T. grandiflora infusions, fractions and isolated substance have potential as antimalarials.

The efficacy of 2-nitrovinylfuran derivatives againstLeishmania in vitro and in vivo

Despite recent advances in the treatment of some forms of leishmaniasis, the available drugs are still far from ideal due to inefficacy, parasite resistance, toxicity and cost. The wide-spectrum antimicrobial activity of 2-nitrovinylfuran compounds has been described, as has their activity against Trichomonas vaginalis and other protozoa. Thus, the aim of this study was to test the antileishmanial activities of six 2-nitrovinylfurans in vitro and in a murine model of leishmaniasis. Minimum parasiticide concentration (MPC) and 50% inhibitory concentration (IC50) values for these compounds against the promastigotes of Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis were determined, as were the efficacies of two selected compounds in an experimental model of cutaneous leishmaniasis (CL) caused by L. amazonensis in BALB/c mice. All of the compounds were active against the promastigotes of the three Leishmania species tested. IC50 and MPC values were in the ranges of 0.8-4.7 µM and 1.7-32 µM, respectively. The compounds 2-bromo-5-(2-bromo-2-nitrovinyl)-furan (furvina) and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan (UC245) also reduced lesion growth in vivo at a magnitude comparable to or higher than that achieved by amphotericin B treatment. The results demonstrate the potential of this class of compounds as antileishmanial agents and support the clinical testing of Dermofural(r) (a furvina-containing antifungal ointment) for the treatment of CL.

A novel pediatric biventricular assist device: in vitro test results.

Most ventricular assist devices (VADs) currently used in infants are extracorporeal. These VADs require long-term anticoagulation therapy and extensive surgery, and two devices are needed for biventricular support. We designed a biventricular assist device based on shape memory alloy that reproduces the hemodynamic effects of cardiomyoplasty, supporting the heart with a compressing movement, and evaluated its performance in a dedicated mockup system. Nitinol fibers are the device's key component. Ejection fraction (EF), cardiac output (CO), and generated systolic pressure were measured on a test bench. Our test bench settings were a preload range of 0-15 mm Hg, an afterload range of 0-160 mm Hg, and a heart rate (HR) of 20, 30, 40, and 60 beats/min. A power supply of 15 volts and 3.5 amperes was necessary. The EF range went from 34.4% to 1.2% as the afterload and HR increased, along with a CO from 180 to 6 ml/min. The device generated a maximal systolic pressure of 25 mm Hg. Cardiac compression for biventricular assistance in child-sized heart using shape memory alloy is technically feasible. Further testing remains necessary to assess this VAD's in vivo performance range and its reliability.

Validation of rotational thromboelastometry during cardiopulmonary bypass : a prospective, observational in-vivo study

Le ROTEM est un test de coagulation réalisable au près du malade qui permet d'objectiver la coagulopathie, de distinguer la contribution des différents éléments du système de coagulation et de cibler les produits procoagulants comme le plasma frais congelé (PFC), les plaquettes, le fibrinogène et les facteurs de coagulation purifiés ou les antifibrinolytiques. 3 des tests disponibles pour le ROTEM sont: EXTEM, INTEM, HEPTEM. Le premier test est stable sous hautes doses d'héparine alors que le deuxième est très sensible à sa présence. Dans le dernier test on rajoute de l'héparinase pour mettre en évidence l'éventuel effet résiduel de l'héparine en le comparant à l'INTEM. Idéalement, le ROTEM devrait être effectué avant la fin du bypass cardiopulmonaire (CEC), donc sous anticoagulation maximale pas héparine, afin de pouvoir administrer des produits pro¬coagulants dans les délais les plus brefs et ainsi limiter au maximum les pertes sanguines. En effet la commande et la préparation de certains produits procoagulants peut prendre plus d'une heure. Le but de cette étude est de valider l'utilisation du ROTEM en présence de hautes concentrations d'héparine. Il s'agit d'une étude observationnelle prospective sur 20 patients opérés électivement de pontages aorto-coronariens sous CEC. Méthode : l'analyse ROTEM a été réalisée avant l'administration d'héparine (TO), 10 minutes après l'administration d'héparine (Tl), à la fin de la CEC (T2) et 10 minutes après la neutralisation de l'anticoagulation avec la protamine (T3). L'état.d'héparinisation a été évalué par l'activité anti-Xa à T1,T2,T3. Résultats : Comparé à TO, la phase de polymérisation de la cascade de coagulation et l'interaction fibrine-plaquettes sont significativement détériorées par rapport à Tl pour les canaux EXTEM et HEPTEM. A T2 l'analyse EXTEM et INTEM sont comparables à celles de EXTEM et HEPTEM à T3. Conclusion: les hautes doses d'héparine utilisées induisent une coagulopathie qui reste stable durant toute la durée de la CEC et qui persiste même après la neutralisation de l'anticoagulation. Les mesures EXTEM et HEPTEM sont donc valides en présence de hautes concentrations d'héparine et peuvent être réalisés pendant la CEC avant l'administration de protamine.

Use of a combined ex vivo/in vivo population approach for screening of human genes involved in the human immunodeficiency virus type 1 life cycle for variants influencing disease progression.

Humans differ substantially with respect to susceptibility to human immunodeficiency virus type 1 (HIV-1). We evaluated variants of nine host genes participating in the viral life cycle for their role in modulating HIV-1 infection. Alleles were assessed ex vivo for their impact on viral replication in purified CD4 T cells from healthy blood donors (n = 128). Thereafter, candidate alleles were assessed in vivo in a cohort of HIV-1-infected individuals (n = 851) not receiving potent antiretroviral therapy. As a benchmark test, we tested 12 previously reported host genetic variants influencing HIV-1 infection as well as single nucleotide polymorphisms in the nine candidate genes. This led to the proposition of three alleles of PML, TSG101, and PPIA as potentially associated with differences in progression of HIV-1 disease. In a model considering the combined effects of new and previously reported gene variants, we estimated that their effect might be responsible for lengthening or shortening by up to 2.8 years the period from 500 CD4 T cells/mul to <200 CD4 T cells/mul.

Encapsulation of packaging cell line results in successful retroviral-mediated transfer of a suicide gene in vivo in an experimental model of glioblastoma.

AIMS: Retroviral-mediated gene therapy has been proposed as a primary or adjuvant treatment for advanced cancer, because retroviruses selectively infect dividing cells. Efficacy of retroviral-mediated gene transfer, however, is limited in vivo. Although packaging cell lines can produce viral vectors continuously, such allo- or xenogeneic cells are normally rejected when used in vivo. Encapsulation using microporous membranes can protect the packaging cells from rejection. In this study, we used an encapsulated murine packaging cell line to test the effects of in situ delivery of a retrovirus bearing the herpes simplex virus thymidine kinase suicide gene in a rat model of orthotopic glioblastoma. MATERIALS AND METHODS: To test gene transfer in vitro, encapsulated murine psi2-VIK packaging cells were co-cultured with baby hamster kidney (BHK) cells, and the percentage of transfected BHK cells was determined. For in vivo experiments, orthotopic C6 glioblastomas were established in Wistar rats. Capsules containing psi2-VIK cells were stereotaxically implanted into these tumours and the animals were treated with ganciclovir (GCV). Tumours were harvested 14 days after initiation of GCV therapy for morphometric analysis. RESULTS: Encapsulation of psi2-VIK cells increased transfection rates of BHK target cells significantly in vitro compared to psi2-VIK conditioned medium (3 x 10(6) vs 2.3 x 10(4) cells; P<0.001). In vivo treatment with encapsulated packaging cells resulted in 3% to 5% of C6 tumour cells transduced and 45% of tumour volume replaced by necrosis after GCV (P<0.01 compared to controls). CONCLUSION: In this experimental model of glioblastoma, encapsulation of a xenogeneic packaging cell line increased half-life and transduction efficacy of retrovirus-mediated gene transfer and caused significant tumour necrosis.

Validation of rotational thromboelastometry during cardiopulmonary bypass: A prospective, observational in-vivo study.

BACKGROUND: Rotational thromboelastometry (ROTEM) is a whole blood point-of-test used to assess the patient's coagulation status. Three of the available ROTEM tests are EXTEM, INTEM and HEPTEM. In the latter, heparinase added to the INTEM reagent inactivates heparin to reveal residual heparin effect. Performing ROTEM analysis during cardiopulmonary bypass (CPB) might allow the anaesthesiologist to anticipate the need for blood products. OBJECTIVE: The goal of this study was to validate ROTEM analysis in the presence of very high heparin concentrations during CPB. DESIGN: Prospective, observational trial. SETTING: Single University Hospital. PARTICIPANTS: Twenty patients undergoing coronary artery bypass grafting. MAIN OUTCOME MEASURE: ROTEM analysis was performed before heparin administration (T0), 10 min after heparin (T1), at the end of CPB (T2) and 10 min after protamine (T3). The following tests were performed: EXTEM, INTEM, and HEPTEM. Heparin concentrations were measured at T1 and at the end of bypass (T2). RESULTS: At T1, EXTEM differed from baseline for coagulation time: +26.7 s (18.4 to 34.9, P < 0.0001), α: -3° (1.0 to 5.4, P = 0.006) and A10: -4.4 mm (2.3 to 6.5, P = 0.0004). INTEM at T0 was different from HEPTEM at T1 for coagulation time: + 47 s (34.3 to 59.6, P >0.0001), A10: -2.3 mm (0.5 to 4.0, P = 0.01) and α -2° (1.0 to 3.0; P = 0.0007). At T2, all parameters in EXTEM and HEPTEM related to fibrin-platelet interaction deteriorated significantly compared to T1. At T3, EXTEM and INTEM were comparable to EXTEM and HEPTEM at T2. CONCLUSION: HEPTEM and EXTEM measurements are valid in the presence of very high heparin concentrations and can be performed before protamine administration in patients undergoing cardiac surgery with CPB. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01455454.

Flow-microfluorometric monitoring of oligoclonal CD8+ T cell responses to an immunodominant Moloney leukemia virus-encoded epitope in vivo.

The TCR repertoire of CD8+ T cells specific for Moloney murine leukemia virus (M-MuLV)-associated Ags has been investigated in vitro and in vivo. Analysis of a large panel of established CD8+ CTL clones specific for M-MuLV indicated an overwhelming bias for V beta4 in BALB/c mice and for V beta5.2 in C57BL/6 mice. These V beta biases were already detectable in mixed lymphocyte:tumor cell cultures established from virus-immune spleen cells. Furthermore, direct ex vivo analysis of PBL from BALB/c or C57BL/6 mice immunized with syngeneic M-MuLV-infected tumor cells revealed a dramatic increase in CD8+ cells expressing V beta4 or V beta5.2, respectively. M-MuLV-specific CD8+ cells with an activated (CD62L-) phenotype persisted in blood of immunized mice for at least 2 mo, and exhibited decreased TCR and CD8 levels compared with their naive counterparts. In C57BL/6 mice, most M-MuLV-specific CD8+ CTL clones and immune PBL coexpressed V alpha3.2 in association with V beta5.2. Moreover, these V beta5.2+ V alpha3.2+ cells were shown to recognize the recently described H-2Db-restricted epitope (CCLCLTVFL) encoded in the leader sequence of the M-MuLV gag polyprotein. Collectively, our data demonstrate a highly restricted TCR repertoire in the CD8+ T cell response to M-MuLV-associated Ags in vivo, and suggest the potential utility of flow-microfluorometric analysis of V beta and V alpha expression in the diagnosis and monitoring of viral infections.