Effect of follicular wave synchronization on in vitro embryo production in heifers

Aiming to achieve the ideal time of ovum pick-up (OPU) for in vitro embryo production (IVP) in crossbred heifers, two Latin square design studies investigated the effect of ovarian follicular wave synchronization with estradiol benzoate (EB) and progestins. For each experiment, crossbred heifers stage of estrous cycle was synchronized either with a norgestomet ear implant (Experiment 1) or a progesterone intravaginal device (Experiment 2) for 7d, followed by the administration of 150 mu g D-cloprostenol. On Day 7, all follicles >3 mm in diameter were aspirated and implants/devices were replaced by new ones. Afterwards, implant/device replacement was conducted every 14 d. Each experiment had three treatment groups. In Experiment I (n = 12), heifers in Group 2X had their follicles aspirated twice a week and those in Groups 1X and 1X-EB were submitted to OPU once a week for a period of 28 d. Heifers from Group 1X-EB also received 2 mg EB i.m. immediately after each OPU session. In Experiment 2 (n = 11), animals from Group 0EB did not receive EB while heifers in Groups 2EB and 5EB received 2 and 5 mg of EB respectively, immediately after OPU. The OPU sessions were performed once weekly for 28 d. Therefore, in both experiments, four OPU sessions were performed in heifers aspirated once a week and in Experiment 1, eight OPU sessions were done in heifers aspirated twice a week. Additionally, during the 7-d period following follicular aspiration, ovarian ultrasonography examinations were conducted to measure diameter of the largest follicle and blood samples were collected for FSH quantification by RIA. In Experiment 1, all viable oocytes recovered were in vitro matured and fertilized. Results indicated that while progestin and EB altered follicular wave patterns, this treatment did not prevent establishment of follicular dominance on the ovaries of heifers during OPU at 7-d intervals. Furthermore, the proposed stage of follicular wave synchronization strategies did not improve the number and quality of the recovered oocytes, or the number of in vitro produced embryos. (C) 2009 Elsevier B.V. All rights reserved.

New antitumoral agents I: In vitro anticancer activity and in vivo acute toxicity of synthetic 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one and derivatives

This paper describes a new method for the preparation of 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one 1 and its derivatives 2-5. This set of synthetic compounds exhibited high antitumoral activities regarding in vitro screening against several human tumor cell lines as lung carcinoma NCI-460, melanoma UACC-62, breast MCF-7, colon HT-29, renal 786-O, ovarian OVCAR-03 and ovarian expressing the resistance phenotype for adriamycin NCI-ADR/ RES, prostate PC-3, and leukemia K-562. Compounds were also tested against murine tumor cell line B16F10 melanoma and lymphocytic leukemia L1210 as well as to their effect toward normal macrophages. Specific activity against colon cancer cells HT-29 was observed for all tested compounds and suggests further studies with models of colon cancer. Compounds 1, 2, and 4 showed significant cytotoxic activity with IC(50) values <= 2.3 mu M for all human cancer cell lines. Intraperitoneal acute administration of compound 1 and 2 showed very low toxicity rate. (C) 2010 Elsevier Ltd. All rights reserved.

Stability and in vitro penetration study of rutin incorporated in a cosmetic emulsion through an alternative model biomembrane

Rutin is employed as antioxidant and to prevent the capillary fragility and, when incorporated in cosmetic emulsions, it must target the action site. In vitro cutaneous penetration studies through human skin is the ideal situation, however, there are difficulties to obtain and to maintain this tissue viability. Among the membrane models, shed snake skin presents itself as pure stratum corneum, providing barrier function similar to human and it is obtained without the animal sacrifice. The objectives of this research were the development and stability evaluation of a cosmetic emulsion containing rutin and propylene glycol (penetration enhancer) and the evaluation or rutin in vitro cutaneous penetration and retention from the emulsion, employing an alternative model biomembrane. Emulsion was developed with rutin and propylene glycol, both at 5.0% w/w. Active substance presented on the formulation was quantified by a validated spectrophotometric method at 361.0 nm. Rutin Rutin cutaneous penetration and retention was performed in vertical diffusion cells with shed snake skin of Crotalus durissus, as alternative model biomembrane, and distilled water and ethanol 99.5% (1:1), as receptor fluid. The experiment was conducted for six hours, at 37.0 +/- 0.5 degrees C with constant stirring of 300 rpm. Spectrophotometry at 410.0 nm, previously validated, determined the active substance after cutaneous penetration/ retention. Emulsion did not promote rutin cutaneous penetration through C. durissus skin, retaining 0.931 +/- 0.0391 mu g rutin/mg shed snake skin. The referred formulation was chemically stable for 30 days after stored at 25.0 +/- 2.0 degrees C, 5.0 +/- 0.5 degrees C and 45.0 +/- 0.5 degrees C. In conclusion, it has not been verified the active cutaneous penetration through the model biomembrane, but only its retention on the Crotalus durissus stratum corneum, condition considered stable for 30 days.

In Vitro Colonic Fermentation and Glycemic Response of Different Kinds of Unripe Banana Flour

This work aimed to study the in vitro colonic fermentation profile of unavailable carbohydrates of two different kinds of unripe banana flour and to evaluate their postprandial glycemic responses. The unripe banana mass (UBM), obtained from the cooked pulp of unripe bananas (Musa acuminata, Nanico variety), and the unripe banana starch (UBS), obtained from isolated starch of unripe banana, plantain type (Musa paradisiaca) in natura, were studied. The fermentability of the flours was evaluated by different parameters, using rat inoculum, as well as the glycemic response produced after the ingestion by healthy volunteers. The flours presented high concentration of unavailable carbohydrates, which varied in the content of resistant starch, dietary fiber and indigestible fraction (IF). The in vitro colonic fermentation of the flours was high, 98% for the UBS and 75% for the UBM when expressed by the total amount of SCFA such as acetate, butyrate and propionate in relation to lactulose. The increase in the area under the glycemic curve after ingestion of the flours was 90% lower for the UBS and 40% lower for the UBM than the increase produced after bread intake. These characteristics highlight the potential of UBM and UBS as functional ingredients. However, in vivo studies are necessary in order to evaluate the possible benefic effects of the fermentation on intestinal health.

Development and validation of HPLC method for imiquimod determination in skin penetration studies

A simple, rapid and sensitive analytical procedure for the measurement of imiquimod in skin samples after in vitro penetration studies has been developed and validated. In vitro penetration studies were carried out in Franz diffusion cells with porcine skin. Tape stripping technique was used to separate the stratum corneum (SC) from the viable epidermis and dermis. Imiquimod was extracted from skin samples using a 7:3 (v/v) methanol:acetate buffer (100 mm, pH 4.0) solution and ultrasonication. Imiquimod was analyzed by H-PLC using C(8) column and UV detection at 242 ran. The mobile phase used was acetonitrile:acetate buffer (pH 4.0, 100 mM):diethylamine (30:69.85:0.15, v/v) with flow rate 1 mL/min. Imiquimod eluted at 4.1 min and the running time was limited to 6.0 min. The procedure was linear across the following concentration ranges: 100-2500 ng/mL for both SC and tape-stripped skin and 20-800 ng/mL for receptor solution. Intra-day and inter-day accuracy and precision values were lower than 20% at the limit of quantitation. The recovery values ranged from 80 to 100%. The method is adequate to assay imiquimod from skin samples, enabling the determination of the cutaneous penetration profile of uniquimod by in vitro studies. Copyright (C) 2008 John Wiley & Sons, Ltd.

In vitro antioxidant activity and in vivo efficacy of topical formulations containing vitamin C and its derivatives studied by non-invasive methods

Background/purpose: Vitamins C and its derivatives, mainly due to their antioxidant properties, are being used in cosmetic products to protect and to reduce the signs of ageing. However, there are no studies comparing the effects of vitamin C [ascorbic acid (AA)] and its derivatives, magnesium ascorbyl phosphate (MAP) and ascorbyl tetra-isopalmitate (ATIP), when vehiculated in topical formulations, mainly using objective measurements, which are an important tool in clinical efficacy studies. Thus, the objective of this study was to determine the in vitro antioxidant activity of AA and its derivatives, MAP and ATIP, as well as their in vivo efficacy on human skin, when vehiculated in topical formulations. Methods: The study of antioxidant activity in vitro was performed with an aqueous and a lipid system. The in vivo methodology consisted of the application of these formulations on human volunteers` forearm skin and the analysis of the skin conditions after 4-week period daily applications in terms of transepidermal water loss (TEWL), stratum corneum moisture content and viscoelasticity using a Tewameter (R), Corneometer (R) and Cutometer (R), respectively. Results: In vitro experiments demonstrated that in an aqueous system, AA had the best antioxidant potential, and MAP was more effective than ATIP, whereas in the lipid system ATIP was more effective than MAP. In in vivo studies, all formulations enhanced stratum corneum moisture content after a 4-week period daily applications when compared with baseline values; however, only the formulation containing AA caused alterations in TEWL values. The formulations containing MAP caused alterations in the viscoelastic-to-elastic ratio, which suggested its action in the deeper layers of the skin. Conclusion: AA and its derivates presented an in vitro antioxidant activity but AA had the best antioxidant effect. In in vivo efficacy studies, only the formulation containing AA caused alterations in TEWL values and the formulation containing MAP caused alterations in the viscoelastic-to-elastic ratio. This way, vitamin C derivatives did not present the same effects of AA on human skin; however, MAP showed other significant effect-improving skin hydration, which is very important for the normal cutaneous metabolism and also to prevent skin alterations and early ageing.

Excised Porcine Cornea Integrity Evaluation in an in vitro Model of Iontophoretic Ocular Research

Background/Aims: It is a challenge to adapt traditional in vitro diffusion experiments to ocular tissue. Thus, the aim of this work was to present experimental evidence on the integrity of the porcine cornea, barrier function and maintenance of electrical properties for 6 h of experiment when the tissue is mounted on an inexpensive and easy-to-use in vitro model for ocular iontophoresis. Methods: A modified Franz diffusion cell containing two ports for the insertion of the electrodes and a receiving compartment that does not need gassing with carbogen was used in the studies. Corneal electron transmission microscopy images were obtained, and diffusion experiments with fluorescent markers were performed to examine the integrity of the barrier function. The preservation of the negatively charged corneal epithelium was verified by the determination of the electro-osmotic flow of a hydrophilic and non-ionized molecule. Results: The diffusion cell was able to maintain the temperature, homogenization, porcine epithelial corneal structure integrity, barrier function and electrical characteristics throughout the 6 h of permeation experiment, without requiring CO(2) gassing when the receiving chamber was filled with 25 m M of HEPES buffer solution. Conclusion: The system described here is inexpensive, easy to handle and reliable as an in vitro model for iontophoretic ocular delivery studies. Copyright (C) 2010 S. Karger AG, Basel

Analysis and application of an equilibrium model for in vitro bioassay systems with three components: Receptor, hormone and hormone-binding-protein

A simple theoretical framework is presented for bioassay studies using three component in vitro systems. An equilibrium model is used to derive equations useful for predicting changes in biological response after addition of hormone-binding-protein or as a consequence of increased hormone affinity. Sets of possible solutions for receptor occupancy and binding protein occupancy are found for typical values of receptor and binding protein affinity constants. Unique equilibrium solutions are dictated by the initial condition of total hormone concentration. According to the occupancy theory of drug action, increasing the affinity of a hormone for its receptor will result in a proportional increase in biological potency. However, the three component model predicts that the magnitude of increase in biological potency will be a small fraction of the proportional increase in affinity. With typical initial conditions a two-fold increase in hormone affinity for its receptor is predicted to result in only a 33% increase in biological response. Under the same conditions an Ii-fold increase in hormone affinity for receptor would be needed to produce a two-fold increase in biological potency. Some currently used bioassay systems may be unrecognized three component systems and gross errors in biopotency estimates will result if the effect of binding protein is not calculated. An algorithm derived from the three component model is used to predict changes in biological response after addition of binding protein to in vitro systems. The algorithm is tested by application to a published data set from an experimental study in an in vitro system (Lim et al., 1990, Endocrinology 127, 1287-1291). Predicted changes show good agreement (within 8%) with experimental observations. (C) 1998 Academic Press Limited.

Targeting of a cholecystokinin-DNA complex to pancreatic cells in vitro and in vivo

The carboxy terminal octapeptide of cholecystokinin (CCK8) is a hormone that binds high affinity receptors in a number of tissues including pancreas and pancreatic tumours. As part of our studies to develop effective gene therapy for the treatment of pancreatic cancers, we have investigated various gene delivery systems that depend on CCK8 receptor targeting. In this paper,we describe the synthesis of a CCK8-DNA complex designed to deliver foreign DNA to cholecystokinin receptor-positive cells. CCK8 was ligated to avidin and then complexed to linearis biotinylated DNA (pSV-CAT). The uptake of P-32-labelled CCK8-DNA complex by rat pancreatic acini was linear with time over 4 h with 65-70% of uptake inhibited by 100 nM CCK8. The complex appeared to be internalised since it could not be removed by acid wash. When administered intra-arterially, the complex was rapidly removed from the circulation with no evidence of targeted delivery to the pancreas, However, following a single intraperitoneal dose, the pancreas accumulated-5- 8% of the total administered complex by 24 h. These results suggest that peptide-dependent gene delivery to CCK receptor positive cells in vivo is feasible but, when administered directly into the circulation, diffusional barriers across the endothelium may limit distribution to peripheral tissues. Intraperitoneal administration therefore may be a useful alternative for targeting the pancreas.

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on hormone secretion from sheep pituitary cells in vitro

Although vasoactive intestinal polypeptide (VIP) is thought to be a prolactin releasing factor, in vivo studies on sheep suggest that it is inactive in this species. Recent studies, based primarily on the rat, suggest that the related pituitary adenylate cyclase-activating polypeptide (PACAP) is also a hypophysiotrophic factor but again in sheep, this peptide has no in vivo effects on hormone secretion despite being a potent activator of adenylate cyclase in vitro. This lack of response to either peptide in vivo in sheep could be due to the low concentration of peptide that reaches the pituitary gland following peripheral injection. In the present study we therefore adopted an alternative approach of evaluating in vitro effects of these peptides on GH, FSH, LH or prolactin secretion from dispersed sheep pituitary cells. In a time-course study, PACAP (1 mu mol/l) increased GH concentrations in the culture medium between 1 and 4 h and again at 12 h but had no effect in the 6 and 24 h incubations. Prolactin, LH and FSH were not affected by PACAP. The response to various concentrations of PACAP (1 nmol/l-1 mu mol/l) were then evaluated using a 3 h incubation. Again prolactin and LH were not affected by PACAP and there was a small increase in GH concentrations but only at high concentrations of PACAP (0.1 and 1 mu mol/l; P<0.05), PACAP also stimulated FSH secretion in cells from some animals although this effect was small, The GH response to PACAP was inhibited by PACAP(6-38), a putative PACAP antagonist; but not by (N-Ac-Tyr(1), D-Arg(2))-GHRH(1-29)-NH2, a GH-releasing hormone (GHRH) antagonist. The cAMP antagonist Rp-cAMPS was unable to block the GH response to PACAP suggesting that cAMP does not mediate the secretory response to this peptide. At incubation times from 1-24 h, VIP (1 mu mol/l) had no effects on prolactin, LH or GH secretion and, in a further experiment based on a 3 h incubation, concentrations of VIP from 1 nmol/l-1 mu mol/l were again without effect on prolactin concentrations. Interactions between PACAP and gonadotrophin releasing hormone (GnRH), GHRH and dopamine were also investigated. PACAP (1 nmol/l-1 mu mol/l) did not affect the gonadotrophin or prolactin responses to GnRH or dopamine respectively. However, at a high concentration (1 mu mol/l), PACAP inhibited the GH response to GHRH. In summary, these results show that PACAP causes a modest increase in FSH and GH secretion from sheep pituitary cells but only at concentrations of PACAP that are unlikely to be in the physiological range. The present study confirms that VIP is not a prolactin releasing factor in sheep.

In vitro effect of Aloe vera, Coriandrum sativum and Ricinus communis fractions on Leishmania infantum and on murine monocytic cells

In South America, visceral leishmaniasis is a zoonosis caused by the protozoan species Leishmania infantum (syn. L. chagasi) and is primarily transmitted through the bite of the female Lutzomyia longipalpis. Its main reservoir in urban areas is the dog. The application of control measures recommended by health agencies have not achieved significant results in reducing the incidence of human cases, and the lack of effective drugs to treat dogs resulted in the prohibition of this course of action in Brazil. Therefore, it is necessary to search new alternatives for the treatment of canine and human visceral leishmaniasis. The objectives of this study were to evaluate the in vitro effect of fractions from Aloe vera (aloe), Coriandrum sativum (coriander), and Ricinus communis (castor) on promastigotes and amastigotes of L. infantum and to analyze the toxicity against the murine monocytic cells RAW 264.7. To determine the viability of these substances on 50% parasites (IC50), we used a tetrazolium dye (MU) colorimetric assay (bromide 3-4.5-dimethylthiazol-2-yl-2,5-dephenyltetrazolium), and on amastigotes we performed an in situ ELISA. All fractions were effective against L. infantum promastigotes and did not differ from the positive control pentamidine (p > 0.05). However, the R. communis ethyl acetate and chloroform fractions, as well as the C. sativum methanol fraction, were the most effective against amastigotes and did not differ from the positive control amphotericin B (p > 0.05). The R. communis ethyl acetate fraction was the least toxic, presenting 83.5% viability of RAW 264.7 cells, which was similar to the results obtained with amphotericin B (p > 0.05). Based on these results, we intend to undertake in vivo studies with R. communis ethyl acetate fractions due the high effectiveness against amastigotes and promastigotes of L. infantum and the low cytotoxicity towards murine monocytic cells. (C) 2011 Elsevier B.V. All rights reserved.

Oral tolerance attenuates changes in in vitro lung tissue mechanics and extracellular matrix remodeling induced by chronic allergic inflammation in guinea pigs

Oral tolerance attenuates changes in in vitro lung tissue mechanics and extracellular matrix remodeling induced by chronic allergic inflammation in guinea pigs. J Appl Physiol 104: 1778-1785, 2008. First published April 3, 2008; doi:10.1152/japplphysiol.00830.2007.-Recent studies emphasize the presence of alveolar tissue inflammation in asthma. Immunotherapy has been considered a possible therapeutic strategy for asthma, and its effect on lung tissue had not been previously investigated. Measurements of lung tissue resistance and elastance were obtained before and after both ovalbumin and acetylcholine challenges. Using morphometry, we assessed eosinophil and smooth muscle cell density, as well as collagen and elastic fiber content, in lung tissue from guinea pigs with chronic pulmonary allergic inflammation. Animals received seven inhalations of ovalbumin (1-5 mg/ml; OVA group) or saline (SAL group) during 4 wk. Oral tolerance (OT) was induced by offering ad libitum ovalbumin 2% in sterile drinking water starting with the 1st inhalation (OT1 group) or after the 4th (OT2 group). The ovalbumin-exposed animals presented an increase in baseline and in postchallenge resistance and elastance related to baseline, eosinophil density, and collagen and elastic fiber content in lung tissue compared with controls. Baseline and post-ovalbumin and acetylcholine elastance and resistance, eosinophil density, and collagen and elastic fiber content were attenuated in OT1 and OT2 groups compared with the OVA group. Our results show that inducing oral tolerance attenuates lung tissue mechanics, as well as eosinophilic inflammation and extracellular matrix remodeling induced by chronic inflammation.

In vitro antifungal drug susceptibilities of dermatophytes microconidia and arthroconidia

Objectives: Arthroconidia have been considered as the primary cause of infection by dermatophytes. However, the in vitro antifungal testing evaluates the responses mainly of microconidia or hyphae, and dermatophytes in vivo often produce arthroconidia, a cellular structure presumably more resistant to antifungals. The aim of this study was to compare the in vitro susceptibility of microconidia and arthroconidia of Trichophyton rubrum, Trichophyton tonsurans and Trichophyton equinum to griseofulvin, itraconazole, terbinafine, fluconazole, amphotericin B and hygromycin B. Methods: Microconidia and arthroconidia were produced in vitro, and their susceptibility to each drug was evaluated by assessing the CLSI M38-A broth microdilution method. Results: Arthroconidia of all strains analysed appeared to be more resistant to fluconazole, griseofulvin and itraconazole than microconidia. The MIC of terbinafine was the same for microconidia and arthroconidia for all strains, and the MIC of amphotericin B for microconidia and arthroconidia was the same for isolates of T. equinum and T. tonsurans, but differed for T. rubrum. Finally, the level of resistance of microconidia for all strains towards the antibiotic hygromycin B was from 25 to 400 mg/L. Conclusions: The difference in the susceptibility between microconidia and arthroconidia depends on the drug and on the strain, and may be one of the causes of therapeutic failure. Also, the level of resistance to the antibiotic hygromycin B presented by microconidia of these isolates will allow the use of hygromycin resistance as a dominant marker in fungal transformation procedures in future studies of gene function.

Validation of ACB in vitro and in vivo as a biomagnetic method for measuring stomach contraction

Background The aim of this study was to validate a biomagnetic method (alternate current biosusceptometry, ACB) for monitoring gastric wall contractions in rats. Methods In vitro data were obtained to establish the relationship between ACB and the strain-gauge (SG) signal amplitude. In vivo experiments were performed in pentobarbital-anesthetized rats with SG and magnetic markers previously implanted under the gastric serosa or after ingestion of magnetic material. Gastric motility was quantified from the tracing amplitudes and frequency profiles obtained by Fast Fourier Transform. Key Results The correlation between in vitro signal amplitudes was strong (R = 0.989). The temporal cross-correlation coefficient between the ACB and SG signal amplitude was higher (P < 0.0001) in the postprandial (88.3 +/- 9.1 V) than in the fasting state (31.0 +/- 16.9 V). Irregular signal profiles, low contraction amplitudes, and smaller signal-to-noise ratios explained the poor correlation between techniques for fasting-state recordings. When a magnetic material was ingested, there was also strong correlation in the frequency and signal amplitude and a small phase-difference between the techniques. The contraction frequencies using ACB were 0.068 +/- 0.007 Hz (postprandial) and 0.058 +/- 0.007 Hz (fasting) (P < 0.002) and those using SG were 0.066 +/- 0.006 Hz (postprandial) and 0.059 +/- 0.008 Hz (fasting) (P < 0.005). Conclusions & Inferences In summary, ACB is reliable for monitoring gastric wall contractions using both implanted and ingested magnetic materials, and may serve as an accurate and sensitive technique for gastrointestinal motility studies.

Mechanisms of Action of Eicosapentaenoic Acid in Bladder Cancer Cells In Vitro: Alterations in Mitochondrial Metabolism, Reactive Oxygen Species Generation and Apoptosis Induction

Purpose: Eicosapentaenoic acid has been tested in bladder cancer as a synergistic cytotoxic agent in the form of meglumine-eicosapentaenoic acid, although its mechanism of action is poorly understood in this cancer. The current study analyzed the mechanisms by which eicosapentaenoic acid alters T24/83 human bladder cancer metabolism in vitro. Materials and Methods: T24/83 human bladder cancer cells were exposed to eicosapentaenoic acid for 6 to 24 hours in vitro and incorporation profiles were determined. Effects on membrane phospholipid incorporation, energy metabolism, mitochondrial activity, cell proliferation and apoptosis were analyzed Reactive oxygen species and lipid peroxide production were also determined. Results: Eicosapentaenoic acid was readily incorporated into membrane phospholipids with a considerable amount present in mitochondrial cardiolipin. Energy metabolism was significantly altered by eicosapentaenoic acid, accompanied by decreased mitochondrial membrane potential, and increased lipid peroxide and reactive oxygen species generation. Subsequently caspase-3 activation and apoptosis were detected in eicosapentaenoic acid exposed cells, leading to decreased cell numbers. Conclusions: These findings confirm that eicosapentaenoic acid is a potent cytotoxic agent in bladder cancer cells and provide important insight into the mechanisms by which eicosapentaenoic acid causes these changes. The changes in membrane composition that can occur with eicosapentaenoic acid likely contribute to the enhanced drug cytotoxicity reported previously in meglumine-eicosapentaenoic acid/epirubicin/mitomycin studies. Dietary manipulation of the cardiolipin fatty acid composition may provide an additional method for stimulating cell death in bladder cancer. In vivo studies using intravesical and dietary manipulation of fatty acid metabolism in bladder cancer merit further attention.