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PubMed2006

Binding of boar spermatozoa to oviductal epithelium in vitro in relation to sperm morphology and storage time.

In vitro short-term storage of boar semen for up to 72 h before insemination negatively affects fertility, but this often remains undetected during semen quality assessment. One important sperm function is the ability to form the functional sperm reservoir in the oviduct. In the present study, we used the modified oviductal explant assay to study sperm binding to oviductal epithelium in vitro in diluted boar semen stored for 24 or 72 h. First, we determined the kinetics of in vitro sperm binding to oviductal epithelium in relation to co-incubation time of sperm and oviductal tissue pieces. Then, we studied how the binding of sperm to oviductal epithelium was affected by in vitro semen storage and by differences among individual boars. Sperm binding after different incubation times was significantly higher when semen was stored 24 h than after 72-h storage (P < 0.05), and peaked at 30-90 min of incubation. Sperm binding differed between boars (n = 44), and was negatively correlated to the percentage of sperm with cytoplasmic droplets (R = -0.51, P < 0.001). There were no significant changes in motility, acrosome integrity and propidium iodide stainability during the 72-h storage period. However, sperm-binding indices were significantly lower after 72 h in vitro storage than after 24-h storage in sperm from boars with normal semen quality (P < 0.05); in contrast, the binding capacity of sperm from boars with higher percentages of morphologically altered sperm remained at a low level. The sperm-binding capacity of sperm from four of the five boars with known subfertility was lower than the mean binding index minus one standard deviation of the boar population studied here. It is concluded that changes in the plasma membrane associated with in vitro ageing reduce the ability of stored boar sperm to bind to the oviductal epithelium. This study shows the potential of sperm-oviduct binding as a tool to assess both male fertility and changes in sperm function associated with in vitro ageing.

Acrosome↗
PubMed2006

Generation of embryonic stem cell lines from mouse blastocysts developed in vivo and in vitro: relation to Oct-4 expression.

Embryonic stem (ES) cells are the source of all embryonic germ layer tissues. Oct-4 is essential for their pluripotency. Since in vitro culture may influence Oct-4 expression, we investigated to what extent blastocysts cultured in vitro from the zygote stage are capable of expressing Oct-4 and generating ES cell lines. We compared in vivo with in vitro derived blastocysts from B6D2 mice with regard to Oct-4 expression in inner cell mass (ICM) outgrowths and blastocysts. ES cells were characterized by immunostaining for alkaline phosphatase (ALP), stage-specific embryonic antigen-1 (SSEA-1) and Oct-4. Embryoid bodies were made to evaluate the ES cells' differentiation potential. ICM outgrowths were immunostained for Oct-4 after 6 days in culture. A quantitative real-time PCR assay was performed on individual blastocysts. Of the in vitro derived blastocysts, 17% gave rise to ES cells vs 38% of the in vivo blastocysts. Six-day old outgrowths from in vivo developed blastocysts expressed Oct-4 in 55% of the cases vs 31% of the in vitro derived blastocysts. The amount of Oct-4 mRNA was significantly higher for freshly collected in vivo blastocysts compared to in vitro cultured blastocysts. In vitro cultured mouse blastocysts retain the capacity to express Oct-4 and to generate ES cells, be it to a lower level than in vivo blastocysts.

Actins↗
PubMed1993

In-vitro effects of insulin-like growth factor-I on gill Na+,K(+)-ATPase in coho salmon, Oncorhynchus kisutch.

The effect of ovine GH (oGH) in vivo and recombinant bovine insulin-like growth factor-I (rbIGF-I) in vitro on gill Na+,K(+)-ATPase activity was investigated in two seasonal experiments conducted during the parr-smolt transformation period of coho salmon. In 1991, when fish were held under a photoperiod of 12 h light : 12 h darkness, the stimulatory effect of oGH (1 microgram/g) on gill Na+,K(+)-ATPase in vivo decreased at the time of expected parr-smolt transformation. Gill Na+,K(+)-ATPase from control fish was insensitive to rbIGF-I in vitro from February to June, whereas GH treatment induced sensitivity to rbIGF-I (100-1000 micrograms/l) in vitro in February and March, but not later in development. In 1992, when fish were held under natural conditions, oGH (4 micrograms/g) stimulated gill Na+,K(+)-ATPase in vivo from February to July. There was, however, of pronounced developmental change in sensitivity of gill Na+,K(+)-ATPase to rbIGF-I in vitro. In February, gills from control fish were insensitive, but oGH treatment in vivo induced sensitivity to rbIGF-I in vitro (100-1000 micrograms/l). In April and May, control fish were sensitive to rbIGF-I in vitro. This sensitivity was not further potentiated by oGH treatment in vivo. In June, gills from control or oGH-treated fish were not sensitive to rbIGF-I in vitro, but in July exogenous oGH again induced gill tissue sensitivity to rbIGF-I at 1000 micrograms/l. Both studies showed that rbIGF-I stimulates gill Na(+),K(+)-ATPase directly; an ability that may depend on priming by endogenous or exogenous GH. This supports the role of IGF-I as an endocrine mediator for GH action during parr-smolt transformation.

Animals↗
PubMed2006

The activin-follistatin system and in vitro early follicle development in goats.

The aim of the present study was to investigate the effects of activin-A and follistatin on in vitro primordial and primary follicle development in goats. To study primordial follicle development (experiment 1), pieces of ovarian cortex were cultured in vitro for 5 days in minimal essential medium (MEM) supplemented with activin-A (0, 10 or 100 ng/ml), follistatin (0, 10 or 100 ng/ml) or combinations of the two. After culture, the numbers of primordial follicles and more advanced follicle stages were calculated and compared with those in non-cultured tissue. Protein and mRNA expression of activin-A, follistatin, Kit ligand (KL), growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) in non-cultured and cultured follicles were studied by immunohistochemistry and PCR. To evaluate primary follicle growth (experiment 2), freshly isolated follicles were cultured for 6 days in MEM plus 100 ng/ml activin-A, 100 ng/ml follistatin or 100 ng/ml activin-A plus 200 ng/ml follistatin. Morphology, follicle and oocyte diameters in cultured tissue and isolated follicles before and after culture were assessed. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) reactions were performed to study DNA fragmentation in follicles. In experiment 1, it was found that goat primordial follicles were activated to develop into more advanced stages, i.e. intermediate and primary follicles, during in vitro culture, but neither activin-A nor follistatin affected the number of primordial follicles that entered the growth phase. Activin-A treatment enhanced the number of morphologically normal follicles and stimulated their growth during cortical tissue culture. The effects were, however, not counteracted by follistatin. The follicles in cultured goat tissue maintained their expression of proteins and mRNA for activin-A, follistatin, KL, GDF-9 and BMP-15. Fewer than 30% of the atretic follicles in cultured cortical tissue had TUNEL-positive (oocyte or granulosa) cells. Activin-A did not affect the occurrence of TUNEL-positive cells in follicles within cortical tissue. In experiment 2, addition of activin-A to cultured isolated primary follicles significantly stimulated their growth, the effect being counteracted by follistatin. Absence of such a neutralizing effect of follistatin in the cultures with ovarian cortical tissue can be due to lower dose of follistatin used and incomplete blockage of activin in these experiments. In contrast to cortical enclosed atretic follicles, all atretic follicles that had arisen in cultures with isolated primary follicles had TUNEL-positive cells, which points to differences between isolated and ovarian tissue-enclosed follicles with regard to the followed pathways leading to their degeneration. In summary, this in vitro study has demonstrated that cultured goat primordial follicles are activated to grow and develop into intermediate and primary follicles. During in vitro culture, the follicles maintain their ability to express activin-A, follistatin, KL, GDF-9 and BMP-15. The in vitro growth and survival of activated follicles enclosed in cortical tissue and the in vitro growth of isolated primary follicles are stimulated by activin-A.

Activins↗
PubMed2006

Effects of nonstructural carbohydrates and protein sources on intake, apparent total tract digestibility, and ruminal metabolism in vivo and in vitro with high-concentrate beef cattle diets.

To investigate the effects of synchronizing nonstructural carbohydrate (NSC) and protein degradation on intake and rumen microbial fermentation, four ruminally fistulated Holstein heifers (BW = 132.3 +/- 1.61 kg) fed high-concentrate diets were assigned to a 4 x 4 Latin square design with a 2 x 2 factorial arrangement of treatments studied in vivo and in vitro with a dual-flow continuous culture system. Two NSC sources (barley and corn) and 2 protein sources [soybean meal (SBM) and sunflower meal (SFM)] differing in their rate and extent of ruminal degradation were combined resulting in a synchronized rapid fermentation diet (barley-SFM), a synchronized slow fermentation diet (corn-SBM), and 2 unsynchronized diets with a rapidly and a slowly fermenting component (barley-SBM, and corn-SFM). In vitro, the fermentation profile was studied at a constant pH of 6.2, and at a variable pH with 12 h at pH 6.4 and 12 h at pH 5.8. Synchronization tended to result in greater true OM digestion (P = 0.072), VFA concentration (P = 0.067), and microbial N flow (P = 0.092) in vitro, but had no effects on in vivo fermentation pattern or on apparent total tract digestibility. The NSC source affected the efficiency of microbial protein synthesis in vitro, tending to be greater (P = 0.07) for barley-based diets, and in vivo, the NSC source tended to affect intake. Dry matter and OM intake tended to be greater (P > or = 0.06) for corn- than barley-based diets. Ammonia N concentration was lower in vitro (P = 0.006) and tended to be lower in vivo (P = 0.07) for corn- than barley-based diets. In vitro, pH could be reduced from 6.4 to 5.8 for 12 h/d without any effect on ruminal fermentation or microbial protein synthesis. In summary, ruminal synchronization seemed to have positive effects on in vitro fermentation, but in vivo recycling of endogenous N or intake differences could compensate for these effects.

Animal Feed↗
PubMed2006

Influence of the amount of dietary fiber on the available energy from hindgut fermentation in growing pigs: use of cannulated pigs and in vitro fermentation.

Available energy from hindgut fermentation to pigs fed various amounts of dietary fiber was investigated using an in vivo-in vitro methodology. Six growing pigs fitted with a simple T-shaped cannula at the terminal ileum, and following a Latin-square design, were fed 3 diets differing in the content of non-starch polysaccharides (NSP): a low fiber diet (LFD, 77 g/kg of DM), a standard fiber diet (SFD, 160 g/kg of DM), and a high fiber diet (HFD, 240 g/kg of DM). After adaptation to the diet for 10 d, samples from feces and ileum were collected and analyzed for DM, energy, NSP, and chromic oxide; feces were also analyzed for short chain fatty acids (SCFA). Freeze-dried ileal samples (10 g/L) were fermented in vitro in a fecal slurry consisting of an anaerobic mineral salt medium and feces (50 g/L) from cannulated pigs fed the same diets. Available energy was calculated from the amount of SCFA produced in vitro after 48 h of incubation. Nonstarch polysaccharide content in the fermented material was measured to assess the in vitro degradation of this fraction. Increasing dietary NSP from 77 to 240 g/kg of feed DM increased (P < 0.001) ileal flow from 199 to 468 g/kg of feed, leading to a reduction in the energy digested at the terminal ileum, from 15 to 11 MJ/kg of feed DM and an increment in energy digested in the hindgut, from 1.6 to 3.5 MJ/kg of feed DM. Total in vitro production of SCFA/kg of feed DM was dependent on the amount of ileal substrate available for fermentation; that is, increased concentrations of NSP in the diet led to an increase in the SCFA that may be available to the animal (P < 0.001). The molar ratio of SCFA produced in vitro was affected by diet; the high fiber diet showed the greatest (P = 0.004) proportion of acetic acid, and the low fiber diet showed a tendency (P = 0.081) to an increased butyric acid proportion compared with the other 2 diets. Net disappearance of NSP during fermentation in vivo and in vitro were compared and showed a close relationship (P < 0.001, slope = 0.906, r = 0.960). In our experimental conditions, available energy as SCFA to the animal from hindgut fermentation increased with the concentration of dietary NSP (P < 0.001) and provided between 7.1 and 17.6% of the total available energy.

Animal Feed↗
PubMed1999

Comparison of in vivo and in vitro tests of resistance in patients treated with chloroquine in Yaoundé, Cameroon.

The usefulness of an isotopic in vitro assay in the field was evaluated by comparing its results with the therapeutic response determined by the simplified WHO in vivo test in symptomatic Cameroonian patients treated with chloroquine. Of the 117 enrolled patients, 102 (87%) completed the 14-day follow-up, and 95 isolates obtained from these patients (46 children, 49 adults) yielded an interpretable in vitro test. A total of 57 of 95 patients (60%; 28 children and 29 adults) had an adequate clinical response with negative smears (n = 46) or with an asymptomatic parasitaemia (n = 11) on day 7 and/or day 14. The geometric mean 50% inhibitory concentration of the isolates obtained from these patients was 63.3 nmol/l. Late and early treatment failure was observed in 29 (30.5%) and 9 (9.5%) patients, respectively. The geometric mean 50% inhibitory concentrations of the corresponding isolates were 173 nmol/l and 302 nmol/l. Among the patients responding with late and early treatment failure, five isolates and one isolate, respectively, yielded a discordant result (in vivo resistance and in vitro sensitivity). The sensitivity, specificity, and predictive value of the in vitro test to detect chloroquine-sensitive cases was 67%, 84% and 86%, respectively. There was moderate concordance between the in vitro and in vivo tests (kappa value = 0.48). The in vitro assay agrees relatively well with the therapeutic response and excludes several host factors that influence the results of the in vivo test. However, in view of some discordant results, the in vitro test cannot substitute for in vivo data on therapeutic efficacy. The only reliable definition of "resistance" in malaria parasites is based on clinical and parasitological response in symptomatic patients, and the in vivo test provides the standard method to determine drug sensitivity or resistance as well as to guide national drug policies.

Adolescent↗
PubMed1999

Characterization of cultured bladder smooth muscle cells: assessment of in vitro contractility.

PURPOSE: The contractile properties of in vitro cultured bladder smooth muscle cells (SMC) are unknown. This study characterized the in vitro contractile response of human and rat bladder SMC to several pharmacological agonists known to induce in vivo contraction of intact bladder muscle. MATERIALS AND METHODS: Human and rat bladder SMC were seeded separately within attached collagen lattices. Contractility of SMC was analyzed by measuring alterations in lattice diameter after exposure and release to the following contractile agonists: carbachol (10(-7)-10(-3) microM), calcium-ionophore (10 microM), lysophosphatidic acid (LPA) (1 microM), endothelin (0.1 microM), KCl (3.33 mmicroM) angiotensin II (10 microM), and serotonin (100 microM). Results were recorded as a mean reduction of the lattice diameter. In addition, immunohistochemical analysis for phenotypic markers of smooth muscle cell differentiation was performed on bladder SMC cultured within collagen lattices. Human palmar fascia fibroblasts, which have been previously well characterized by in vitro contractility and immunohistochemistry, were tested in parallel and used as controls for all the above experiments. RESULTS: Human SMC had significant contractile responses to calcium-ionophore (31% +/- 4 relative percent contraction, p <0.05), LPA (34% +/- 4, p <0.05), and endothelin (37 +/- 5%, p <05). There was no significant contraction in response to carbachol, angiotensin II, KCl, or serotonin. Rat bladder SMC had a similar contractile response but did not contract in response to endothelin. In contrast to human and rat bladder SMC, fibroblasts did not contract to calcium-ionophore. CONCLUSIONS: In vitro cultured bladder SMC demonstrate loss of contractile response to normal in vivo pharmacologic agonists. Both human and rat bladder SMC can be distinguished in vitro from fibroblasts based upon their lack of contractile response to calcium- ionophore. These results demonstrate the ability to further characterize cultured bladder SMC with in vitro contractility. Further characterization is essential if we are to advance our understanding of the clinical applicability of in vitro studies utilizing cultured bladder SMC.

Animals↗
PubMed2000

Expression and splicing of FGF receptor mRNAs during APRE-19 cell differentiation in vitro.

PURPOSE: The expression and alternative splicing of the four FGF receptor (FGFR) mRNAs are regulated in a developmental- and tissue-specific fashion. Capability of differentiation in vitro of the retinal pigment epithelial cell line ARPE-19 has been previously demonstrated. In this study, the hypothesis that FGF receptor gene expression and the alternative splicing of the FGFR1 mRNA is regulated as a function of ARPE-19 differentiation in vitro was tested. METHODS: ARPE-19 cells were plated at sparse or confluent densities and maintained in culture up to 14 months. The expression of FGF receptors and the ratio of the FGFR1beta to FGFR1alpha splice variants of the FGFR1 transcript were quantified by a published PCR technique. Two in vivo samples of human RPE served as controls. RESULTS: Sparse cultures of ARPE-19 cells predominantly express FGFR1. When these cultures are allowed to differentiate, FGFR2 is also expressed. Samples of mRNA from RPE cells in vivo exhibit FGFR1 and FGFR2 expression as well as FGFR3 expression, a form that is minimally apparent in vitro. The ratio of the FGFR1beta to FGFR1alpha splice variant decreases as a function of cell differentiation in vitro and approaches the ratio observed in human RPE cells in vivo. Stimulation of cultures in vitro with FGF2 as a prototypical differentiation agent does not regulate the ratio of the FGFR1beta to FGFR1alpha splice variant. CONCLUSIONS: Differentiation of the ARPE-19 cell line in vitro recapitulates many but not all the in vivo patterns of FGFR expression and splicing. This in vitro system may be useful for selected studies on how cellular differentiation regulates FGF receptor gene expression and splicing.

Alternative Splicing↗
PubMed1996

Feeding of Hard Ticks In Vitro: New Perspectives for Rearing and for the Identification of Systemic Acaricides.

Experiments on in vitro feeding of the hard ticks Ixodes ricinus, Boophilus microplus, Amblyomma variegatum and Amblyomma hebraeum are described. An in vitro test for systemic acaricides is presented. Work started with a feeding chamber continuously supplied with new blood. It proved too susceptible to fungal contamination and not efficient enough. Simple feeding units made of honey jars with an inner glass tube were far more practical. Reinforced silicone membrances or baudruche membrances impregnated with glue were stretched across the bottom end of the tube. Weekly open-collected bovine blood from the slaughter house, where it is considered as discard, served as nutrient and changed 1-3 times a day. The feeding jars were held in a waterbath (38 degrees C). The blood contained microbicides. Attachment of ticks on the membranes was induced with combinations of host hair, conspecific tick faeces, bovine hair extract and synthetic aggregation attachment pheromone mixtures (Amblyomma). Larvae of the one-host cattle tick A. microplus were bred in vitro through the nymphal to the unfed adult stage. Females gained half of the natural body mass and laid eggs. The life-cycle of A. hebraeum was completed in vitro by consecutive feeding of all life-stages (larvae, nymphs, adults) on silicone membranes. In adult B. microplus, A. variegatum and A. hebraeum the reproductive capacity was strongly impaired, possibly in consequence of the low quality of the blood. A suboptimal in vitro nutrition was more fatal for the fertility than for the moult of ticks, as demonstrated by the good results for larvae and nymphs, particularly those of A. hebraeum. The toxicity of the systemic acaricide Ivermectin(R) for nymphs of A. hebraeum was confirmed using the in vitro feeding method. This method offers new in vitro possibilities for tick rearing as well as for tests with systemic acaricides, antifeedants and repellents. It allows experiments on transmission of pathogens and with radioactive markers.

Journal Article↗
PubMed2001

Prediction of human hepatic clearance from in vivo animal experiments and in vitro metabolic studies with liver microsomes from animals and humans.

We investigated the quantitative prediction of human hepatic metabolic clearance from in vitro experiments focusing on cytochrome P450 metabolism with eight model compounds, FK1052, FK480, zolpidem, omeprazole, nicardipine, nilvadipine, diazepam, and diltiazem. For the compounds, in vivo human hepatic extraction ratios ranged widely from 0.03 to 0.87. In vitro and in vivo hepatic intrinsic clearance (CL(int)) values for each compound were measured and calculated in rats and/or dogs and humans. CL(int,in vitro) was determined from a substrate disappearance rate at 1 microM in hepatic microsomes, which was a useful method. CL(int,in vivo) was calculated from in vivo pharmacokinetic data using three frequent mathematical models (the well stirred, parallel-tube, and dispersion models). The human scaling factor values (CL(int,in vivo)/CL(int,in vitro)) showed marked difference among the model compounds (0.3-26.6-fold). On the other hand, most of the animal scaling factors were within 2-fold of the values in humans, suggesting that scaling factor values were similar in the different animal species. When human CL(int,in vitro) values were compared with the actual CL(int,in vivo), correlation was not necessarily good. By contrast, using human CL(int,in vitro) corrected with the rat and/or dog scaling factors yielded better predictions of CL(int,in vivo) that were mostly within 2-fold of the actual values. Furthermore, successful predictions of human CL(oral) and hepatic extraction ratio (E(H)) were obtained by use of the human CL(int,in vitro) corrected with animal scaling factors. The new variant method is a simple one, incorporating additional information from animal studies and providing a more reliable prediction of human hepatic clearance.

Animals↗
PubMed1999

[In vitro cleavage action of c-erbB-2 oncogene-specific ribozyme].

OBJECTIVE: To construct in vitro transcription vectors of genes of c-erbB-2 specific ribozyme and its substrate and to probe its in vitro cleavage action. METHODS: According to the computer design, a specific restriction site EcoR V was added to the 3' end of the ribozyme gene (RZ1). Then, the RZ1 gene and its substrate gene were cloned into the in vitro transcription vector pGEM3Zf(-) separately. The recombinants containing RZ1 gene were first screened by agrose ge1 electrophoresis through EcoR V digestion and was identified by automatic sequencing. The products of in vitro transcription were labeled with 32P. In vitro cleavage reaction was performed at 37 degrees C for 1 h under the presence of Mg++. The cleavage product was analyzed by polyacrilamide gel electrophoresis. After autoradiography, the cleavage rate was counted by image analysis. RESULTS: The recombinants containing the RZ1 gene were successfully selected by the EcoR V digestion and were designated as pGM3Z-RZ1. The automatic sequence analysis proved that the synthesized RZ1 gene was correct. The target gene was also cloned into the pGEM3Zf(-) under SP6 promoter. After in vitro transcription, the cleavage reaction was shown to have cut off 79.3% target RNA in 1 h. CONCLUSION: The c-erbB-2 oncogene-specific ribozyme has a high activity in vitro. It lays a foundation for the study of the therapeutic use of ribozyme in gene therapy of cancer.

Genes, erbB-2↗
PubMed2000

[Ultrastructure differences of in vitro cotton fiber and native cotton fiber].

Native cotton fiber and in vitro cotton fiber that was induced from cotton ovule callus by suspension culture were observed using transmission electron microscope and scanning electron microscope. The ovule surface on the first day preanthesis was quite smooth. On the anthesis, it had a lot of protuberances. Two kinds of callus, smooth and rough were found. The microfibrils of callus was vertical to the cell long axis and they changed their orientations with the development of the in vitro cotton fiber: from the vertical to shallow spiral and then to parallel to the cell long axis. So was the native cotton fiber. It suggests that in vitro cotton fiber and native cotton fiber have similar development process. Compared with the ovule surface cell, most callus cells had smaller nuclear. During the development of the fiber, the plasm of native cotton fiber was denser than that of in vitro fiber, and it has more cellular organ than in vitro fiber. The cell wall of native cotton fiber was thicker and denser than that of the in vitro cotton fiber too. It suggests that the physiological activity of in vitro cotton fiber was less active than native cotton fiber.

Cotton Fiber↗
PubMedDate not supplied

In vitro diagnostic evaluation of patients with inhalant allergies: summary of probability outcomes comparing results of CLA- and CAP-specific immunoglobulin E test systems.

For the diagnosis of allergy, presence of allergen-specific immunoglobulin E (IgE) usually is established either by allergen skin tests or by in vitro allergen-specific IgE measurements. However, in vitro assays of specific IgE often are modified as manufacturers improve allergens or change reagents to optimize test performance, affecting the diagnostic performance of in vitro allergen-specific IgE assays. This investigation compares the diagnostic outcomes of the Hitachi Chemical Diagnostics chemiluminescent assay (CLA) and Pharmacia, capsulated hydrophilic carrier polymer (CAP) in vitro allergen-specific IgE test methods in patients with inhalant allergy to a panel of selected allergens. Sera were obtained from 60 consecutive patients who had a clinical history suggesting inhalant allergy and were evaluated by allergen skin-prick test (SPT). Only patients with clinical findings of allergic asthma or rhinoconjunctivitis were included. Sera from patients with at least one positive SPT, which clinically correlated with the case history, were used for specific IgE measurements. Sensitivity and specificity were defined as conditional probabilities describing performances of the CAP system and the CLA system in reference to a standard composed of a combination of allergen-specific symptoms and a positive SPT. A test concordance of 79% was found between the CLA and CAP test results with a correlation coefficient of 0.8. Allergen-specific IgE assay sensitivity of the CLA and CAP systems was similar and allergen dependent, ranging from 67 to 100%. Assay specificity ranged from 39 to 86% for the CLA system and from 36 to 81% for the CAP system. When comparing the specific IgE results with allergen SPTs, 75% (+/- 3%) of CLApositive patients had a positive SPT, and 92% (+/- 4%) of CAPpositive patients had a positive SPT. Eighty-four percent (+/- 4%) of CLAnegative patients had a negative SPT, whereas 69% (+/- 5%) of CAPnegative patients had a negative SPT. The overall concordance between skin tests and in vitro tests was 76% for CLA and 67% for CAP. CLA and CAP score values showed good correlation and both tests may be useful when skin tests cannot be performed to identify subjects with IgE-mediated allergy. The CLA and CAP assays for allergen-specific IgE may be useful as part of an initial allergy evaluation because of the high negative predictive value of negative test results. For the majority of allergens the sensitivity was high. However, the specificity of both in vitro tests was low, indicating that positive in vitro test results should be evaluated carefully in conjunction with clinical symptoms and allergen-specific skin tests to determine the clinical relevance of the allergen sensitization.

Adult↗
PubMedDate not supplied

Preparation and in vitro transfection efficiency of chitosan microspheres containing plasmid DNA:poly(L-lysine) complexes.

PURPOSE: Studies on DNA complexes with cationic polymers are prompted by the search for nonviral DNA carriers for gene therapy. Among them, poly(L-Lysine) (PLL) has been extensively studied. On the other hand, these systems deliver DNA as a bolus without long-term release. The aims of this study were to encapsulate plasmid DNA:poly(L-lysine) (pDNA:PLL) complexes into chitosan microspheres as an alternative to the PLL based gene delivery and investigate its in vitro release and transfection characteristics as well as plasmid DNA integrity and stability against serum and DNase I challenge. METHODS: pUC18 plasmid DNA that encoded beta-galactosidase was used as a model. The microspheres were prepared by complex coacervation method and the release and in vitro transfection properties were investigated. pDNA:PLL complexes were prepared at two different mass ratios. In vitro release studies were performed at 37 +/- 0.5 degrees C and drug release was monitored both spectrophotometrically and fluorometrically. Structural integrity of the pDNA:PLL complexes were determined by Southern blotting analysis. Protective effect of encapsulation of pDNA:PLL complexes against DNase I and serum treatment were also studied. In vitro transfection studies were performed by using 3T3 cell line. RESULTS: According to our in vitro release data, the mass ratio of pDNA:PLL significantly affected the release of pDNA:PLL complexes from chitosan microspheres, and the structure of the plasmid DNA did not change during the experiments. pDNA:PLL-loaded chitosan microspheres indicated high stability against fetal bovine serum and DNase I treatment for a week. In vitro transfection data showed that pDNA:PLL-loaded chitosan microspheres could be effectively transfected 3T3 cells in vitro. CONCLUSION: As a conclusion, pDNA:PLL complexes could be encapsulated into chitosan microspheres with maintaining their structural and functional integrity and this system may be a good alternative for polycation based gene carriers.

3T3 Cells↗
PubMedDate not supplied

In situ and in vitro effects of bleaching with carbamide peroxide on human enamel.

This study evaluated in vitro and in situ the potential adverse effects of 10% carbamide peroxide on human enamel using microhardness, calcium loss and surface morphology analysis. Twenty-four enamel slices (4 mm2) were obtained from recently extracted premolars. The specimens were polished under water-cooling down to 1,200-grade sandpaper. After initial microhardness readings (100 g), the specimens were randomly divided into two groups for in situ and in vitro conditions. The specimens were covered with 10% carbamide peroxide for eight hours. After removing the bleaching gel, the in vitro specimens were stored in deionized water and the in situ specimens, included in an intra-oral appliance, were placed in the oral cavity of four volunteers. These cycling sequences took place for 14 days. Upon conclusion of the bleaching treatment, new microhardness readings were performed on all specimens. Calcium dosage was assessed from the bleaching gel collected after initial exposure on day one, then from gel collected between days two and seven and gel collected between day eight and 14 using an atomic absorption spectrophotometer. Surface morphology was observed from two non-treated control specimens and two specimens of each experimental bleached group under SEM evaluation. Statistical analysis (ANOVA and Tukey tests) disclosed that specimens bleached in situ showed similar microhardness to unbleached specimens and had statistically higher (p < 0.01) hardness than in vitro bleached specimens. The loss of calcium in the in vitro situation at 14 days was 2.5 times higher than the in situ condition. SEM micrographs demonstrated that surface alterations were more pronounced in the in vitro condition. The adverse effects of carbamide peroxide on enamel were evident in specimens bleached in vitro but were not seen in situ. The presence of saliva could prevent the demineralizing effect of bleaching gel in situ.

Analysis of Variance↗
PubMed2004

Cultivation and characterization of a bovine in vitro model of the cornea.

The aim of this study was to develop an in vitro model of the cornea of bovine cells, to characterise the model by histochemical methods and to investigate permeation of ophthalmic drugs through the model. As in the in vivo situation, an in vitro model of the cornea should consist of all three different types of cells. In the current study, the construction of the in vitro cornea was performed using cells prepared from primary cultures. To investigate the state of the cells in the cultures, growth curves were established. Immunocytochemical determination of keratin and vimentin was performed for all three isolated and sub-cultivated cell types of the bovine cornea. To further simulate the in vivo conditions, corneal epithelial cells were seeded onto the collagen-gel base containing the stromal cells with an underlying sheet of endothelium. Permeation experiments were performed with pilocarpine hydrochloride and timolol hydrogen maleate as model drugs and excised bovine cornea and the in vitro cornea as permeation barriers. The immunohistochemical investigations show that excised bovine cornea and the in vitro model of the cornea are comparable with respect to the expression of keratin K3, indicating that the primarily isolated cells correspond to the different cell types of the cornea. Culturing of the epithelial cells on the complex basis has led to the formation of a corneal epithelium with several layers, closely resembling the morphology of the in vivo epithelium. Although the permeation rates of the drug through the in vitro cornea were always higher, the sequence in which the drugs permeate through the two types of barriers was the same. The drug permeation through the in vitro cornea may therefore be a useful predictive tool to estimate the permeability coefficients of drugs through excised cornea.

Adrenergic beta-Antagonists↗
PubMed2006

[Growth differentiation factor-9 gene expression in in vitro cultured oocytes in mice].

OBJECTIVE: To explore the relation between oocyte maturation and growth differentiation factor-9 (GDF-9) gene expression. METHODS: Ovariectomy was performed in 50 Kunming female mice of 10 days old, and the preantral follicles were isolated from the ovaries and cultured in medium drops for 12 days. Oocytes and somatic cells were mechanically isolated. The oocytes cultured in vitro for 2, 4, 6, 8, 10, and 12 days constituted the in vitro cultured group and the oocytes obtained from female mice of 12, 14, 16, 18, 20, and 22 days old served as the in vivo group. Semi-quantitative RT-PCR and agar gel electrophoresis were performed to quantify GDF-9 gene expression in each oocyte. RESULTS: Follicle survival, antrum formation and maturation rate was 89.5%, 51.8% and 56.6% in the in vitro cultured follicles, respectively. GDF-9 gene expression on days 2, 4, 6, 8, 10, and 12 in in vitro cultured oocytes was 0.83-/+0.08, 0.52-/+0.09, 0.45-/+0.13, 0.49-/+0.09, 0.49-/+0.09, and 0.68-/+0.08, respectively; GDF-9 gene expression in in vivo grown oocytes of 12, 14, 16, 18, 20, and 22 days were 0.64-/+0.35, 0.48-/+0.10, 0.52-/+0.10, 0.66-/+0.08, 0.72-/+0.09, and 0.91-/+0.11, respectively. Between days 8 and 12, GDF-9 gene expression in in vitro cultured oocyte was significantly lower than that in in vivo grown oocytes (P<0.05). CONCLUSION: MII oocytes can be obtained from in vitro culture of the preantral follicles. GDF-9 gene expression in the oocytes varies with their growth stages. Between days 8 and 12 of in vitro culture, GDF-9 gene expression in the cultured oocytes is different from that in in vivo grown oocytes.

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