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PubMed1998

Ex vivo and in vitro testis and ovary explants: utility for identifying steroid biosynthesis inhibitors and comparison to a Tier I screening battery.

Testis and ovary explants have been proposed as in vitro screens for identifying potential inhibitors of steroid biosynthesis. The goals of the current study were to optimize the conditions of the two assays, to characterize these assays using several compounds with well-defined endocrine activity, and to compare the responses from the explant assays with an in vivo male battery currently undergoing validation using the Crl:CD BR rat in order to evaluate their utility as test systems for screening unknown compounds for possible steroid biosynthesis inhibition activity. There were two components to the testis/ovary assays: ex vivo and in vitro. The ex vivo component used testes/ovaries from animals dosed with the test compounds in vivo, and the in vitro component used testes/ovaries from control animals. For the testis assays, decapsulated testis explants (50 mg) were placed into glass scintillation vials, +/-1.0 IU/ml hCG for 3 h in a shaking water bath (34 degrees C). Following the incubation period, medium was removed, centrifuged, and frozen until assayed for hormone concentrations. A similar procedure was used for the ovary explant assay except that each ovary was incubated separately. The testis explants were evaluated using the following compounds: ketoconazole (KETO), a testosterone biosynthesis inhibitor; aminoglutethimide (AG) (only in vitro) and anastrozole (ANA), aromatase inhibitors; finasteride (FIN), a 5alpha-reductase inhibitor; 17beta-estradiol (17beta-E2), an estrogen receptor agonist; flutamide (FLUT), an androgen receptor antagonist; ICI-182,780 (ICI), an estrogen receptor antagonist; haloperidol (HALO), a D2 receptor antagonist; and reserpine (RES), a dopamine depletor. In the ovary assay, AG (only in vitro), ANA, ICI, and HALO (only in vitro) were evaluated. Addition of fetal calf serum to the medium allowed measurement of estradiol (E2) in the testis assay, but production was not inhibited by ANA or AG. In the ovary explant assay, only AG was identified as inhibiting E2 production in vitro. Hence, both the testis and ovary explant assays appear to have limited utility for detecting aromatase inhibitors. Screening of these nine diverse endocrine-active compounds resulted in all of them being identified as altering the endocrine system when assessed by ex vivo and in vitro testis explants. Using only the in vitro assessment with the criteria of steroid biosynthesis inhibition, four of nine compounds were correctly identified in the testis explant assay (17beta-E2, KETO, FLUT, and HALO). The predictability of both the in vitro and ex vivo ovary assay was 50%, suggesting a 50% false positive or negative rate with unknown compounds. However, of the seven compounds assessed to date (17beta-E2, ICI, ANA, KETO, FLUT, HALO, and RES), all were correctly identified using an in vivo male battery, which also has the capability to detect other endocrine activities. Therefore, the testis and ovary explant assay would not be necessary if one were using an in vivo male battery, since this screen would identify steroid biosynthesis inhibitors and would also identify several other endocrine activities. Because of the difficulties in assessing cytotoxicity and the high false positive/negative rates, the ovary and testis explant assays are not useful as routine screening procedures for detecting steroid biosynthesis inhibitors; however, they may have utility in confirming in vivo findings.

Animals↗
PubMed2001

Establishment of neuronal in vitro models of ischemia in 96-well microtiter strip-plates that result in acute, progressive and delayed neuronal death.

Using 96-well microtiter strip-plates we established in vitro ischemia models with acute, progressive and delayed neuronal death onset. In vitro ischemia was induced by washing neuronal cultures with a balanced salt solution with (acute/delayed models) or without (progressive model) 25 mM 2-deoxy-D-glucose and incubating in an anaerobic chamber. Reperfusion was performed by removing cultures from the anaerobic chamber and washing and/or adding Dulbecco's modified Eagle medium containing N2 supplement. Acute neuronal death resulted in cell swelling during in vitro ischemic incubation with the majority of neurons appearing swollen and necrotic within 3 h post-insult. Progressive neuronal death was characterized by cell shrinkage during and immediately following in vitro ischemia with increasing neuronal degeneration resembling both necrosis and apoptosis over a 24-h period post-in vitro ischemia. Delayed neuronal death was induced by glutamate-receptor blockade during in vitro ischemia. Neurons appeared morphologically normal immediately following and up to 6 h after in vitro ischemia and then started to degenerate over the next 42 h by a process resembling apoptosis. We monitored oxygen consumption during in vitro ischemia and found it to be similar for the three models and have shown that plastic culture wells store oxygen. The establishment of acute, progressive and delayed in vitro models of ischemia using 96-well microtiter strip-plates will provide useful tools to further investigate ischemic neuronal death/survival mechanisms and provide a high-throughput system to evaluate potential neuroprotective agents. Oxygen storage in plastic culture wells is likely to contribute to the extended oxygen- and oxygen-glucose-deprivation times required to induce significant neuronal injury in vitro.

Animals↗
PubMed1997

Contribution of serum protein association to discrepancy between the in vivo and in vitro UDS results for 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843).

U-89843 has been shown to undergo biotransformation, both in vitro and in vivo, to form U-97924 as a major primary metabolite. U-89843 was found to be positive in an in vitro UDS mutagenesis screen conducted with primary rat hepatocytes in serum-free media. In contrast to in vitro results, no evidence of genetic toxicity of U-89843 was observed in rats in the in vivo/in vitro version of the UDS test with single oral doses up to 1400 mg/kg. The negative results may be related to more robust in vivo detoxification mechanisms or relatively lower exposure to reactive metabolites formed by bioactivation of U-89843 as compared to that observed in the serum-free in vitro hepatocyte test system. Further studies showed rat serum suppressed the in vitro metabolism of U-89843 as well as the formation of the corresponding hydroxylated metabolite, U-97924, the putative precursor of proposed reactive electrophilic metabolite. The measured in vivo systemic clearance of U-89843 (0.53 l/h/kg) in rats was about 1000-fold slower than the in vitro intrinsic clearance (606 l/h/kg) estimated by measuring the formation of U-97924 in rat liver microsomal incubations. Since U-89843 is extensively associated with serum proteins a poor extraction ratio into the liver may account for the slower biotransformation of U-89843 in vivo as compared to that exhibited in in vitro serum-free hepatocyte incubations. Addition of bovine serum albumin (1-40 mg/ml) to the in vitro UDS assay medium decreased the UDS mean net grains per nucleus response of U-89843. These results suggest that the effect of serum protein should be considered when comparing serum-free in vitro UDS and in vivo UDS results for highly serum protein bound compounds.

Animals↗
PubMed2003

Evaluation of the in vivo disintegration of solid dosage forms of a bile acid sequestrant in dogs using gamma-scintigraphy and correlation to in vitro disintegration.

PURPOSE: [corrected] To evaluate the in vivo disintegration behavior of tablets and capsules of a bile acid sequestrant, DMP 504, in beagle dogs and to assess the significance of the in vitro disintegration of the dosage forms on subsequent in vivo behavior in order to draw possible in vitro-in vivo correlations. METHODS: Tablet and capsule formulations of a bile acid sequestrant, DMP 504, were formulated with samarium oxide and neutron activated to produce radioactive 53Sm to noninvasively evaluate their in vivo behavior in beagle dogs by gamma-scintigraphy. A four-way crossover design was completed (n = 4) in which (a) tablets from two different batches were administered under the fasted condition and manufactured using different lots of drug substance where one batch exhibited relatively faster in vitro disintegration time (30 min) than the other tablet batch, which resulted in slower disintegration (45 min), (b) a capsule formulation was administered to fasted beagles, and (c) the tablet having slower in vitro disintegration was also administered in the fed state, and its in vivo disintegration was compared to that observed in the fasted state. RESULTS: Tablets manufactured using a lot of DMP 504 having relatively fast in vitro disintegration (approximately 30 min) resulted in relatively rapid in vivo disintegration time (15 min) in the fasted condition. This in vivo disintegration time was comparable to the in vivo disintegration of the capsules (17 min) even though the in vitro capsule disintegration time was considerably faster (2 min). Tablets prepared using a drug substance that provided a longer in vitro disintegration time (approximately 45 min) resulted in a slower in vivo disintegration (63 min). There was no difference observed in the in vivo disintegration behavior in fasted and fed dogs for the tablets that provided slower in vitro disintegration. CONCLUSION: In vivo disintegration of tablets of the bile acid sequestrant DMP 504 correlated with in vitro disintegration times. Gamma-Scintigraphy continues to be a good tool to use during early stages of product development to investigate in vivo performance of dosage forms. The results of this study provided evidence that the physical chemical specifications of the drug substance may not always be indicative of in vitro or in vivo performance of tablet dosage form, even when formulation and process are not changed.

Animals↗
PubMed1997

Comparison of DNA adduct formation by aristolochic acids in various in vitro activation systems by 32P-post-labelling: evidence for reductive activation by peroxidases.

Aristolochic acid I (AAI) and aristolochic acid II (AAII), the two major components of the carcinogenic plant extract aristolochic acid (AA), are known to be mutagenic and to form DNA adducts in vivo. According to the structures of the major DNA adducts identified in animals and humans, nitroreduction is the crucial pathway in the metabolic activation of these naturally occurring nitroarenes to their ultimate carcinogenic species. Using the nuclease P1-enhanced version of the 32P-post-labelling assay we investigated the formation of DNA adducts by AAI and AAII in different in vitro activation systems in order to determine the most suitable in vitro system mimicking target tissue activation. Although DNA adducts resulting from oxidative activation of AAs have not yet been identified both reductive and oxidative in vitro systems were employed. In vitro incubations were conducted under standardized conditions (0.3 mM AAs; 4 mM dNp as calf thymus DNA) using rat liver microsomes, xanthine oxidase (a mammalian nitroreductase), horseradish peroxidase, lactoperoxidase and chemical reduction by zinc. Enzymatic incubations were performed under aerobic and anaerobic conditions. A combination of two independent chromatographic systems (ion-exchange chromatography and reversed-phase HPLC) with reference compounds was used for the identification of DNA adducts detected by the 32P-post-labelling assay. The two known major adducts of AAI or AAII found in vivo were generated by all in vitro systems except for incubations with AAII and horseradish peroxidase where two unknown adducts predominated. Irrespective of the in vitro activation system used, the majority of adduct spots obtained were identified as the previously characterized four AA-DNA adducts: dA-AAI, dA-AAII, dG-AAI and dG-AAII. This indicates that both reductive and peroxidative activation of AAI or AAII resulted in chromatographically indistinguishable DNA adducts. Thus, peroxidase mediated activation of AAs led to the formation of the same adducts that had been observed in vivo and upon reductive activation in several in vitro systems. Quantitative analyses of individual adducts formed in the various in vitro systems revealed relative adduct labelling (RAL) values over a 100,000-fold range from 4 in 10(3) for activation of AAII to deoxyadenosine adducts by zinc to only 3 in 10(8) for activation of AAII by lactoperoxidase. The extent of DNA modification by AAI was higher than by AAII in all enzymatic in vitro systems. Only activation by zinc resulted in higher total binding to exogenous DNA by AAII than by AAI. Aerobic incubations with rat liver microsomes generated AAI- and AAII-DNA adduct profiles reproducing profiles in target tissue (forestomach) of rats, thus providing the most appropriate activation among the in vitro systems tested.

Animals↗
PubMed2002

A cytogenetic study of in-vitro matured murine oocytes after ICSI by human sperm.

BACKGROUND: The purpose of this study was to investigate the chromosomal complement and developmental potential of in-vitro matured murine oocytes following ICSI by human sperm. METHODS: Heterologous ICSI fertilization between mouse oocytes and human sperm was employed in order to overcome the reduced fertilization rates observed after conventional IVF due to zona hardening during in-vitro maturation, and to assess separately maternal and paternal chromosome complements. Cytogenetic analyses were performed in four types of oocytes: (i) in-vitro matured metaphase II (MII) oocytes; (ii) in-vivo matured MII oocytes; (iii) in-vitro matured oocytes after ICSI; (iv) in-vivo matured oocytes after ICSI. RESULTS: Activation rates after ICSI of in-vitro matured oocytes was lower than that of in-vivo matured oocytes (69.9 versus 97.2%, P < 0.01), and premature chromosomal condensation was only observed in in-vitro matured oocytes. However, there were no significant differences in developmental rates after successful activation between in-vivo and in-vitro matured ICSI oocytes (69.7 versus 76.6%). The incidences of aneuploidy and structural aberrations were similar between the ICSI embryos and non-ICSI (MII) oocytes. Furthermore, the frequency of chromosomal aberrations was not associated with in-vitro or in-vivo maturation. Similar analyses of paternal chromosomes indicated that there were no significant differences in the incidence of chromosomal aberrations between the embryos derived from in-vitro and in-vivo matured oocytes. CONCLUSIONS: These results suggest that in-vitro matured oocytes following ICSI do not lead to an increase in the frequency of aneuploidy and structural aberrations when human sperm are injected into mouse oocytes.

Aneuploidy↗
PubMed2001

Possible explanation of the disparity between the in vitro and in vivo measurements of Rubisco activity: a study in loblolly pine grown in elevated pCO2.

Rubisco activity can be measured using gas exchange (in vivo) or using in vitro methods. Commonly in vitro methods yield activities that are less than those obtained in vivo. Rubisco activity was measured both in vivo and in vitro using a spectrophotometric technique in mature Pinus taeda L. (loblolly pine) trees grown using free-air CO2 enrichment in elevated (56 Pa) and current (36 Pa) pCO2. In addition, for studies where both in vivo and in vitro values of Rubisco activity were reported net CO2 uptake rate (A) was modelled based on the in vivo and in vitro values of Rubisco activity reported in the literature. Both the modelling exercise and the experimental data showed that the in vitro values of Rubisco activity were insufficient to account for the observed values of A. A trichloroacetic acid (TCA) precipitation of the protein from samples taken in parallel with those used for activity analysis was co-electrophoresed with the extract used for determining in vitro Rubisco activity. There was significantly more Rubisco present in the TCA precipitated samples, suggesting that the underestimation of Rubisco activity in vitro was attributable to an insufficient extraction of Rubisco protein prior to activity analysis. Correction of in vitro values to account for the under-represented Rubisco yielded mechanistically valid values for Rubisco activity. However, despite the low absolute values for Rubisco activity determined in vitro, the trends reported with CO2 treatment concurred with, and were of equal magnitude to, those observed in Rubisco activity measured in vivo.

Carbon Dioxide↗
PubMed2003

Estimates of starch digestion in the rat small intestine differ from those obtained using in vitro time-sensitive starch fractionation assays.

The objectives of this study were as follows: 1) to determine the rate and extent of starch disappearance from the small intestine of the rat fed selected starch sources, 2) to determine the ratios of the major starch fractions [rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS)] in those starch sources using two in vitro methods and 3) to compare the two data sets to determine the accuracy of the in vitro methods. Diets were prepared using cornstarch, potato starch, amylomaize, maltodextrin, modified maltodextrin or pullulan. Starch sources and diets were analyzed for starch fractions by two in vitro methods. Diets were fed to rats, intestinal contents were collected and the ethanol-induced precipitate from the contents was analyzed to obtain a digestion curve that was mathematically modeled for comparison to results obtained using the two in vitro methods. Only the cornstarch diet had a defined amount of RDS, SDS and RS. The RDS concentration obtained from the intestinal contents of the rats fed the cornstarch diet differed (P < 0.05) from that determined by one in vitro method but was consistent with the value obtained using the other in vitro method. All other digestible starch values obtained differed (P < 0.05) among methods except for that of amylomaize. Starch fractions in starch sources obtained using in vitro procedures differed (P < 0.05) from values obtained for diets. The rate of disappearance differed (P < 0.05) between in vivo and in vitro procedures. There was minimal agreement between in vitro methods tested, and there was also minimal agreement between in vitro and in vivo results. Classification of starch into RDS and SDS components cannot be accomplished for a variety of starch sources, with cornstarch being the major exception.

Animals↗
PubMed2006

Relationships between leaf conductance to CO2 diffusion and photosynthesis in micropropagated grapevine plants, before and after ex vitro acclimatization.

In vitro-cultured plants typically show a low photosynthetic activity, which is considered detrimental to subsequent ex vitro acclimatization. Studies conducted so far have approached this problem by analysing the biochemical and photochemical aspects of photosynthesis, while very little attention has been paid to the role of leaf conductance to CO(2) diffusion, which often represents an important constraint to CO(2) assimilation in naturally grown plants. Mesophyll conductance, in particular, has never been determined in in vitro plants, and no information exists as to whether it represents a limitation to carbon assimilation during in vitro growth and subsequent ex vitro acclimatization. In this study, by means of simultaneous gas exchange and chlorophyll fluorescence measurements, the stomatal and mesophyll conductance to CO(2) diffusion were assessed in in vitro-cultured plants of the grapevine rootstock '41B' (Vitis vinifera 'Chasselas'xVitis berlandieri), prior to and after ex vitro acclimatization. Their impact on electron transport rate partitioning and on limitation of potential net assimilation rate was analysed. In vitro plants had a high stomatal conductance, 155 versus 50 mmol m(-2) s(-1) in acclimatized plants, which ensured a higher CO(2) concentration in the chloroplasts, and a 7% higher electron flow to the carbon reduction pathway. The high stomatal conductance was counterbalanced by a low mesophyll conductance, 43 versus 285 mmol m(-2) s(-1), which accounted for a 14.5% estimated relative limitation to photosynthesis against 2.1% estimated in acclimatized plants. It was concluded that mesophyll conductance represents an important limitation for in vitro plant photosynthesis, and that in acclimatization studies the correct comparison of photosynthetic activity between in vitro and acclimatized plants must take into account the contribution of both stomatal and mesophyll conductance.

Acclimatization↗
PubMed1999

Review of the efficacy of individualized chemotherapy selected by in vitro drug sensitivity testing for patients with cancer.

PURPOSE: The purpose of this study is to review the potential efficacy of individualized chemotherapy selected by in vitro drug sensitivity testing (DST) compared with empiric regimens for patients with cancer. METHODS: MEDLINE and CANCERLIT were searched for articles published (in English) about prospective clinical trials in which patients were treated with chemotherapy that was selected with the use of in vitro DST. RESULTS: We identified 12 prospective studies that examined the benefit of chemotherapy selected by DST. Five hundred six patients (33%) were treated with chemotherapy that was selected with the use of in vitro DST. The mean response rate for patients treated with in vitro-selected therapy was 27% (range, 10% to 100%; n = 12 studies) compared with 18% (range, 0% to 100%; n = 7 studies) for patients treated with empiric therapy. Five studies (only one randomized) evaluated the impact of chemotherapy selected by in vitro DST on patient survival. Three studies showed that survival was 1 to 4 months longer for the 238 patients treated with empiric chemotherapy compared with that of the 65 patients treated with chemotherapy that was selected by in vitro testing. Two nonrandomized studies showed that survival was 4 or 19 months longer for 27 patients treated with chemotherapy selected by in vitro testing compared with that of 80 patients who were treated with empiric chemotherapy. CONCLUSION: Only one third of patients entered in prospective trials of in vitro DST were actually treated with an in vitro best regimen. The response rates seem to be better with in vitro selected chemotherapy regimens than with empiric regimens, but the impact on survival has not been adequately addressed.

Carcinoma, Small Cell↗
PubMed1988

A murine model to evaluate the ability of in vitro clonogenic assays to predict the response to tumors in vivo.

The use of the human tumor cloning assay as a predictor of clinical response of human tumors to drugs is predicated on the hypothesis that the in vivo response of a tumor to a drug can be correlated with the in vitro response of cells derived from the tumor. To test this hypothesis, we utilized a murine tumor model in which the in vivo and in vitro responses of a tumor can be accurately and reproducibly compared. Drug activity was assessed in P388 leukemia with the standard in vivo antitumor assay (i.p. tumor/i.p. drug administration) and an in vitro assay wherein the ascites tumor cells are removed from mice, treated with a drug, and directly cloned in soft agar to measure clonogenic capacity. The response of P388 cells to analogues within four separate classes of antitumor agents, anthracyclines, anthraquinones, platinum(II) coordination complexes, and phosphinogold(I) complexes was evaluated. The clonogenic assay failed to discriminate between highly active in vivo antitumor agents and analogues with only marginal in vivo efficacy (i.e., doxorubicin and daunorubicin versus rhodomycins A and B, ametantrone versus NSC 276740, cisplatin versus transplatin, [Au(dppe)2]Cl versus [Au(depe)2]PF6. Furthermore, the in vitro clonogenic assay failed to detect carboplatin which was a highly active agent in vivo. The basis for these discrepancies was explored by a more detailed comparison of doxorubicin and rhodomycin B. In vivo or in vitro drug exposure with subsequent measurement of cell kill by the in vitro clonogenic and in vivo tumorigenic assay demonstrated that the in vitro assay overestimated the cytotoxic potency of the drugs relative to the tumorigenic assay. Treatment of tumors in vivo with doxorubicin at doses below the maximally tolerated dose in mice resulted in multiple log cell kill as measured in vitro or in vivo, whereas rhodomycin B was cytotoxic only at dose levels exceeding its maximally tolerated dose. The results indicate that a subset of tumor stem cells capable of forming colonies in soft agar are significantly more sensitive to the cytotoxic effects of anthracyclines than are in vivo tumorigenic stem cells. Cytotoxic potency as measured by an in vitro soft agar clonogenic assay is not an accurate predictor of in vivo antitumor efficacy even in a model in which ascites tumor cells are directly exposed to i.p. drug. The in vitro cytotoxicity assay is useful only as a nonselective prescreen and must be used in combination with other indicators of tumor cell selectivity and dose-limiting organ toxicity.

Animals↗
PubMed1981

Chemoimmunotherapy of a Friend leukemia with cells secondarily sensitized in vitro: effect of culture duration on therapeutic efficacy.

Although in vivo-primed cells are known to be rendered more effective in adoptive tumor therapy by secondary sensitization in vitro, they were tested only at the time of maximum in vitro cytolytic reactivity. Since the requirements for in vitro cytotoxicity and tumor therapy differ, the present study was designed to evaluate and compare the influence of culture duration on these two effector functions. Spleen cells from inbred C57BL/6 mice primed in vivo with the Friend virus-induced leukemia FBL-3 were secondarily sensitized in vitro by culture with tumor and tested for cytotoxicity in vitro in a 4-hour 51Cr release assay and for therapeutic efficacy in vivo against established tumor. In vivo-primed cells tested directly without prior culture were effective in therapy but were not cytotoxic in vitro. Culture of primed cells for 3 days rendered them cytotoxic as measured in vitro. This cytotoxicity increased through day 5, then it plateaued. Cultured duration modified the in vivo efficacy of primed cells differently. Cells cultured for 3 days with secondary sensitization by tumor became more effective in tumor therapy than noncultured primed cells. Increasing the duration of in vitro sensitization from 3 to 5 days did not enhance in vivo therapeutic efficacy, despite a concurrent increase in in vitro cytotoxicity. However, further lengthening of the culture duration to 7 days rendered cells more effective in vivo. These discrepancies presumably reflect different effector cell requirements and/or regulation operative for tumor lysis during a short-term in vitro assay and for tumor eradication following adoptive transfer of immune cells in vivo.

Animals↗
PubMed1977

[Radiation sensitivity of lymphocyte stimulation. 1. Experiments on in-vitro sensitivity of cell number and mitosis activity].

Radiation-induced transformations of stimulated lymphocytes are not to be regarded as characteristical responses of lymphocyte populations as such, but represent general radiobiologic principles of the stimulated proliferation. Thus, basic problems of stimulated proliferation in comparison with a spontaneous quick proliferation of normal cellular systems in vitro have chiefly been studied by means of two stimulation models in vivo, the model of liver regeneration following partial hepatectomy and the model of beta stimulation of the salivary glands. The radiosensitivity of the lymphocyte stimulation in vitro as well as in vivo has been analyzed especially with regard to its quality as a model. The task was approached methodically by: a) Determination in vitro of the number of cells (66 cultures), b) morphological analysis of the mitotic activity in vitro (270 cultures), c) measurements in vitro with 3h-thymidine of the DNA synthesis using liquid scintillation counts (more than 3000 cultures), d) measurements in vitro with 3H-thymidine of the DNA synthesis using autoradiography (66 cultures), e) determination in vivo of the DNA synthesis by means of the incorporation of 131I-desoxyuridine into the mouse spleen (investigation with 100 mice). For a model of lymphocyte stimulation mainly that of PHA stimulation in vitro and in vivo of T-lymphocytes was chosen, supplemented by the one of PWM stimulation of T- and B- lymphocytes for measuring in vitro the DNA synthesis. In particular, influence of in vitro irradiation upon several variables of the stimulation model was investigated in order to examine the methodics critically: a) During the determination of the mitotic index too short an acbition by partial synchronization effects within the dose range of 10 to 500 R. The chronological analysis of the process during determination of the mitotic activity is indispensable in order to differentiate the mitotic inhibition from a mitotic delay in the case of high doses, b) For the determination of the DNA synthesis the concentration of stimulative PHA or PWM has no substantial influence on the radiation-induced inhibition effect; the constancy of serum percentage in the culture system, however, is an important condition for comparability of different experiments. Moreover, only a short period of labeling with 3H-thymidine, lasting 1.5 to 12 hours, interferes with radiation-induced inhibition of the DNA synthesis, this probably being also due to partial synchronization effects within the dose range of 10 to 500 R. c) In order to determine the DNA synthesis wether in vitro or in vivo, a chronological analysis is a condition indispensable for differentiation between the three factors involved: Delay of DNA synthesis, blockage of DNA synthesis, and inhibition of the proliferation...

Adult↗
PubMed1993

Reversal of gallium arsenide-induced suppression of the antibody-forming cell response by vehicle supernatants. I. Pharmacokinetics after in vitro and in vivo exposure.

Exposure of splenocytes in vivo or in vitro to gallium arsenide (GaAs) dose-dependently suppresses the ability of these cells to produce antibody after in vitro immunization with sheep red blood cells. In addition, it has been demonstrated that GaAs exerts immunosuppressive effects early (36 hr) in the generation of a primary antibody-forming cell (AFC) response. The objective of this study was to determine if the GaAs-induced suppression was produced as a result of a GaAs-induced alteration in the secretion of soluble mediators. Supernatants from in vivo and in vitro vehicle (VH)-exposed splenocyte cultures time-dependently reversed GaAs-induced suppression of the in vitro-generated primary AFC response produced by both in vitro (50 microM) and in vivo (200 mg/kg) exposure to GaAs. Supernatants from in vitro GaAs-exposed cells suppressed the VH response 40, 89 and 93% at 24, 36 and 48 hr, respectively. Using the arsenic-binding compound meso-2,3-dimercaptosuccinic acid (100 microM), it was determined that the suppression of the VH response by supernatants from in vitro GaAs-exposed cultures was confounded by the presence of free arsenic in the in vitro GaAs-exposed culture supernatant. In contrast, suppression of in vivo VH-exposed AFC responses by supernatants from in vivo GaAs-exposed cells was not seen. The time-dependent reversal of immunosuppression produced by in vivo or in vitro exposure to GaAs, by supernatants from in vivo and in vitro VH-exposed cells mimics the reported kinetics of suppression by addition of GaAs to antibody cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

In-vitro matured metaphase-I oocytes have a lower fertilization rate but similar embryo quality as mature metaphase-II oocytes after intracytoplasmic sperm injection.

About 4% of all the oocytes denuded prior to intracytoplasmic sperm injection (ICSI) are in metaphase-I (MI). Frequently, these oocytes achieve meiosis after a few hours of in-vitro culture and are available for ICSI on the day of oocyte retrieval. In this retrospective study, the aim was to evaluate the fertilization rate and the developmental capacity of these in-vitro matured MI oocytes. After controlled ovarian stimulation using human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) in 896 ICSI cycles, 1210 MI-to-MII-matured oocytes were injected approximately 4 h after in-vitro culture and 8803 MII oocytes were injected immediately, or later, after denudation. The fertilization rate of in-vitro matured oocytes was significantly lower than that of mature MII oocytes (52.7 and 70.8% respectively, P < 0.00l). Embryo quality was only slightly different as regards the numbers of good quality embryos: 47.4% good quality embryos were obtained in the in-vitro matured oocyte group, whereas 53.2% good quality embryos were obtained in the MII oocyte group (P < 0.05). The same proportions of excellent (5.7 and 7.0%, NS) and fair quality (17.6 and 15.3%, NS) embryos were obtained for in-vitro matured and mature oocytes respectively. Embryos derived from in-vitro matured oocytes were transferred only if they were of better quality or if there were not enough mature oocyte derived embryos available. Fifteen transfers involved only embryos derived from in-vitro matured oocytes: 11 single embryo transfers and four transfers of two embryos, resulting in one singleton pregnancy and the birth of a healthy baby. It may be concluded that in cycles with few MII oocytes it might be worthwhile to inject in-vitro matured MI oocytes in order to increase the number of embryos available for transfer.

Cell Differentiation↗
PubMed2002

Effects of oocyte maturation regimen on the relative abundance of gene transcripts in bovine blastocysts derived in vitro or in vivo.

Bovine embryos produced in vitro differ substantially from embryos produced in vivo in the mRNA expression patterns of genes important for development. Several factors in the in vitro production systems have profound effects on embryonic mRNA expression patterns. The effects of the type of maturation on the expression pattern of genes important for development in blastocysts produced in vitro have not yet been investigated. The aim of the present study was to investigate the effects of various maturational protocols on the relative abundance of a panel of six marker genes, indicative of compaction and cavitation, metabolism, stress susceptibility and RNA processing, in bovine blastocysts produced in vitro. Four groups of blastocysts were analysed by a sensitive semi-quantitative RT-PCR assay. Blastocysts were produced in vitro from oocytes of different origin from: (1) 3-8 mm follicles; (2) preovulatory follicles before the LH surge; and (3) preovulatory follicles 24 h after the LH surge. The first two groups were matured in vitro, whereas the third group had undergone maturation in vivo. A fourth group comprised blastocysts developed entirely in vivo. Expression of glucose transporter 1 was significantly (P < 0.05) higher, and expression of desmocollin 2 and plakophilin tended to be higher (P < 0.1) for in vivo (group 4) compared with in vitro blastocysts (group 1), whereas no differences were found for heat shock protein 70.1, E-cadherin and poly(A) polymerase. Expression of the six transcripts did not differ among blastocysts produced in vitro from oocytes of groups 1, 2 and 3. Results indicate that alterations in the relative abundance of these transcripts in blastocysts produced in vitro cannot primarily be attributed to the origin of the oocyte, but are likely to have been induced by post-maturation or fertilization culture conditions.

Animals↗
PubMed1987

In vitro fertilization with normal development in the sheep.

Ovine tubal (n = 87) and ovarian in vitro matured oocytes (n = 99) were fertilized in vitro with ejaculated spermatozoa capacitated for 8 h in modified defined medium buffered with Hepes. High levels of fertilization were obtained as assessed by development to two- to six-cell stage within 40 h (75.8% for ovulated and 62.6% for in vitro matured oocytes). Electron microscope analysis of oocytes 20-22 h after insemination indicated that in vitro fertilization approximated the in vivo events. Embryos (two- to six-cell) were transferred surgically to the oviducts of pseudopregnant rabbits. Three days later, 42 (from ovulated oocytes) and 15 (from in vitro matured oocytes) embryos were recovered; 26 (61.9%) and 10 (66.6%), respectively, had cleaved at least once. Embryos incubated in vivo (n = 20 from ovulated oocytes; n = 9 from in vitro matured oocytes) were transferred surgically to the uteri of seven and four recipient ewes resulting in four and two pregnancies, respectively, from which three and one, respectively, have been maintained (greater than 3 months). The first lamb resulting from the in vitro fertilization of an ovulated oocyte was born. In addition, six embryos (two- to four-cell) from tubal oocytes and ten embryos (two- to six-cell) from in vitro matured oocytes were directly transferred to the oviducts of two and three ewes, respectively. Two pregnancies resulting from in vitro matured fertilized oocytes are in progress (greater than 3 months).

Animals↗
PubMed1997

Convolution-based approaches for in vivo-in vitro correlation modeling.

One approach to in vivo-in vitro correlation (IVIVC) for extended release (ER) oral dosage forms is to directly model the relationship between the time courses of in vitro release and plasma drug concentrations. For drugs that exhibit linear, time-invariant disposition this can be done using models based on the convolution integral. Advantages of this approach relative to deconvolution-based IVIVC approaches include the following: The relationship between measured quantities (in vitro release and plasma drug concentrations) is modeled directly in a single stage rather than via an indirect two stage approach. The model directly predicts the plasma concentration time course. As a result: The modeling focuses on the ability to predict measured quantities (not indirectly calculated quantities such as the cumulative amount absorbed). The results are more readily interpreted in terms of the effect of in vitro release on conventional bioequivalence metrics. It is easier to construct methods that do not require the administration of an IV, oral solution, or IR reference dose. A variety of convolution-based IVIVC models and modeling strategies are possible depending on the relationship between in vivo and in vitro release, the existence of nonlinear absorption or presystemic biotransformation, and the in vivo study design. The simplest approach is applicable to the case where the in vitro release rate equals the in vivo release (or absorption) rate and the study design includes the administration of an IV, oral solution, or IR dose. That basic convolution-based method can be extended to adjust for differences between the in vitro and in vivo release rates. This is accomplished by formally modeling those differences. Potential models include time-scaling and convolution. The extent of drug absorption may sometimes depend upon the release rate. This may be due to phenomena such as saturable presystemic biotransformation or truncated absorption due to intestinal transit past the sites of absorption. The relationship between the in vitro release rate and extent of absorption may be modeled empirically or mechanistically. Such models may be coupled with convolution to construct an overall IVIVC model for the relationship between in vitro release and plasma drug concentrations. It is also possible to apply convolution-based IVIVC models to study designs in which no IV, oral solution, or IR dose has been administered. Details of the various modeling approaches listed above are presented. Selected approaches are illustrated by examples of their application to real data.

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