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PubMed2006

In vitro study assessing the response of plasmodium falciparum malaria to chloroquine, sulfadoxine/pyrimethamine, quinine and mefloquine in Wad Medani District, Sudan.

OBJECTIVE: To assess the in vitro response of Plasmodium falciparum malaria to chloroquine (CQ), sulfadoxine/pyrimethamine (SDX/PYR), Quinine (QU) and Mefloquine (MQ) and monitoring their resistance. METHODS: In 1999 to 2000, an in vitro study was carried out in Wad Medani district in Sudan. The standard protocol of the WHO in vitro micro-test Mark II was used for the selection of the subjects, the collection of blood samples, the culture techniques, the examination of the post-culture blood slides and the interpretation of the results. RESULTS: In vitro micro-test Mark II were performed on 62 Plasmodium falciparum isolates. Of these isolates, 42 produced successful schizonts growth. The data obtained showed that 29 of 42 isolates (69%) were CQ resistant with an effective concentrations (EC); EC50 = 399.621 nM, EC90 = 2754.145 nM and EC99 = 13284.967 nM to inhibit the schizont maturation, the values of SDX/PYR showed a flat regression line as an indication of in vitro reduced response with an EC50 = 0.262 nM, EC90 = 147.390 nM and EC99 = 25722.296 nM, and the response to the QU indicated only one of the 42 isolates (2.4%) was resistant with an EC50 = 150.085 nM, EC90 = 822.825 nM and EC99 = 3293.667 nM, while all the 42 isolates were sensitive to MQ with an EC50 = 190.763 nM, EC90 = 615.125 nM and EC99 = 1597.504 nM. CONCLUSION: The results of this study revealed a high degree of in vitro resistance to CQ and reduced in vitro response to SDX/PYR and QU, while MQ was fully sensitive. The effective concentrations to inhibit 50, 90 and 99% of the parasite maturation were determined for antimalarial drugs efficacy monitoring surveillance in Sudan.

Animals↗
PubMedDate not supplied

Introduction to in vitro estimation of metabolic stability and drug interactions of new chemical entities in drug discovery and development.

Determination of metabolic properties of a new chemical entity (NCE) is one of the most important steps during the drug discovery and development process. Nowadays, in vitro methods are used for early estimation and prediction of in vivo metabolism of NCEs. Using in vitro methods, it is possible to determine the metabolic stability of NCEs as well as the risk for drug-drug interactions (DDIs) related to inhibition and induction of drug metabolic enzymes. Metabolic stability is defined as the susceptibility of a chemical compound to biotransformation, and is expressed as in vitro half-life (t(1/2)) and intrinsic clearance (CL(int)). Based on these values, in vivo pharmacokinetic parameters such as bioavailability and in vivo half-life can be calculated. The drug metabolic enzymes possess broad substrate specificity and can metabolize multiple compounds. Therefore, the risk for metabolism-based DDIs is always a potential problem during the drug development process. For this reason, inhibition and induction in vitro screens are used early, before selection of a candidate drug (CD), to estimate the risk for clinically significant DDIs. At present, most pharmaceutical companies perform in vitro drug metabolism studies together with in silico prediction software and automated high-throughput screens (HTS). Available data suggest that in vitro methods are useful tools for identification and elimination of NCEs with unappreciated metabolic properties. However, the quantitative output of the methods has to be improved. The aim of this review is to highlight the practical and theoretical basis of the in vitro metabolic methods and the recent progress in the development of these assays.

Animals↗
PubMed1991

Tumor necrosis factor and c-fos expression in human peripheral-blood monocytes: expression is dependent on stage of in vitro differentiation.

The differentiation of monocytes and macrophages both in vitro and in vivo can be characterized by the modulation of specific functional and molecular phenotypes. We have determined in human peripheral-blood monocytes (HPBM) the role of in vitro differentiation on the expression of nonspecific tumoricidal activity, induction of soluble tumor necrosis factor (TNF) activity and TNF-specific mRNA transcription. HPBM were activatable by bacterial lipopolysaccharide (LPS; 1 microgram/ml) for nonspecific cytotoxicity to A375M (human malignant melanoma cell line) only during the first 24 h of in vitro differentiation. Activated supernatants of HPBM were found to be partially neutralizable (75 +/- 7%) by rabbit polyclonal anti-TNF antibody and, in freshly isolated HPBM, the release of soluble TNF activity determined by the L929 assay was found to occur only after activation with LPS. Maximal TNF release occurred at 8 h of LPS stimulation, and required both protein and RNA synthesis as evidenced by the ability of both actinomycin D and cycloheximide to inhibit its release. Neither control untreated HPBM nor recombinant interferon-gamma (rIFN-gamma; 1 U/ml)-treated HPBM alone released soluble TNF activity. Further in vitro culture determined that HPBM were activatable for TNF release out to 72 h of culture after which HPBM became resistant to LPS-mediated TNF release. The expression of TNF and c-fos mRNA was also determined during in vitro differentiation. Both TNF and c-fos mRNA were expressed in freshly isolated HPBM, and returned to baseline by 24 h of in vitro culture. Treatment of HPBM with LPS induced TNF transcription as late as 5 days of in vitro culture with maximal induction occurring during the first 48 h. rIFN-gamma significantly induced TNF transcription at 24 h in the absence of soluble TNF activity, but did not increase transcription at later times. The expression of nonspecific cytolytic activity, the release of soluble bioactive TNF and the induction of TNF and c-fos mRNA are regulated in HPBM by differentiation-determined processes.

Cell Differentiation↗
PubMed1990

Beta-lactam antibiotic-induced platelet dysfunction: evidence for irreversible inhibition of platelet activation in vitro and in vivo after prolonged exposure to penicillin.

beta-Lactam antibiotics cause platelet dysfunction with bleeding complications. Previous in vitro studies documented reversible inhibition of agonist-receptor interaction. This mechanism is inadequate to explain the effect of beta-lactam antibiotics in vivo. Platelet function does not return to normal immediately after drug treatment, implying irreversible inhibition of platelet function. We report here evidence of irreversible platelet functional and biochemical abnormalities after in vitro and in vivo exposure to beta-lactam antibiotics. Irreversible binding of [14C]-penicillin (Pen) occurred in vitro. After 24 hours' in vitro incubation with 10 to 20 mmol/L Pen, or ex vivo after antibiotic treatment, irreversible functional impairment occurred; but no irreversible inhibition of alpha 2 adrenergic receptors, measured with [3H]-yohimbine, or high-affinity thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors, measured with agonist [3H]-U46619 and antagonist [3H]-SQ29548, occurred. However, low-affinity platelet TXA2/PGH2 receptors were decreased 40% after Pen exposure in vitro or in vivo, indicating irreversible membrane alteration. Two postreceptor biochemical events were irreversibly inhibited in platelets incubated with Pen for 24 hours in vitro or ex vivo after antibiotic treatment. Thromboxane synthesis was inhibited 28.3% to 81.7%. Agonist-induced rises in cytosolic calcium ([Ca2+]i) were inhibited 40.1% to 67.5% in vitro and 26.6% to 52.2% ex vivo. Therefore, Pen binds to platelets after prolonged exposure, resulting in irreversible dysfunction attributable to inhibition of TXA2 synthesis and impairment of the rise in [Ca2+]i. The loss of low-affinity TXA2/PGH2 receptors suggests that the primary site of action of these drugs is on the platelet membrane.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1986

Bacterial adherence to respiratory tract cells. Relationships between in vivo and in vitro pH and bacterial attachment.

Alterations in in vitro pH have been shown to have a significant effect on the bacterial binding capacity of epithelial cells for certain organisms. We investigated the effect of in vivo pH and in vitro changes in pH on the adherence of Pseudomonas aeruginosa to buccal and tracheal cells of 19 chronic tracheostomy patients. In addition, airway pH was measured in 5 normal volunteers. Oropharyngeal and endobronchial pH were measured with a flexible electrode that could be passed through a bronchoscope under direct visualization. In vitro adherence of Pseudomonas to respiratory epithelial cells was determined at pH 6.5 and 7.2. Colonization status of the patients was determined by culture of oropharyngeal and tracheal secretions. The pH value of each site in the respiratory tract and its secretions were different in the tracheostomy patients (buccal pH 6.3; tracheal, 6.9; sputum, 7.5). No difference was noted in pH of the 2 sites in control subjects (buccal, 6.7; tracheal, 6.7). Changes in in vitro pH had a significant effect (p less than 0.004) on bacterial binding to epithelial cells from both sites. The pH had its greatest effect on tracheal cells of patients colonized with Pseudomonas. Increased in vitro binding of these organisms was noted at the more alkaline pH. The magnitude of the effect of in vitro pH alteration on bacterial binding correlated directly with the degree of binding. These results demonstrated that pH has a significant effect on in vitro Pseudomonas adherence and the effect is most marked on cells obtained from the lower respiratory tract of patients colonized with this organism.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMedDate not supplied

Cytogenetic effects of amsacrine on human lymphocytes in vivo and in vitro.

Amsacrine (m-AMSA) is presently being utilized in phase I-II studies at the Medicine Branch, National Cancer Institute, National Institutes of Health (Bethesda, MD), and is being administered as a continuous infusion to patients with progressive malignancy after conventional therapy. In the present study, we examined the effects of this drug, in vivo and in vitro, on chromosomal morphology and the frequency of sister chromatid exchange (SCE) induction in human peripheral blood lymphocytes. In the in vivo studies, eight patients receiving 30 mg/m2/day of m-AMSA by continuous infusion showed increased levels of chromosomal aberrations, up to a maximum of 14% (median; range, 10%-24%) at 96 hours compared to 1% (median; range, 0%-4%) in the control group; no increase was noted in SCE frequencies. In vitro studies of normal human lymphocytes with various concentrations of m-AMSA, including doses comparable to those used in vivo, showed both increased levels of chromosomal aberrations, ranging from 8% to 100%, and increased SCEs, ranging from 1.5 times the normal at the lowest concentration studied (0.005 microgram/ml) to 12 times the normal (0.25 microgram/ml). In correlating SCEs with breaks in the in vitro studies, it was found that two-thirds of the total breaks occurred at SCE points; this indicates a close relationship between the events leading to these two types of aberrations. In vitro isotope incorporation studies showed inhibition of both RNA and DNA syntheses but no significant inhibition of protein synthesis; at a dose of 5.0 micrograms/ml, RNA synthesis was 80% inhibited and DNA synthesis was 60% inhibited after 36 hours of incubation with m-AMSA. The difference in the results between in vitro and in vivo SCE studies may be due to possible DNA repair in the in vivo-treated lymphocytes in m-AMSA-free medium in vitro, a process not available to the in vitro-treated cells.

Adolescent↗
PubMed1994

Comparison between the effects of treatment in vitro and in vivo with lipopolysaccharide on responsiveness of rat thoracic aorta.

Effects of treatment in vivo and in vitro with lipopolysaccharide (LPS) on the responsiveness of rat thoracic aorta were examined. The endothelium-denuded aortic strips isolated 6 hr after intraperitoneal (i.p.) administration of LPS (20 mg/kg), which could produce hemodynamic changes, relaxed in response to L-arginine. The same amplitude of relaxation was produced by L-arginine in the aortic strips incubated with a lower dose of LPS (1 microgram/ml) in vitro for 6 hr. Both relaxing responses were inhibited by NG-nitro-L-arginine (L-NOARG). The contractile responses to phenylephrine and KCl were reduced by LPS treatment in vivo or in vitro, but the extent of inhibition was greater in vivo than in vitro. Further, the attenuation of contractile responses was completely reversed by L-NOARG in the strips treated in vitro, whereas the reversal by L-NOARG was incomplete in the strips treated with LPS in vivo. Endothelium-dependent relaxation induced by acetylcholine was attenuated by LPS in vivo but not in vitro. These results suggest that the hyporesponsiveness of rat thoracic aorta after treatment in vitro with LPS, which can produce hemodynamic changes, may be related to an enhanced NO production in the smooth muscle cells, while not only the NO pathway but also additional factors may be involved in the vascular hyporesponsiveness of the sepsis rats.

Acetylcholine↗
PubMedDate not supplied

Hardening of zona pellucida of mouse oocytes and embryos in vivo and in vitro.

OBJECTIVE: To evaluate the effect of in vitro culture on zona pellucida resistance in mouse oocytes and embryos. METHOD: Zona pellucida resistance was assessed by comparing duration of zona lysis in the presence of alpha-chymotrypsin. The effects of artificial or physiological conditions of development were evaluated by comparing embryos in vitro with those left to reach the same stage of development in vivo. RESULTS: The time required for zona lysis of oocytes increased after 2, 24, and 48 hours in vitro (P < .001). The same observation holds true for oocytes left in vivo during 24 hours. Fertilization both in vivo and in vitro induced a major increase in zona resistance. At the two-cell stage, in vitro culture did not harden the zona pellucida. At the morula stage and beyond, enzymatic lysis was slightly longer in vitro as compared to that of similar stages recovered from the genital tract. CONCLUSIONS: Our data indicate that in vitro culture conditions do not modify zona hardening in oocytes and only slightly increased zona resistance from the morula stage on.

Animals↗
PubMed1997

Prediction of species differences (rats, dogs, humans) in the in vivo metabolic clearance of YM796 by the liver from in vitro data.

The bioavailability after oral administration of (S)-(-)-2,8-dimethyl-3-methylene-1-oxa-8-azaspiro [4,5] decane-L-tartarate monohydrate (YM796), which is being developed as an antidementia drug, at a dose of 1 mg/kg was very low (3.4%) in rats, but considerably higher (16.1%) in dogs. The oral clearances (CLoral, Dose/AUCoral) in rats and dogs were, respectively, 300 and 18 times more than that already reported in humans. We have previously reported successful attempts to predict the in vivo hepatic metabolic clearance of YM796 from in vitro data in humans. In our study, the in vitro metabolism of YM796 was determined using liver microsomes prepared from both rats and dogs and we also investigated if the species difference observed in vivo could be quantitatively reproduced in vitro. In rats, total metabolite formation could be described by single component kinetics with a Km of 13.4 microM and a Vmax of 520 nmol/min/g liver. However, in dogs, total metabolite formation could be described by three components, as also reported for humans. The Km and Vmax values for the high-affinity, low-capacity component (Km1 and Vmax1) in dogs and humans were, respectively, 8.1 and 1.7 microM, and 10.9 and 1.2 nmol/min/g liver. The overall intrinsic metabolic clearances estimated from the in vitro studies (CLint,in vitro) for rats and dogs were 38.8 and 2.6 ml/min/g liver, respectively, being approximately 40 and 3 times more than that previously reported for humans (0.94 ml/min/g liver). The overall intrinsic hepatic clearances (CLint,in vivo) calculated from in vivo CLoral were 30.4, 3.4 and 0.73 ml/min/g liver for rats, dogs and humans, respectively, indicating that the in vivo hepatic clearance of YM796 can be predicted from in vitro metabolism data in each species. Thus, the pronounced species difference in the metabolic clearance observed in vivo can be quantitatively predicted from in vitro metabolic data using liver microsomes, and was predominantly due to the large difference in the Vmax values.

Animals↗
PubMed2006

In vitro fluoroquinolone resistance in staphylococcal endophthalmitis isolates.

OBJECTIVE: To evaluate the in vitro susceptibility and cross-resistance of gatifloxacin and moxifloxacin vs older fluoroquinolones among coagulase-negative staphylococci recovered from patients with clinical endophthalmitis. METHODS: A combination of E tests and disk diffusion methods was used to determine in vitro susceptibility and cross-resistance for 111 coagulase-negative staphylococci isolates recovered during a 15-year period (January 1, 1990, to December 31, 2004) against 5 fluoroquinolones. RESULTS: In vitro susceptibilities (percentage sensitive) in descending order were as follows: gatifloxacin, 74.5%; moxifloxacin, 72.1%; levofloxacin, 69.3%; ciprofloxacin, 65.6%, and ofloxacin, 60.4%. More than 65% of the coagulase-negative staphylococci resistant to ciprofloxacin (n = 38) demonstrated in vitro cross-resistance to gatifloxacin (25 [65.8%] of 38) and moxifloxacin (27 [71.1%] of 38). During the initial 5 years (January 1, 1990, to December 31, 1994), 96.6% of the coagulase-negative staphylococci were sensitive to gatifloxacin and moxifloxacin, with minimal inhibitory concentration required to inhibit or kill 90% of the isolates of 0.19 microg/mL and 0.12 microg/mL, respectively. During the last 5-year period (January 1, 2000, to December 31, 2004), the percentage of sensitive coagulase-negative staphylococci declined to 65.4% for gatifloxacin and moxifloxacin (P=.02). Minimal inhibitory concentration required to inhibit or kill 90% of the isolates was 32 microg/mL or greater for both drugs. CONCLUSIONS: Gatifloxacin and moxifloxacin demonstrated an in vitro efficacy of less than 80% for coagulase-negative staphylococci endophthalmitis in the present study. Ciprofloxacin resistance may serve as a surrogate for concurrent in vitro resistance for gatifloxacin and moxifloxacin. Resistance increased significantly during the last 5 years. Declining in vitro susceptibility to gatifloxacin and moxifloxacin may have important implications for the prevention and treatment of postoperative endophthalmitis.

Anti-Bacterial Agents↗
PubMed1998

Dual labeling of the cytoskeleton and DNA strand breaks in porcine embryos produced in vivo and in vitro.

In vitro-produced embryos exhibit decreased cell numbers, small inner cell masses and reduced pregnancy rates after transfer. Evaluation of intracellular components of in vitro-produced or -manipulated embryos will lead to improved methodology for embryo production. Whole mount techniques were developed to utilize terminal deoxynucleotidyl-transferase 3' nick end labeling (TUNEL) to detect broken DNA. Subsequent labeling of either tubulin or actin filaments provides further evidence of cytological damage. Porcine embryos produced in vitro or in vivo were evaluated throughout the cleavage and preimplantation stages of development. Early cleavage stages up to the 8-cell stage never contained TUNEL-labeled nuclei. However, TUNEL labeling of in vitro-produced morula revealed some blastomeres with broken DNA. Nearly all in vitro-produced blastocysts displayed some TUNEL positive cells, whereas in vivo-collected embryos at a similar stage displayed few, if any, TUNEL-labeled nuclei. The ratio of TUNEL-labeled DNA to total DNA area of in vitro-derived blastocysts was significantly greater than their in vivo counterparts (P < 0.05). Microtubule and microfilament labeling identified blastomeres of unequal size and shape that were losing cellular integrity. These data suggest that the combination of these labeling techniques may be useful in evaluating cellular damage in embryos produced under in vitro conditions.

Animals↗
PubMed2000

Oocyte metabolism predicts the development of cat embryos to blastocyst in vitro.

Current methods for detecting complete oocyte maturation and developmental competence are inadequate. The objectives of this study were to (1) examine the relationship between cat oocyte energy metabolism and development in vitro after fertilization and (2) determine if cumulus cell metabolism could be used to predict development of individual oocytes after fertilization in vitro. The hanging drop method was used to assess metabolism of three different types of cat oocytes: immature (IMO), in vitro matured (IVM), and in vivo matured (IVOM). Stage of oocyte nuclear maturation or developmental competence was assessed after metabolic analysis. Glycolysis and oxidation of glucose, glutamine, palmitate, and lactate increased with the resumption of oocyte meiotic maturation (P<0.05). Pyruvate was the preferred substrate, but uptake was not linked to maturation. IVM oocytes had impaired glucose and palmitate metabolism compared to IVOM oocytes (P<0.05). Oocyte glycolytic activity and oocyte glucose oxidation correlated well with embryo development after insemination in vitro (P<0.05). Furthermore, oocytes that had similar glucose metabolism and that were grouped together for culture on this basis had higher (P<0.05) overall rates of development than oocytes grouped randomly. There was no correlation (P>0.05) between cumulus cell metabolism and individual oocyte development after in vitro fertilization. The data reveal that energy metabolism is linked to oocyte maturation in the cat and that glucose metabolic activity can indicate those oocytes most likely to fertilize and develop in vitro. Measuring cumulus cell metabolism does not accurately predict individual oocyte development after insemination in vitro.

Animals↗
PubMed2000

Normal detrusor is more sensitive than hypertrophied detrusor to in vitro ischemia followed by re-oxygenation.

Partial outlet obstruction results in marked metabolic as well as contractile alterations. Specifically, the ratio of anaerobic to oxidative metabolism is significantly greater in hypertrophied than normal bladder smooth muscle, lactate dehydrogenase (LDH) and lactic acid production are increased, and the contractile apparatus is altered to allow for metabolically more efficient tension generation. In addition, contractile responses of hypertrophied bladder are apparently more resistant than those of normal bladder to hypoxia. In the current experiment, we studied the effects of in vitro ischemia (hypoxia + substrate deprivation) followed by an in vitro model of reperfusion (re-oxygenation + substrate replacement) on contractile responses of normal and hypertrophied urinary bladder strips. We used repetitive field stimulation (FS) during the hypoxic period as a model for hyperreflexia. The purpose of the current study was to compare the responses of normal and hypertrophied bladder smooth muscle to repetitive stimulation in the presence of in vitro ischemia followed by re-oxygenation and substrate replacement. Thirty-two rats were separated into four groups of eight each. The rats in groups 1 and 3 were subjected to partial outlet obstruction. Two weeks later, all rats were anesthetized; their bladders were isolated and cut into four strips. Each strip was mounted in an isolated bath, and after 1-hour incubation in Tyrode's solution containing glucose (in the presence of O(2)), contractile responses to FS, carbachol, and KCl were determined. After this first set of stimulations, the strips were incubated without glucose and in the presence of N(2) for 30 minutes and 1 hour (groups 1 and 2); and for 2 and 4 hours (groups 3 and 4). For groups 1 and 2, the tissues were stimulated at 5-minute intervals with FS at 32 Hz, 1-millisecond duration, 3-second trains (in vitro model of hyperreflexia). For groups 3 and 4, no stimulations were performed during the ischemic period. At the end of the ischemic period, all strips were washed and incubated for 1 hour in the presence of O(2) and with glucose. At the end of this incubation, all strips received a second set of stimulations. a) Partial outlet obstruction resulted in a significant increase in bladder weight. b) Responses to in vitro ischemia: After in vitro ischemia, contractile responses of both normal and hypertrophied tissues to FS were reduced to a significantly greater degree than were responses to carbachol and KCl. The rate of development of contractile dysfunction was significantly greater in normal bladder tissue strips than in hypertrophied bladder strips. c) Responses to repetitive stimulation: The rate of development of contractile dysfunction was significantly greater in all strips subjected to repetitive stimulation than in those not repetitively stimulated; in addition, normal bladder strips were more sensitive than hypertrophied strips to hypoxia and substrate deprivation-induced contractile dysfunction. The rate of contractile failure induced by in vitro ischemia followed by re-oxygenation and substrate replacement was significantly greater for normal bladder strips than for hypertrophied bladder strips. These results indicate that, after partial outlet obstruction, the hypertrophied tissue is more resistant than normal tissue to hypoxia and substrate deprivation.

Animals↗
PubMed1977

Chronic lymphocytic leukemia: correlation of clinical course and therapeutic response with in vitro testing and morphology of lymphocytes.

Forty-two patients with chronic lymphocytic leukemia (CLL) were studied for morphology of lymphocytes by light and electron microscopy (EM), in vitro responses of lymphocytes to a battery of physical and chemical agents, overall clinical status, immunologic status, course, and response to therapy. CLL lymphocytes could be classified by EM into four groups on the basis of cell size and nuclear contour and by light microscopy into two groups, small cells and large cells (lymphosarcoma cells). Patient survival did not vary with cell size or morphology as determined by light or electron microscopy. In vitro testing of CLL lymphocytes following exposure to X-ray, PHA, DMSO 2 hr at 43 degrees C, prednisolone, glutaminase, and asparaginase permitted a separation of patients into categories of normal and abnormal in vitro responses. A normal in vitro response predicted a good response to therapy but an abnormal in vitro response did not preclude a good response to therapy. Following therapy, normalization of abnormal EM morphology and in vitro response was seen in some patients. Most patients tested had decreased serum immunoglobulins and abnormal PHA responses. There was a high incidence of infections and second neoplasms. Immunologic deficits could not be correlated with variations in lymphocyte morphology or in vitro response.

Asparaginase↗
PubMed1984

Effect of the 21-bp repeat upstream element on in vitro transcription from the early and late SV40 promoters.

The role of the 21-bp repeat region [simian virus 40 (SV40) coordinates 40-103] on early and late SV40 promoter functions has been investigated in vitro using a variety of mutated templates. Using either a HeLa whole cell extract or a S100 extract, we analyzed the transcripts by quantitative S1 nuclease mapping. GC-rich motifs contained in the 21-bp direct repeat constituted an essential element for efficient early transcription in vitro in agreement with previous in vivo results. These GC-rich motifs act in a non-polar fashion, since inversion of the 21-bp region did not reduce early transcription. Some point mutations in the 22-bp imperfectly repeated sequence, that drastically reduce initiations from the early promoter in vivo, had little effect in vitro, indicating that all the functions of these GC-rich motifs cannot be reproduced in vitro at present. The requirement for the 21-bp repeat region was less stringent when the concentration of the early promoter sequence was increased, which suggests that its function may be to facilitate the recognition of the 'weak' SV40 early TATA box. The multiple late start sites were accurately used in vitro and the GC-rich motifs contained in the 21-bp repeat region were an important element for efficient in vitro initiation of transcription from the late promoter, irrespective of their orientation. However, the effect of the 21-bp repeat region on late initiations decreased strikingly with increasing distance to the start sites, although it was still detectable over a distance of 220 bp. Under the present in vitro conditions, the 72-bp repeat region stimulates weakly both early and late transcription.

Chromosome Deletion↗
PubMedDate not supplied

The kinetics of baboon fibrinogen adsorption to polymers: in vitro and in vivo studies.

Fibrinogen adsorption on polymers from blood may mediate or potentiate thrombosis because of its involvement in both the intrinsic clotting system and the formation of platelet aggregates. While the kinetics of fibrinogen adsorption from plasma in vitro have previously been found to be very different on polar and nonpolar surfaces [T. A Horbett, "The kinetics of adsorption of plasma proteins to a series of hydrophilic-hydrophobic copolymers," ACS Org. Coat. Plas. Chem. 40, 642-646 (1979)] the significance of this difference with respect to thrombogenesis in vivo has not been clarified. In this study, the kinetics of deposition of baboon 125I fibrinogen from plasma in vitro or from blood in vivo on a series of polymers was measured. The polymers chosen for this study had previously been found to have a large range in surface polarity and reactivity in the in vivo baboon shunt model. The kinetics of fibrinogen adsorption in vitro were observed to be of three types, depending on the polymer: high initial adsorption decreasing to a lower steady state value; constant throughout the time course; low initial adsorption rising steadily to a plateau value. In vivo, fibrinogen deposition kinetics were of two types: low, constant deposition throughout the time course, independent of heparinization; low deposition initially followed by a second phase of greatly increased deposition (probably as fibrin) which was prevented or greatly decreased by heparinizing the animals. Polymers for which fibrinogen adsorption increased to a plateau in vitro were found to have a heparin inhibitable second phase of enhanced in vivo fibrinogen deposition. These polymers also have been found in previous studies to enhance the rate of platelet destruction when used as in vivo shunts on baboons. Conversely, most polymers with high initial in vitro fibrinogen adsorption followed by a decrease had low fibrinogen deposition behavior in vivo and were also minimally destructive of platelets. The adsorption kinetics of fibrinogen to polymers from blood in vivo and in vitro and the consumption of platelets in vivo induced by the polymers all vary with polymer polarity. More polar polymers had in vitro fibrinogen kinetics characterized by a rise to a plateau, in vivo fibrinogen deposition characterized by a second stage of great increase inhibitable by heparin, and enhanced platelet consumption. The correlation of three separate indicators of surface thrombogenicity with surface polarity suggests that more polar materials may be more thrombogenic because of an influence on the way in which fibrinogen interacts with these surfaces.

Adsorption↗
PubMed2004

Lysolecithin induces demyelination in vitro in a cerebellar slice culture system.

Demyelination is a hallmark of several human diseases, including multiple sclerosis. To understand better the process of demyelination and remyelination, we explored the use of an in vitro organotypic cerebellar slice culture system. Parasagittal slices of postnatal Day 10 (P10) rat cerebella cultured in vitro demonstrated significant myelination after 1 week in culture. Treatment of the cultures at 7 days in vitro (DIV) with the bioactive lipid lysolecithin (lysophosphatidylcholine) for 15-17 hr in vitro produced marked demyelination. This demyelination was observed by immunostaining for the myelin components myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). After a transient demyelinating insult with lysolecithin in vitro, the cultures recovered with oligodendrocyte differentiation recapitulating a normal time course; there was initially re-expression of CNPase and MBP during this recovery, and this was followed by MOG. In addition, there seemed to be some limited remyelination during the recovery phase. Lysolecithin thus induces demyelination in an in vitro organotypic cerebellar slice culture system, providing a model system for studying myelination, demyelination, and remyelination in vitro.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed2004

Ritonavir-PEG 8000 amorphous solid dispersions: in vitro and in vivo evaluations.

Ritonavir is a large, lipophilic molecule that is practically insoluble in aqueous media and exhibits an exceedingly slow intrinsic dissolution rate. Although it has favorable lipophilicity, in vitro permeability studies have shown that ritonavir is a substrate of P-glycoprotein. Thus, the oral absorption of ritonavir could be limited by both dissolution and permeability, thereby making it a Class IV compound in the Biopharmaceutics Classification System. Because formulations rarely exert direct influence on local intestinal permeability, the effect of enhanced dissolution rate on oral absorption was explored. More specifically, poly(ethylene glycol) (PEG)-amorphous ritonavir solid dispersions were prepared with different drug loadings, and the in vitro and in vivo performances of the dispersions were evaluated. In vitro dissolution was conducted in 0.1N HCl with a USP Apparatus I. A crossover design was used to evaluate the oral bioavailability of amorphous dispersions relative to crystalline drug in beagle dogs. Intrinsic dissolution measurements of the two solid phases indicated a 10-fold improvement in intrinsic dissolution rate for amorphous ritonavir compared with the crystalline counterpart. In vitro dissolution of ritonavir depended on the solid phase as well as drug loading of the dispersion. In vivo study results indicate that amorphous solid dispersions containing 10-30% drug exhibited significant increases in area under the curve of concentration versus time (AUC) and maximum concentration (C(max)) over crystalline drug. For example, 10% amorphous dispersion exhibited increases of 22- and 13.7-fold in AUC and C(max), respectively. However, both in vitro dissolution and bioavailability decreased with increasing drug load, which led to the construction of a multiple Level C in vitro-in vivo relationship for this Class IV compound. The established relationship between in vitro dissolution and in vivo absorption can help guide formulation development.

Absorption↗