Biodegradable polymer film as a source for formation of human fetal retinal pigment epithelium spheroids.
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PURPOSE: To evaluate the attachment of human fetal rctinal pigment epithelial (HFRPE) cells to a biodegradable polymer film with subsequent formation of spheroids in vitro. METHODS: Ten biodegradable polymer films with different compositions were examined for their physical properties and ease of manipulation under a dissecting microscope. The film with the most suitable handling characteristics was chosen, and a purely isolated sheet of HFRPE cells was attached to it. The purity of the cells was assessed by their pigmentation and expression of cytokeratin. Proliferation was assessed by incorporation of 5-bromo-2'-deoxyuridine (BrdtJ). Cellular structure was analyzed under light and electron microscopes, and the functional capability of the cells was evaluated by rod outer segment (ROS) phagocytosis. RESULTS: The polymer film with composition 50:50 poly (DL-lactide) (PLA)/poly (DL-lactide-co-glycolide) (PLG) with an inherent viscosity of 1.03 dl/g was found to be the most suitable for handling under the microscope. Sheets of HFRPE cells attached to the polymer films within 48 hours and began to form spheroids. All the isolated cells were pigmented and expressed cytokeratin. They possessed a cuboidal morphology, numerous apical microvilli, and no sign of dedifferentiation. HFRPE cells produced extracellular matrix (collagen filaments) on their basal side, filling the cavities of the polymer film. The cells subsequently proliferated, incorporated BrdU, migrated onto the culture plate to form monolayers, and phagocytized ROS. CONCLUSIONS: Biodegradable polymer films can be used as a scaffold for the adhesion of the HFRPE sheet and formation of spheroids. Spheroids represent a source of high density and well-differentiated HFRPE cells that are easy to transfer. Furthermore, the stricture of the membrane makes it suitable for additional applications. (+info)
Fetal hemoglobin (HbF) synthesis in baboons, Papio cynocephalus. Analysis of fetal and adult hemoglobin synthesis during fetal development.
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Fetal hemoglobin (HbF) and adult hemoglobin (HbA) synthesis was studied in fetal baboons, Papio cynocephalus, to determine the normal pattern of hemoglobin production during fetal development. Fetuses ranging from 53 to 180 days gestation (term gestation 184 days) were used. Erythroid cells were incubated with 3H-L-leucine, and the rates of globin chain synthesis and the distribution of radioactivity into hemoglobin intermediates and completed hemoglobin molecules were determined. Gamma chain synthesis accounted for approximately 97% of the total nonalpha chain synthesis up to 140 days gestation; beta chain synthesis accounted for the remainder. After 140 days gestation, approximately equal quantities of gamma and beta chain were synthesized in the bone marrow. Prior to 140 days gestation, total alpha chain synthesis was 30% greater than total non-alpha chain synthesis, while there was balanced chain synthesis after 140 days gestation. During the period of excess alpha chain synthesis, fetal erythrocytes contained a large pool of alpha-hemoglobin (alpha chain with heme attached) molecules uncombined with beta or gamma chains. In view of the possibility that alpha chains may have a lower affinity for gamma chains than beta chains, excess alpha chain synthesis may be required to maintain low levels of free gamma chains. (+info)
The proliferation of transplanted haematopoietic cells derived from bone marrow and fetal liver.
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The proliferation of transplanted murine haematopoietic cells, derived from the femoral bone marrow of young adults and from the liver on the sixteenth day of gestation, has been compared under standardized conditions. Suspensions of bone marrow cells contained 9-6+/-0-9 CFU per 10(5) cells (sm) and suspensions of fetal liver cells contained 3-7+/-0-5 CFU per 10(5) cells (Sl).Sl/Sm = 0-4. The mean diameter of colonies established by cells derived from bone marrow was 1-3+/-0-3 mm (established mean volume, Vm = 1-2 mm3) and the mean diameter of colonies established by cells derived from fetal liver was 1-7+/-0-1 mm (estimated mean volume, Vl ;-6 mm3). Vl/Vm ;-2. The relationship between the weight of the spleen and the number of bone marrow cells injected into lethally irradiated recipients 10 days previously has been confirmed, and a similar relationship between the weight of the spleen and the number of fetal liver cells injected has been demonstrated. An arbitrarily defined difference of 50 mg between the weight of the spleen in untreated irradiated controls and the weight of the spleen in irradiated recipients of haematopoietic cells has been observed 10 days after the administration of 2-2 x 10(6) bone marrow cells (Dm) or 3-0 x 10(6) fetal liver cells (Dl). Dm/Dl = 0-7. The calculated values of Dm/Dl (relative proliferative capacity), Sl/Sm (relative stem cell content) and Vl/Vm (relative clone size) are in good agreement with the values estimated using the equation Dm/Dl = Sl/Sm x Vl/Vm. (+info)
Inability of the smallest light chain to bind to fetal fast muscle myosin.
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1. The smallest light chain of myosin, g3, was not transferred from adult HMM to fetal myosin in alkali (pH 10.5) under conditions when the light chains dissociated from myosin. 2. The g3 isolated from adult myosin did not bind to fetal myosin at either pH 7.8 or 10.5. (+info)
Carcinogenesis: a late effect of irreversible toxic damage during development.
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Intrauterine and early postnatal life are periods of exceptionally high susceptibility to certain kinds of chemical carcinogens. The most potent known transplacental carcinogens are direct acting alkylating agents. Most nonreactive compounds, which require enzymes for metabolic conversion into chemically reactive "proximate carcinogens," are less effective because the required enzymes are present at low levels in the fetus, and many proximate carcinogens are too reactive to reach the fetus when formed in maternal tissues. Despite this, many carcinogens which require metabolic activation are very active transplancentally, as the intrinsic susceptibility of rapidly dividing fetal cells compensates effectively for comparatively low tissue levels of reactive metabolites. Transplacental carcinogens of all kinds are most effective late in gestation, generally after organogenesis has begun and after the period of greatest susceptibility to teratogens. Only a small number of known carcinogens have been tested for transplacental carcinogenic activity. The great majority of tumors induced transplacentally in the well-studied rodent and lagomorph species (mouse, rat, Syrian hamster, and rabbit) have morphologic features of adult, rather than embryonal, tissues. A given agent tends to induce in a given species largely the same types of tumor when given transplacentally as when administered directly to postweaning animals, unless its carcinogenic effect in the latter is ascribable to some peculiarity of distribution, metabolism, or physiology. In a second species, the spectrum of tumors induced either before of after birth may be quite different. For bioassay of suspected carcinogens, the significance of perinatal carcinogenesis lies in the facts that the fetal and preweaning rodent is an extremely sensitive indicator of carcinogenic activity, and that the facile adaptibility of fetal cells to tissue culture and their rapid expression in vitro of properties of neoplastic transformation make possible a rapid in vivo/in vitro screening system for chemical carcinogens. (+info)
Techniques for assessment of teratogenic effects: developmental enzyme patterns.
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Most studies designed for assessing teratogenic effects focus on only three of four types of final manifestations of abnormal development; namely intrauterine death, malformations, and growth retardation. Developmental toxicity evaluations generally do not include functional deficits. Current techniques are inadequate for assessing functional capability during perinatal development, and there is a need for improved measures. Thus, measurement of developmental enzyme patterns is proposed as an approach that directly evaluates acquisition of metabolic competence of fundamental organ systems. During ontogenesis most organs acquire their full complement of enzyme activities in a programmed sequence which corresponds to attainment of complete functional capability. The usual alterations in enzyme activity that characterize these patterns occur at one of three time periods, namely, late fetal, early neonatal, or late suckling. Qualitative or quantitative changes in these patterns at any time by one or more key enzymes of a tissue could be indicative of developmental toxicity. Factors are outlined relating to consideration of developmental enzyme profiles as indices of maturation capable of reflecting the action of toxic agents. This presentation: (1) reviews the current state of the art for evaluating effects on development, (2) considers the applicability of enzyme patterns for biochemical assessment of development, (3) characterizes the target tissues and those metabolic pathways and/or specific enzymes most sensitive and adaptable to practical toxicity evaluations, and (4) describes the steps being taken to validate this system. The ultimate objective of this approach is to determine whether alterations in developmental enzyme profiles will provide a technique with improved capability for assessing developmental toxicity. (+info)
Behavioral testing as a method for assessing risk.
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Behavioral effects have been found to result from the prenatal administration of substances known to be teratogenic to the CNS. These effects occur at dose levels lower than those producing gross malformations and when the agent is administered at times other than that optimal for CNS relevant technique for detecting adverse consequences of prenatal exposure to drugs and chemicals. Behavioral testing, however, also appears to have attributes that dictate a thoughtful approach to its role as a method for assessing risk, and additional research is needed to obtain usable techniques. The need for such research is intensified by the present inability to identify potential behavioral teratogens by means other than laboratory investigation. (+info)
Research strategies for behavioral teratology studies.
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Several compelling aruguments have been advanced in support of expanding the use of "behavioral teratology" evaluations as routine components of toxicologic screening procedures. As a basis for development of effective behavioral teratology screening approaches, a conceptual framework is presented which interrelates: (1) changes in relative functional brain capacity with age, (2) possible times and durations of exposures to environmental insults, and (3) various types of toxicity testing procedures carried out at appropriate time points in relation to different exposure period. Within the context, several research strategies for behavioral teratology studies are concisely posed and evaluated. These include: (1) clinical hypothesis testing, where particular effect(s) of a given agent are evaluated based on hypotheses derived from clinical or epidemiological observations; (2) comprehensive screening approaches, where multifaceted, long-term longitudinal neurobehavioral evaluations are employed to assess whether any of a large number of possible deletarious effects are exerted by an agent and at what threshold exposure levels; (3) alternative screening heuristics, by which adequate assessments of neurobehavioral toxicity of various agents may be accomplished without completion of more exhaustive, but also more expensive and time-consuming comprehensive screening protocols. (+info)