In vitro aging of human bone marrow derived stromal cells.

We have studied the in vitro cellular aging process in human bone marrow derived stromal cells. Two cell types, here called type I and type II, can be distinguished in the primary cell population. During subcultivation, resulting in aging in vitro, the type I cells gradually disappear, whereas the type II cells form an increasing fraction of the population. This observation is confirmed by cloning experiments, revealing that type I cells act as progenitor cells, giving rise to non-dividing type II cells. These results are best explained by the terminal differentiation theory as proposed by Martin et al.

Variations in the stromal cell population of human bone marrow during aging.

Age-related variations in the stromal cell population of human bone marrow were studied by means of a cloning assay. It was found that these cells are heterogeneous both in division capacity and in morphology. Fibroblast-like progenitor cells could be distinguished from epithelial-like cells, having only a very limited division potential. A significant inverse correlation between the donor age and the number of progenitor cells could be demonstrated. This loss of progenitor cells during aging was compensated by an increase in the number of epithelial-like cells.

Similarity between in vitro and in vivo cellular aging.

In vivo and in vitro cellular aging were compared by determining the division capacity of individual, cloned cells of (1) the primary stromal population of human bone marrow, obtained from donors of various ages, and (2) the population at various passage levels of an in vitro subcultivated culture of the same origin. We find a strong similarity between the two series of data. This observation provides a further argument that cellular aging in vitro represents a biologically relevant phenomenon.

Blockade of LH and FSH secretion by LH-releasing hormone, by the LH-releasing hormone analogue, buserelin, and by combined treatment with LH-releasing hormone and oestradiol benzoate.

The LH and FSH release-stimulating (experiment 1) and -blocking (experiment 2) effects of LH-releasing hormone (LHRH) and of the LHRH analogue D-Ser(But)6-des-Gly10-LHRH-ethylamide (buserelin), as well as the effect of combined treatment with LHRH and oestradiol benzoate (OB; experiment 3) on the 'supra-maximally' LHRH-stimulated release of LH and FSH were studied in rats ovariectomized for 2 weeks. Pretreatment with LHRH (250 or 500 ng/h) or buserelin (250 ng/h) for 6 days was effected by means of subcutaneously implanted Alzet osmotic minipumps; control rats received a 'sham pump', i.e. a piece of silicone elastomer with the dimensions of a minipump. Oestradiol benzoate (3 micrograms/injection) or solvent was injected subcutaneously 75 and 27 h before the induction of LH/FSH responses. Experiment 1 revealed that after infusion of LHRH and buserelin, both at the rate of 1 microgram/h, plasma LHRH concentrations were established which were about twice as low as the plasma buserelin concentrations. This might suggest that buserelin has a longer half-life than LHRH. As an LH and FSH release-stimulating substance, however, it appeared that buserelin was about as effective as LHRH. Experiment 2, however, suggested that as an LH/FSH release-blocking agent buserelin was much more effective than LHRH. In addition, after buserelin pretreatment the pituitary glands contained much less LH and FSH than after LHRH pretreatment at both dose levels used.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of luteolysis during late pregnancy on pituitary responsiveness to gonadotrophin-releasing hormone in the rat.

We investigated whether the increase in the gonadotrophin response to gonadotrophin-releasing hormone (GnRH) during the last days of pregnancy and the occurrence of parturition on day 22 of pregnancy in rats are due to the increase in the plasma concentrations of oestradiol-17 beta after luteolysis, which occurs around day 20. In a first series of experiments we studied the effects of s.c. implantation of two capsules containing oestradiol on basal and GnRH-stimulated secretion of LH and FSH before and after luteolysis. Before luteolysis, ovariectomy increased basal LH and FSH; oestradiol treatment prevented this increase partly (FSH) or completely (LH). Ovariectomy also lowered the LH response to the infusion of GnRH (100 ng/h). Oestradiol treatment on the other hand, increased the LH and FSH responses of both intact and ovariectomized rats above the level in intact non-treated control rats. After luteolysis, ovariectomy increased basal FSH only. Treatment with oestradiol did not prevent the increase in basal FSH and ovariectomy diminished the LH response to GnRH infusion. Oestradiol treatment maintained the LH response in ovariectomized rats at the control level and increased the FSH responses of both intact and ovariectomized rats to a higher level than in control rats. Furthermore, the LH and FSH responses of the oestradiol-treated groups of intact and ovariectomized rats were higher after luteolysis than before. In a second series of experiments two capsules containing progesterone were s.c. implanted before or after luteolysis. Progesterone treatment suppressed the plasma concentration of oestradiol and the gonadotrophin responses to infusion of GnRH on the expected day of parturition in both groups of rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Pituitary responsiveness to LHRH during pregnancy in the rat: effect of progesterone.

The LH and FSH responses to a standard infusion of LHRH were studied on days 8, 12, 15, 17, 19, 21 and 22 after conception as well as on day 23, i.e. after parturition. Groups of rats were also killed on days 8, 15, 19, 22 and 23 and on the day of pro-oestrus of the 4-day cycle for the assay of progesterone, 20 alpha-dihydroprogesterone (DHP), oestradiol-17 beta, LH and FSH. Finally, the post-partum surges of LH and FSH were compared with those at pro-oestrus in 4-day cyclic rats. The LH and FSH responses to LHRH were relatively low on days 8 and 12, twice as high on days 15, 17 and 19, had increased further on day 21 and reached maximal values on day 22. The gonadotrophin responses were low on day 23. The post-partum surges of LH and FSH were much higher than the pro-oestrous surges. Pituitary contents of LH and FSH were higher than on pro-oestrus of the 4-day cycle. On day 23, however, the pituitary contents had declined by 60-80%. No apparent relationship was found between plasma concentrations of LH and FSH and LHRH responsiveness during pregnancy. Concentrations of oestradiol-17 beta on day 22 were higher than on all other days of pregnancy, but lower than on pro-oestrus of the 4-day cycle. Concentrations of progesterone were high until day 19 and low on days 22 and 23; the concentration of DHP was low until day 19 and very high on days 22 and 23.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of follicle-stimulating hormone secretion by steroid-free bovine follicular fluid in the ovariectomized rat.

The effects of steroid-free bovine follicular fluid (bFF) and sodium phenobarbitone on spontaneous LH releasing hormone (LHRH)-induced secretion of FSH and LH were studied in ovariectomized rats. Luteinizing hormone releasing hormone was administered by infusion to rats anaesthetized with phenobarbitone. Bovine follicular fluid reduced FSH release and synthesis. Luteinizing hormone release remained unaffected after bFF treatment. Phenobarbitone reduced both FSH and LH release. The observed suppressive effects of bFF and phenobarbitone on FSH secretion were additive, suggesting that the basal release of FSH has an LHRH-dependent and an LHRH-independent component. Furthermore, bFF did not affect pituitary responsiveness of LH secretion to LHRH and reduced the responsiveness of FSH secretion only when administered some time before the LHRH challenge. The present observations support the view that in the ovariectomized rat the pituitary gland is the only site of action of inhibin-like activity as present in bFF.

A comparison of the LH-releasing activities of LH-RH and its agonistic analogue buserelin in the ovariectomized rat.

LH-RH and the potent agonistic analogue (D-Ser(But)6-des-Gly10)-LH-RH(1-9)-ethylamide (HOE-766 or buserelin) were at several doses either infused or injected intravenously in 5-weeks-ovariectomized rats, which had been treated with either 3 micrograms estradiol-benzoate (EB) or with oil, 24 h previously. Blood samples for assay of LH were taken during the subsequent 24 h. Pituitary glands were removed at the end of the experiments. Buserelin, when infused, was slightly more effective than LH-RH on releasing LH. When injected, however, buserelin was at the higher dose ranges increasingly more effective as an LH-releasing agent than LH-RH. EB-treatment increased the LH response of the pituitary gland to both peptides in an identical way. It was concluded that buserelin derives its high potency not from its intrinsic LH-releasing activity, which is only slightly greater than that of LH-RH, but from a longer duration of action.

Glucose and insulin responses during mixed meals or infusion of glucose in pregnant and lactating rats.

We studied the glucose tolerance in freely moving rats throughout pregnancy and lactation and during the first week after weaning. Dioestrous virgin rats served as controls. Basal glucose and insulin levels were determined after a 2-hr fasting period. Subsequently, the changes of the insulin and the glucose levels were determined during ingestion of a mixed ad lib meal or a 2 g mixed test meal, or during infusion of glucose (7.4 mg/min for 20 min) into the vena cava. Basal glucose levels were high during early pregnancy, low during late pregnancy, and in the normal range throughout lactation and after weaning. Basal insulin levels were decreased at the end of lactation. The results of the ad lib meal and test meal experiments were essentially the same. Glucose tolerance during meals was somewhat decreased early in pregnancy. The corresponding insulin responses greatly increased during the last week of pregnancy. Glucose tolerance during IV infusion of glucose was normal during pregnancy, but increased during lactation. Insulin responses to the infusion were increased during pregnancy and decreased during lactation. We concluded that glucose tolerance is hardly affected by pregnancy and even increases in the course of lactation. This is effected by an increased responsiveness of the B-cells to glucose during late pregnancy and by an increased turnover of glucose during lactation. We discuss to what extent the actions of progesterone, placental lactogen and prolactin may explain these adaptions of maternal metabolism.

Fatty acid chain length combinations in ascitic fluid triglycerides containing lymphatic absorbed medium-chain fatty acids.

Information on the fatty acid chain length combinations in lymph triglycerides containing medium-chain fatty acids was obtained by combining the fatty acid composition and the carbon number composition of triglycerides from ascitic fluid of patients on medium-chain triglyceride-containing diets. In these triglycerides, the major part of the medium-chain fatty acids was present in combination with long-chain fatty acids. These results indicate that part of the lymphatic absorbed medium-chain fatty acids are absorbed as triglycerides which also contain long-chain fatty acids.

Prenatal ultrasound diagnosis of an intracranial teratoma influencing management: case report and review of the literature.

Of seventeen cases of prenatally diagnosed intracranial teratomas, fourteen babies were delivered through a cesarean section. All children died before or shortly after delivery. In this report another case is presented of ultrasound diagnosis of an intracranial teratoma. The teratoma filled the whole fetal cranium, and eroded through the skull base into the left maxillary region. In full agreement with the wishes of the parents, conservative management resulted in the spontaneous vaginal delivery of a fetus that died during labor as a result of brain compression and acidosis during the second stage. In contrast with other cases in the literature, no encephalocentesis was performed. In order to minimize maternal morbidity, it is advised to induce or accept labor as soon as the diagnosis is clear and to pursue a vaginal delivery.

The identification of atypical reserve cells in smears of patients with premalignant and malignant changes in the squamous and glandular epithelium of the uterine cervix.

Atypical reserve cells can be recognized in cervical smears by their characteristic exfoliation pattern and the fragility of their cytoplasm. The frequency of cases with such cells in 30,000 cervical smears is given, as well as that in 440 cases of dysplasia, epidermoid carcinoma in situ and invasive carcinoma. The presence of atypical reserve cells was found to increase in frequency with the increasing severity of the lesions. The bipotential character of these cells is discussed in relation to the possible progression to carcinoma in situ of the adenomatous type in two of the 42 cases of pure atypical reserve cell hyperplasia, and the presence of these cells in the majority of the 43 cases of adenocarcinoma of the uterine cervix in the present series.

[Diet treatment of the diabetic patient: yesterday and today. What has changed? What can be learned from it?]. / De dieetbehandeling van de diabeticus: vroeger en nu. Wat is er veranderd? Wat is eruit geleerd?

The initial treatment in the past of diabetes was one "of trial and error" as easily understandable. Carbohydrate restriction and their replacement by fats was followed, happily, last century already, by global caloric restriction. Around the fourties, after the introduction of insulin in 1922, the principle of the carbohydrate tolerance was introduced as an objective+ measure, followed by the proposal of "bread equivalencies" in the dietetic practice, assuring in this manner a figured evaluation. Around the seventies years the diet fiber came into its own and seemed very important for the evolution of the postprandial glycemia and insulinemia. Next to this, complex polysaccharides looked also of prime impact on this two biochemical parameters. The nature of this complex character is due to their liaison with other diet components (lectins, phytins, tannins etc.). So the concept of the glycemic index was born which explains why next to the presence of a given carbohydrate quantity the evolution of the glycemia is different from what is expected, just because the absorption is accelerated or retarded by the structure or the manipulation of these polysaccharides. This novel data are so much the more important because actually the hyperglycemia on the long run seems so important for the development of micro-angiopathy, at the base of the famous diabetic triad (nephropathy, retinopathy, neuropathy). The hyperlipemia is also beneficially influenced by the diet fiber and at the same level by the complex polysaccharides. All this statements are at the base of novel ways for the dietetic treatment of diabetes, as well on the quantitative as on the qualitative level and equally so for the diabetes of type I as of type II.

The Most Probable Limit of Detection (MPL) for rapid microbiological methods.

Classical microbiological methods have nowadays unacceptably long cycle times. Rapid methods, available on the market for decades, are already applied within the clinical and food industry, but the implementation in pharmaceutical industry is hampered by for instance stringent regulations on validation and comparability with classical methods. Equivalence studies become less relevant when rapid methods are able to detect only one single microorganism. Directly testing this capability is currently impossible due to problems associated with preparing a spiked sample with low microbial counts. To be able to precisely estimate the limit of detection of rapid absence/presence tests, the method of the most probable limit is presented. It is based on three important elements; a relatively precise quantity of microorganisms, a non-serial dilution experiment and a statistical approach. For a set of microorganisms, a limit of detection of one was demonstrated using two different rapid methods.