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.

Production of soluble human alpha3-fucosyltransferase (FucT VII) by membrane targeting and in vivo proteolysis.

The rational design of fucosyltransferase (FucT VII) inhibitors as potential medication in the treatment of rheumatoid arthritis requires the three-dimensional structure of this member of the glycosyltransferase family. Structure determination by X-ray diffraction analysis needs purified, soluble enzyme protein. For this purpose we developed a novel method for the high-yield production of soluble FucT VII by in vivo proteolysis. To obtain a soluble form of FucT VII a mammalian expression construct was made encoding an N-terminal portion of FucT VI (amino acids 1-63) fused with the stem region and catalytic domain of FucT VII (amino acids 39-342). Chinese hamster ovary cells stably transfected with this construct produced FucT activity in the supernatant, which has the same catalytic properties as wild-type FucT VII. This soluble form of FucT VII can be obtained in high amounts (1 mg/L) and can be efficiently purified by GDP-hexanolamine affinity chromatography. In conclusion, it was demonstrated that the intrinsic properties of FucT VII could be transferred to secreted FucT VII constructs, which may open possibilities for production of soluble forms of other members of the glycosyltransferase family as well.

The magnetic immuno polymerase chain reaction assay for direct detection of salmonellae in fecal samples.

Direct polymerase chain reaction (PCR)-based detection with fecal specimens is hampered by inhibitory compounds, such as bilirubin and bile salts. These fecal compounds showed significant inhibition of PCR at low concentrations (10 to 50 micrograms/ml). For direct PCR analysis, fecal samples must be diluted 500-fold to overcome inhibition. Therefore, the magnetic immuno PCR assay (MIPA), which combines immunomagnetic separation by using specific monoclonal antibodies and PCR, was used to directly detect salmonellae in feces from humans. Immunomagnetically extracted stool samples needed to be diluted only 10-fold when 1 microgram of T4 gene 32 protein was added to the PCR. The MIPA sensitivity obtained was 10(5) CFU/ml of feces. A panel of monoclonal antibodies specific for Salmonella serogroups A to E was used to extract salmonellae from clinical samples. MIPA detection of salmonellae occurred with 11 out of 14 stool samples stored at 4 degrees C for 2 months. MIPA detection of salmonellae in stool samples is a promising, fast method for detection and identification.

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.

Nontoxigenic strains of Clostridium difficile lack the genes for both toxin A and toxin B.

A total of 39 toxigenic and 20 nontoxigenic strains of Clostridium difficile were tested for the presence of either toxin A or toxin B by the polymerase chain reaction (PCR). All toxigenic strains produced cytotoxin as assayed by using highly sensitive fetal lung fibroblasts and were positive for toxin A as well as toxin B in the PCR assay. All nontoxigenic strains failed to produce toxin and were negative in the PCR assay. This study shows that nontoxigenic strains of Clostridium difficile lack the toxin A as well as the toxin B gene.

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)

[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.

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.

Minor interference of low-dose pulses of GnRH with the positive effect of oestradiol on the pituitary gland.

In the ovariectomized rat we investigated the effects of prolonged treatment with oestradiol and 'physiological' pulses of exogenous GnRH on the GnRH-responsiveness of the pituitary gland. Rats were for 20 h pre-treated with either GnRH (3-ng pulses of 3 min duration every 20 min) or saline and for 19 h with either oestradiolbenzoate (OB; 3 micrograms/sc injection) or oil. Then LH and FSH responses were evoked by continuous test infusions of GnRH at the rates of either 60, 100, 150 or 1000 ng/h, lasting 10 h. It appeared that, irrespective of pre-treatment with either GnRH or saline, OB caused an increase of the maximal LH and FSH responses (that is the responses to the maximal stimulus of 1000 ng GnRH/h), and an increase of the GnRH-sensitivity of the pituitary gland, as far as the FSH secretion is concerned. GnRH pulses caused, irrespective of pre-treatment with OB or oil, a decrease of the GnRH-sensitivity of the pituitary gland, as well as a decrease of the maximal FSH response and of the pituitary FSH content. It was concluded that low-dose pulses of exogenous GnRH desensitize the pituitary gland and deplete the pituitary FSH stores, but do not change the positive effect of oestradiol on the maximal gonadotropin response.

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)

Decidua and the control of corpus luteum function, follicular development and pituitary LHRH-responsiveness in pseudopregnant and pregnant rats.

The mid-pregnancy rescue of corpora lutea can be mimicked in the pseudopregnant rat by induction of decidual tissue in the uterus: in such rats, around day 10, there is neither luteolysis, nor resumption of follicle-development or increase of the pituitary responsiveness to LHRH. The results suggest that the mid-pregnancy rescue of corpora lutea is caused by a maternal factor.

Studies on the modulation of the desensitization of the pituitary gland by luteinizing hormone-releasing hormone in the ovariectomized rat.

In ovariectomized rats the desensitization of the LH cells in vivo, which develops during constant rate infusion of LHRH, 1) does not depend on a concomitant depletion of the pituitary LH stores, 2) proceeds normally when the hypothalamo-pituitary connection has been severed and 3) is a process in which LH itself is not involved.

Effect of supplementing medium chain triglycerides with linoleic acid-rich monoglycerides on severely disturbed serum lipid fatty acid patterns in patients with cystic fibrosis.

7 patients with cystic fibrosis, having serum lipids with fatty acid compositions (FACs) skewed in the direction of essential fatty acid deficiency, were given a mixture of medium chain triglycerides (MCTs) with linoleic acid-rich monoglycerides (LAMs) as food fat (about 1-1.25 g/kg body weight/day). Blood was sampled before and after a short period (4-11 days) of experimental fat feeding. The FACs of the serum triglycerides (TGs), cholesterol esters (CEs) and phospholipids (PLs) were determined before and after dietary treatment. Dietary treatment resulted in a significant increase of the previously reduced fraction of linoleic acid in all lipid classes. In TGs, the increase in the fraction of linoleic acid was accompanied by a reduction of the previously increased fraction of palmitoleic acid to normal values, by a reduction in the fraction of oleic acid to subnormal values and by an increase in the fraction of stearic acid which, however, remained normal. In PLs, the increase in the fraction of linoleic acid was accompanied by an increase in the fraction of dihomogammalinoleic acid which remained normal, however. In CEs, concomitant with the increase of the previously reduced linoleic acid fraction, there was a reduction of previously elevated palmitoleic and oleic fractions, the latter to a normal value. Thus, supplementing a MCT-containing diet with LAMs in cystic fibrosis patients results in a considerable amelioration of the previously disturbed FACs of the major serum lipid classes.

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)