Impact of Biometric Patient Data, Probiotic Supplementation, and Selected Gut Microorganisms on Calprotectin, Zonulin, and sIgA Concentrations in the Stool of Adults Aged 18-74 Years.

Alterations to the intestinal barrier may be involved in the pathogenesis of various chronic diseases. The diagnosis of mucosal barrier disruption has become a new therapeutic target for disease prevention. The aim of this study was to determine whether various patient demographic and biometric data, often not included in diagnostic analyses, may affect calprotectin, zonulin, and sIgA biomarker values. Stool markers' levels in 160 samples were measured colorimetrically. The analysis of twenty key bacteria (15 genera and 5 species) was carried out on the basis of diagnostic tests, including cultures and molecular tests. The concentrations of selected markers were within reference ranges for most patients. The sIgA level was significantly lower in participants declaring probiotics supplementation (p = 0.0464). We did not observe differences in gastrointestinal discomfort in participants. We found significant differences in the sIgA level between the 29-55 years and >55 years age-related intervals groups (p = 0.0191), together with a significant decreasing trend (p = 0.0337) in age-dependent sIgA concentration. We observed complex interdependencies and relationships between their microbiota and the analyzed biomarkers. For correct clinical application, standardized values of calprotectin and sIgA should be determined, especially in elderly patients. We observed a correlation between the composition of the gut community and biomarker levels, although it requires further in-depth analysis.

Efferent projections of Vglut2, Foxp2, and Pdyn parabrachial neurons in mice.

The parabrachial nucleus (PB) is a complex structure located at the junction of the midbrain and hindbrain. Its neurons have diverse genetic profiles and influence a variety of homeostatic functions. While its cytoarchitecture and overall efferent projections are known, we lack comprehensive information on the projection patterns of specific neuronal subtypes in the PB. In this study, we compared the projection patterns of glutamatergic neurons here with a subpopulation expressing the transcription factor Foxp2 and a further subpopulation expressing the neuropeptide Pdyn. To do this, we injected an AAV into the PB region to deliver a Cre-dependent anterograde tracer (synaptophysin-mCherry) in three different strains of Cre-driver mice. We then analyzed 147 neuroanatomical regions for labeled boutons in every brain (n = 11). Overall, glutamatergic neurons in the PB region project to a wide variety of sites in the cerebral cortex, basal forebrain, bed nucleus of the stria terminalis, amygdala, diencephalon, and brainstem. Foxp2 and Pdyn subpopulations project heavily to the hypothalamus, but not to the cortex, basal forebrain, or amygdala. Among the few differences between Foxp2 and Pdyn cases was a notable lack of Pdyn projections to the ventromedial hypothalamic nucleus. Our results indicate that genetic identity determines connectivity (and therefore, function), providing a framework for mapping all PB output projections based on the genetic identity of its neurons. Using genetic markers to systematically classify PB neurons and their efferent projections will enhance the translation of research findings from experimental animals to humans.

Exogenous and endogenous determinants of vitamin K status in cystic fibrosis.

Cystic fibrosis (CF) patients are at high risk for vitamin K deficiency. The effects of vitamin K supplementation are very ambiguous. Therefore, we aimed to define the determinants of vitamin K deficiency in a large cohort of supplemented - 146 (86.9%) and non-supplemented - 22 (13.1%) CF patients. Vitamin K status was assessed using prothrombin inducted by vitamin K absence (PIVKA-II) and undercarboxylated osteocalcin (u-OC). The pathological PIVKA-II concentration (≥ 2 ng/ml) and abnormal percentage of osteocalcin (≥ 20%) were found in 72 (42.8%) and 60 (35.7%) subjects, respectively. We found that liver involvement, diabetes, and glucocorticoid therapy were potential risk factors for vitamin K deficiency. Pathological concentrations of PIVKA-II occurred more frequently in patients with pancreatic insufficiency and those who have two severe mutations in both alleles of the CFTR gene. Pathological percentage of u-OC was found more frequently in adult CF patients and those not receiving vitamin K. However, it seems that there are no good predictive factors of vitamin K deficiency in CF patients in everyday clinical care. Early vitamin K supplementation in CF patients seems to be warranted. It is impossible to clearly determine the supplementation dose. Therefore, constant monitoring of vitamin K status seems to be justified.

Hormone secretion in transgenic rats and electrophysiological activity in their gonadotropin releasing-hormone neurons.

Expression of GFP in GnRH neurons has allowed for studies of individual GnRH neurons. We have demonstrated previously the preservation of physiological function in male GnRH-GFP mice. In the present study, we confirm using biocytin-filled GFP-positive neurons in the hypothalamic slice preparation that GFP-expressing somata, axons, and dendrites in hypothalamic slices from GnRH-GFP rats are GnRH1 peptide positive. Second, we used repetitive sampling to study hormone secretion from GnRH-GFP transgenic rats in the homozygous, heterozygous, and wild-type state and between transgenic and Wistar males after ~4 yr of backcrossing. Parameters of hormone secretion were not different between the three genetic groups or between transgenic males and Wistar controls. Finally, we performed long-term recording in as many GFP-identified GnRH neurons as possible in hypothalamic slices to determine their patterns of discharge. In some cases, we obtained GnRH neuronal recordings from individual males in which blood samples had been collected the previous day. Activity in individual GnRH neurons was expressed as total quiescence, a continuous pattern of firing of either low or relatively high frequencies or an intermittent pattern of firing. In males with both intensive blood sampling (at 6-min intervals) and recordings from their GnRH neurons, we analyzed the activity of GnRH neurons with intermittent activity above 2 Hz using cluster analysis on both data sets. The average number of pulses was 3.9 ± 0.6/h. The average number of episodes of firing was 4.0 ± 0.6/h. Therefore, the GnRH pulse generator may be maintained in the sagittal hypothalamic slice preparation.

PIVKA-II plasma levels as markers of subclinical vitamin K deficiency in term infants.

BACKGROUND: As the vitamin K content of human milk is low and the newborn infant's stores of vitamin K are small, vitamin K deficiency with hemorrhage in the newborn is a worldwide problem. Proteins Induced by Vitamin K Absence (PIVKA-II) are the inactive under-γ-carboxylated forms of vitamin K-dependent clotting factors and they could be useful in predicting subclinical vitamin K deficiency (VKD). OBJECTIVES: To demonstrate that PIVKA-II are earlier markers of subclinical VKD than Prothrombin time (PT) in exclusively breast-fed newborns. METHODS: A prospective, controlled, randomized study, including 53 term newborns receiving vitamin K prophylaxis (0.5 mg i.m.) at birth, was performed. At 30 days newborns were divided into three groups (G) receiving respectively: 25 µg/die of vitamin K (G I), 12 µg/die (G II) or placebo (G III). PIVKA-II and PT were measured on 30th and 90th days of life. RESULTS: G III and GII showed a significant increase in PIVKA-II from 30 to 90 days of life respectively from 2.6 to 4.7 (p = 0.001) and from 2.3 to 3.5 (p < 0.001). No significant changes were found in GI. PT showed no significant changes among groups. CONCLUSIONS: PT is a less sensitive marker than PIVKA II. Oral supplementation with 25 µg/die avoids an increase of PIVKA-II. Despite increased PIVKA-II do not mean an impending occurrence of bleeding, they highlight a subclinical VKD and its relative risk.

Effects of isoflavones on the coagulation and fibrinolytic system of postmenopausal women.

OBJECTIVE: We evaluated the effects of soy isoflavone supplementation on hemostasis in healthy postmenopausal women. METHODS: In this double-blinded, placebo-controlled study, 47 postmenopausal women 47-66 y of age received 40 mg of soy isoflavone (n = 25) or 40 mg of casein placebo (n = 22) once a day for 6 mo. Levels of factors VII and X, fibrinogen, thrombin-antithrombin complex, prothrombin fragments 1 plus 2, antithrombin, protein C, total and free protein S, plasminogen, plasminogen activator inhibitor-1, and D-dimers were measured at baseline and 6 mo. Urinary isoflavone concentrations (genistein and daidzein) were measured as a marker of compliance and absorption using high-performance liquid chromatography. Baseline characteristics were compared by unpaired Student's t test. Within-group changes and comparison between the isoflavone and casein placebo groups were determined by a mixed effects model. RESULTS: The levels of hemostatic variables did not change significantly throughout the study in the isoflavone group; however, the isoflavone group showed a statistically significant reduction in plasma concentration of prothrombin fragments 1 plus 2; both groups showed a statistically significant reduction in antithrombin, protein C, and free protein S levels. A significant increase in D-dimers was observed only in the isoflavone group. Plasminogen activator inhibitor-1 levels increased significantly in the placebo group. However, these changes were not statistically different between groups. CONCLUSION: The results of the present study do not support a biologically significant estrogenic effect of soy isoflavone on coagulation and fibrinolysis in postmenopausal women. However, further research will be necessary to definitively assess the safety and efficacy of isoflavone.

Regulation of calprotectin expression by interleukin-1alpha and transforming growth factor-beta in human gingival keratinocytes.

BACKGROUND AND OBJECTIVE: Calprotectin, a heterodimer of S100A8 and S100A9 with antimicrobial properties, is expressed in gingival keratinocytes and plays an important role in innate immunity. Because calprotectin expression is localized in the spinous cell layer of the gingival epithelium, we hypothesized that the expression of calprotectin in keratinocytes is related to the differentiation stage. The aim of the present study was to investigate the relationship between calprotectin expression and keratinocyte differentiation using some factors that regulated its differentiation. MATERIAL AND METHODS: Normal human gingival keratinocytes were isolated from gingival tissues obtained at the extraction of wisdom teeth, and were cultured in serum-free keratinocyte medium supplemented with interleukin-1alpha or calcium, which promote keratinocyte differentiation, and transforming frowth factor-beta (TGF-beta) or retinoic acid, which suppress its differentiation. The expression of S100A8/A9 mRNA and the production of calprotectin in normal human gingival keratinocytes were examined by northern blotting and enzyme-linked immunosorbent assay, respectively. The expression of cytokeratin 14, involucrin and filaggrin (marker proteins of keratinocyte differentiation) was investigated by immunohistochemical staining, and the DNA-binding activity of CCAAT/enhancer binding protein alpha (C/EBPalpha), a transcription factor, was examined by electrophoretic mobility shift assay. RESULTS: The expression of S100A8/A9 mRNA and the production of calprotectin were increased by interleukin-1alpha and calcium, but decreased by TGF-beta. RA inhibited the expression of S100A8/A9 and keratinocyte differentiation, which were induced by interleukin-1alpha. C/EBPalpha DNA-binding activity in normal human gingival keratinocytes was enhanced by interleukin-1alpha and calcium, but suppressed by TGF-beta. CONCLUSION: The present study suggests that calprotectin expression is related to keratinocyte differentiation and that C/EBPalpha is a regulator of calprotectin expression in keratinocytes.

Sleep-wake disturbances in sporadic Creutzfeldt-Jakob disease.

BACKGROUND: The prevalence and characteristics of sleep-wake disturbances in sporadic Creutzfeldt-Jakob disease (sCJD) are poorly understood. METHODS: Seven consecutive patients with definite sCJD underwent a systematic assessment of sleep-wake disturbances, including clinical history, video-polysomnography, and actigraphy. Extent and distribution of neurodegeneration was estimated by brain autopsy in six patients. Western blot analyses enabling classification and quantification of the protease-resistant isoform of the prion protein, PrPSc, in thalamus and occipital cortex was available in four patients. RESULTS: Sleep-wake symptoms were observed in all patients, and were prominent in four of them. All patients had severe sleep EEG abnormalities with loss of sleep spindles, very low sleep efficiency, and virtual absence of REM sleep. The correlation between different methods to assess sleep-wake functions (history, polysomnography, actigraphy, videography) was generally poor. Brain autopsy revealed prominent changes in cortical areas, but only mild changes in the thalamus. No mutation of the PRNP gene was found. CONCLUSIONS: This study demonstrates in sporadic Creutzfeldt-Jakob disease, first, the existence of sleep-wake disturbances similar to those reported in fatal familial insomnia in the absence of prominent and isolated thalamic neuronal loss, and second, the need of a multimodal approach for the unambiguous assessment of sleep-wake functions in these patients.

Cloning and hypothalamic distribution of the chicken thyrotropin-releasing hormone precursor cDNA.

In this paper we report the cloning of the chicken preprothyrotropin-releasing hormone (TRH) cDNA and the study of its hypothalamic distribution. Chicken pre-proTRH contains five exact copies of the TRH progenitor sequence (Glu-His-Pro-Gly) of which only four are flanked by pairs of basic amino acids. In addition, the amino acid sequence contains three sequences that resemble the TRH progenitor sequence but seem to have lost their TRH-coding function during vertebrate evolution. The amino acid sequence homology of preproTRH between different species is very low. Nevertheless, when the tertiary structures are compared using hydrophobicity plots, the resemblance between chicken and rat prepro-TRH is striking. The cloning results also showed that the chicken preproTRH sequence includes neither a rat peptide spacer 4 (Ps4) nor a Ps5 connecting peptide. Comparison of the cDNA sequence with the chicken genome database revealed the presence of two introns, one in the 5' untranslated region, and another downstream from the translation start site. This means that the gene structure of chicken preproTRH resembles the gene stucture of this precursor in mammals. Based on the cDNA sequence, digoxigenin-labelled probes were produced to study the distribution of preproTRH in the chicken brain. By means of in situ hybridization, preproTRH mRNA was detected in the chicken paraventricular nucleus (PVN) and in the lateral hypothalamus (LHy).

Dietary soy containing phytoestrogens does not activate the hemostatic system in postmenopausal women.

The soybean is rich in isoflavone phytoestrogens, which are ligands for estrogen receptors, but it is unknown whether soy/phytoestrogens have similar procoagulant effects to estrogen. In this randomized double-blind trial, 40 healthy postmenopausal women of age 50-75 yr received soy protein isolate (40 g soy protein, 118 mg isoflavones) (n = 19) or casein placebo (n = 21). Plasma markers of coagulation, fibrinolysis, and endothelial dysfunction were measured at baseline and 3 months. The baseline characteristics of the two groups were similar. Compared with casein placebo, soy decreased triglycerides (P < 0.005) and low-density lipoprotein/high-density lipoprotein ratio (P < 0.001) and increased lipoprotein (a) (P < 0.05). Activity of coagulation factor VII (VIIc) decreased similarly in both groups (P < 0.005). Prothrombin fragments 1 + 2 (a marker of thrombin generation) decreased in the soy group (P < 0.005), but the change was not different from the casein group. There was no effect of soy on soluble fibrin (a marker of fibrin production), plasminogen activator inhibitor-1 (a marker of fibrinolytic inhibitory potential), D-dimer (a marker of fibrin turnover), or von Willebrand factor (a marker of endothelial damage). In conclusion, the results of the current study do not support biologically significant estrogenic effects of soy/phytoestrogens on coagulation, fibrinolysis, or endothelial function.

Echis time, under-carboxylated prothrombin and vitamin K status in intensive care patients.

Vitamin K deficiency is a known cause of coagulopathy in hospitalized patients, but the extent of the problem has not been well assessed. This noninterventional, prospective observational study of 35 adults was undertaken in the intensive care unit (ICU) and examined the incidence of and the methods for diagnosing vitamin K deficiency. Measurements of prothrombin time, Echis time and plasma concentrations of under-carboxylated prothrombin (proteins induced in vitamin K absence or antagonism, PIVKA-II), vitamin K1 and ferritin were made during the 48 h after admission to the unit and repeated if coagulopathy developed later. Plasma vitamin K1 was low in 15 admissions (43%), in 11 cases of patients with coagulopathy and in four cases without coagulopathy. PIVKA-II was present in 12 cases (34%), of whom four had low vitamin K1 levels. All of the eight patients with raised PIVKA-II but normal vitamin K concentration were hyperferritinaemic. We conclude that low plasma vitamin K levels, suggestive of low tissue stores, are common in intensive care patients with or without coagulopathy. As 34% of patients had a raised PIVKA-II, this suggests that vitamin K stores may be insufficient to maintain full gamma-carboxylation of prothrombin and emphasize the need to anticipate vitamin K deficiency in the ICU setting by appropriate supplementation.

Posttranslational processing of progrowth hormone-releasing hormone.

The prepro-GH-releasing hormone (prepro-GHRH; 12.3 kDa) precursor, like other neuropeptide precursors, undergoes proteolytic cleavage to give rise to mature GHRH, which is the primary stimulatory regulator of pituitary GH secretion. In this study we present the first model of in vitro pro-GHRH processing. Using pulse-chase analysis, we demonstrate that at least five peptide forms in addition to GHRH are produced. The pro-GHRH (after removal of its signal peptide, 10.5 kDa) is first processed to an 8.8-kDa intermediate form that is cleaved to yield two products: the 5.2-kDa GHRH and GHRH-related peptide (GHRH-RP; 3.6 kDa). GHRH-RP is a recently described peptide derived from proteolytic processing of pro-GHRH that activates stem cell factor, a factor known to be essential for hemopoiesis, spermatogenesis, and melanocyte function. Further cleavage results in a 3.5-kDa GHRH and a 2.2-kDa product of GHRH-RP. Like GHRH, there is GHRH-RP immunostaining in hypothalamic neurons in the median eminence as detected by immunohistochemistry and immunoelectron microscopy. Based on deduced amino acid sequences of the pro-GHRH processing products, several peptides were synthesized and tested for their ability to stimulate the cAMP second messenger system. GHRH, GHRH-RP, and one of these peptides [prepro-GHRH-(75-92)-NH2] all significantly stimulated the PKA pathway. This work delineates a new model of pro-GHRH processing and demonstrates that novel peptides derived from this processing may have biological action.

Atrial natriuretic-like peptide and its prohormone within metasequoia.

Metasequoia glyptostroboides was one of the dominant conifers in North America, Asia, and Europe for more than 100 million years since the late Cretaceous Albian Age, but Quaternary glaciations drove the Metasequoia population to apparent extinction. A small pocket of Metasequoia, however, was found in central China in the 1940s representing the only surviving population of this "living fossil" species. Atrial natriuretic peptide, a 28-amino-acid peptide hormone that causes sodium and water excretion in animals, has been found to be part of the first peptide hormonal system in lower plants. The existence of this hormonal system has never been examined within trees of any genus. High-performance gel permeation chromatography of the leaves and stems (i.e., branches) of Metasequoia followed by atrial natriuretic peptide radioimmunoassay revealed an ANP-like peptide and its prohormone (i.e., approximately 13,000 mol wt) were present in both leaves and stems of this conifer. The elution profile of ANP-like peptide in stems of Metasequoia had a shoulder to the left of where pure synthetic ANP elutes suggesting the possibility of a slightly larger peptide eluting within this shoulder secondary to alternate processing of the ANP-like prohormone and similar to what occurs with the kidney of animals. The elution profile of ANP-like peptide in the leaves of Metasequoia revealed two peaks; one where ANP elutes and a second peak suggesting a smaller peptide that has been metabolically processed. The presence of the ANP-like prohormone strongly suggests that ANP-like gene expression is occurring in both leaves and stems of Metasequoia since this prohormone is the gene product of this hormonal system. The presence of the ANP-like hormonal system in trees implies that this hormonal system may have been present early in land plant evolution to allow trees to reach heights of greater than 30 feet where a water flow-enhancing substance is absolutely necessary for water flow to occur to these heights.

Ongoing prothrombotic state in patients with antiphospholipid antibodies: a role for increased lipid peroxidation.

We measured the urinary excretion of Isoprostane F2alpha-III and Isoprostane-F2alpha-VI, two markers of in vivo lipid peroxidation, and the circulating levels of the prothrombin fragment F1+2, a marker of thrombin generation, in 18 antiphospholipid antibodies-positive patients, in 18 antiphospholipid antibodies-negative patients with systemic lupus erythematosus, and in 20 healthy subjects. Furthermore, 12 patients positive for antiphospholipid antibodies were treated with (n = 7) or without (n = 5) antioxidant vitamins (vitamin E at 900 IU/d and vitamin C at 2, 000 mg/d) for 4 weeks. Compared with antiphospholipid antibodies-negative patients, antiphospholipid antibodies-positive patients had higher urinary values of Isoprostane-F2alpha-III (P =. 0001), Isoprostane-F2alpha-VI (P =.006), and plasma levels of the prothrombin fragment F1+2 (P =.0001). In antiphospholipid-positive patients, F1+2 significantly correlated with Isoprostane-F2alpha-III (Rho =.56, P =.017) and Isoprostane-F2alpha-VI (Rho =.61, P =.008). After 4 weeks of supplementation with antioxidant vitamins, we found a significant decrease in F1+2 levels (P <.005) concomitantly with a significant reduction of both Isoprostane-F2alpha-III (P =.007) and Isoprostane-F2alpha-VI (P <.005). No change of these variables was observed in patients not receiving antioxidant treatment. This study suggests that lipid peroxidation might contribute to the activation of clotting system in patients positive for antiphospholipid antibodies.

Serum des-gamma-carboxy prothrombin levels determined by a new generation of sensitive immunoassays in patients with small-sized hepatocellular carcinoma.

OBJECTIVE: Des-gamma-carboxy prothrombin (DCP), also called protein induced by vitamin K absence or antagonist II (PIVKA-II), is a tumor marker complementary to AFP for the diagnosis of hepatocellular carcinoma (HCC). Currently available immunoassays for DCP are not sensitive enough to detect HCC at an early stage. Recently, two new immunoassays with enhanced sensitivity were developed. The aim of this study was to assess the diagnostic values of the new methods in patients with small-sized HCC. METHODS: Coded serum samples obtained from 36 patients with small-sized and single-nodular HCC (< or = 3 cm in diameter) and 49 patients with posthepatitic cirrhosis not carrying HCC were analyzed. DCP levels were determined in three different ways: 1) conventional EIA; 2) a new immunoassay using the electrochemiluminescence (ECLIA) detection system; and 3) a new immunoradiometric assay (IRMA). Lectin-reactive profiles of AFP (AFP-L3) were also determined. RESULTS: In 36 patients with small-sized HCC, the rates of abnormal values obtained by the conventional, ECLIA, and IRMA methods were 2.7%, 27.8%, and 16.7%, respectively. An ROC analysis of the two new methods (ECLIA vs IRMA) revealed a better performance by the ECLIA method (p < 0.05). The true positive rate of AFP-L3 was 22.2%, whereas a combination assay of ECLIA for DCP and AFP-L3 resulted in a 41.7% sensitivity with a specificity of 90%. CONCLUSIONS: Compared with the conventional method, the sensitivity in detecting small-sized HCC was increased in the two new DCP immunoassays (ECLIA and IRMA). The overall performance as evaluated by an ROC analysis was significantly better in ECLIA than in IRMA.

The role of gonadectomy and testosterone replacement on thymic luteinizing hormone-releasing hormone production.

We and others have identified luteinizing hormone-releasing hormone (LHRH) in cells of the immune system in both animals and humans. LHRH is an immunostimulant, and testosterone is an immunosuppressant. Because testosterone is known to modulate the concentrations of hypothalamic LHRH, we wondered whether testosterone might also alter the concentrations of rat thymic LHRH. Two weeks after castration or sham castration, adult male rats were implanted with either vehicle or testosterone capsules. All animals were killed 4 days after capsule implantation. Thymic LHRH concentration increased significantly in castrated animals. Testosterone replacement prevented this increase. The concentration of the LHRH precursor, proLHRH, decreased significantly, but testosterone replacement prevented this decrease. Steady-state concentrations of LHRH mRNA were not changed by castration or by hormonal replacement. In contrast to the post-castration increase in thymic LHRH, LHRH content of the hypothalamus decreased significantly. Whereas concentrations of LHRH were lower in the thymus than in the hypothalamus, proLHRH concentrations were much greater in the thymus. These data suggest that gonadal manipulation modulates LHRH molecular processing and its tissue concentration in the thymus in addition to those in the hypothalamus, and that the regulation of LHRH molecular processing by testosterone in the hypothalamus is different from that in the thymus.

Glucocorticoids suppress corticotropin-releasing hormone and vasopressin expression in human hypothalamic neurons.

Glucocorticoids are widely used in clinical practice in a variety of immune-mediated and neoplastic diseases, mostly for their immunosuppressive, leukopenic, antiedematous, or malignancy-suppressive actions. However, their usage is limited because of serious and sometimes life-threatening side-effects. Endogenous glucocorticoids are secreted by the adrenal cortex under the control of the hypothalamus and the pituitary gland. This hypothalamo-pituitary-adrenal axis, in turn, is under the negative feedback control of glucocorticoids. Although the suppression of adrenocortical and pituitary gland functions by glucocorticoids has been shown in humans, a feedback effect at the level of the hypothalamus, as shown in rat, has not been reported to date. The present study shows for the first time that glucocorticoids suppress both CRH and vasopressin (AVP) in the human hypothalamus. We studied immunocytochemically the postmortem hypothalami of nine corticosteroid-exposed subjects and eight controls. The number of CRH-expressing cells in the hypothalamic paraventricular nucleus of glucocorticoid-exposed patients was only 3.3% of that in the controls, and the total immunoreactivities for AVP were 31% and 33% of that in the controls in the supraoptic nucleus and the paraventricular nucleus, respectively, whereas the immunoreactivity for oxytocin did not differ between the two groups. Suppression of hypothalamic CRH and AVP neurons by glucocorticoids may have important consequences for neuroendocrinological mechanisms such as the disturbance of water balance during the treatment as well as the immunological processes in the brain and the pathogenesis of the withdrawal syndrome after discontinuation of corticosteroid treatment. In addition, as both AVP and CRH neurons also project to other brain structures and influence memory, mood, and behavior, their suppression by glucocorticoids may be responsible for at least part of the central nervous system side-effects of glucocorticoids.

Simultaneous quantitation of substance P-encoding preprotachykinin alternatively spliced mRNAs and substance P receptor NK-1 mRNA by an RNase protection assay.

Tachykinins form a family of peptides with neurotransmitter/neuromodulator function. Four tachykinins, substance P, neurokinin A, neuropeptide gamma and neuropeptide K, are encoded by the same PreProTachykinin (PPT) gene. Alternatively spliced mRNAs encode different combinations of these peptides (Brown, E.R., Harlan, R.E., Krause, J.E., Gonadal steroid regulation of substance P (SP) and SP-encoding mRNA in the rat anterior pituitary and hypothalamus, Endocrinology, 126 (1990) 330-340; Krause, J.E., Chirgwin, J.M., Carter, M.S., Xu, Z.S., Hershey, A.D., Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A, Proc. Natl. Acad. Sci. USA, 84 (1987) 881-885). The proportion of PPT mRNAs varies from tissue to tissue (Carter, M.S., Krause, J.E., Structure, expression, and some regulatory mechanisms of the rat preprotachykinin gene encoding substance P, neurokinin A, neuropeptide K, and neuropeptide gamma, J. Neurosci., 10 (1990) 2203-2214), and within the rat hypothalamus according to the estrous cycle-related hormonal status (Gautreau, A., Duval, P., Kerdelhué, B., Variations in substance P-encoding preprotachykinin and substance P receptor NK-1 mRNA transcripts in the rat hypothalamus throughout the estrous cycle: a correlation between amounts of beta-preprotachykinin and NK-1 mRNA, Mol. Brain Res., (1997) in press). Tachykinin receptors as well as tachykinins are regulated at the mRNA level. A fully quantitative method is needed to deal with the complex physiological regulation of the tachykinin system. Here, we describe an RNase protection assay that allows the simultaneous quantitation of alternatively spliced PPT mRNAs, Substance P receptor NK-1 mRNA, and glyceraldehyde-3-phosphodehydrogenase (GAPDH) mRNA as an internal control, in the rat hypothalamus. The advantages of this method are its high sensitivity (0.1 pg) and a wide range of linearity (more than 3 orders of magnitude). Moreover, this protocol provides guidelines to set up a quantitative multiprobe RNase protection assay for other genes.

Attenuation of the polypeptide 7B2, prohormone convertase PC2, and vasopressin in the hypothalamus of some Prader-Willi patients: indications for a processing defect.

7B2 is a neuroendocrine chaperone interacting with the prohormone convertase PC2 in the regulated secretory pathway. Its gene is located near the Prader-Willi syndrome (PWS) region on chromosome 15. In a previous study we were able to show 7B2 immunoreactivity in the supraoptic nucleus (SON) or the paraventricular nucleus (PVN) in only three of five PWS patients. Here we report that in contrast with five other PWS patients, the neurons in the hypothalamic SON and PVN of the two 7B2-immunonegative PWS patients also failed to show any reaction using two antibodies directed against processed vasopressin (VP). On the other hand, even these two cases reacted normally with five antibodies that recognize different parts of the VP precursor. This finding pointed to a processing defect. Indeed, the same patients had no PC2 immunoreactivity in the SON or PVN, whereas PC1 immunoreactivity was only slightly diminished. In conclusion, in the VP neurons of two PWS patients, greatly reduced amounts of 7B2 and PC2 are present, resulting in diminished VP precursor processing.

A new mixed micellar preparation for oral vitamin K prophylaxis: randomised controlled comparison with an intramuscular formulation in breast fed infants.

OBJECTIVE: To compare a new oral preparation of vitamin K1 (Konakion MM) containing lecithin and glycocholic acid with a standard intramuscular (IM) preparation during the first 8 weeks of life in exclusively breast fed infants. METHODS: Infants were randomised at birth to the IM group (1 mg vitamin K) or the oral group (2 mg given at birth and repeated at 7 and 30 days of life). Prothrombin time (INR), plasma vitamin K1, and PIVKA II (undercarboxylated prothrombin) were monitored at 14, 30, and 56 days of age. RESULTS: Seventy nine infants were randomised to the oral group and 77 to the IM group. Sixty seven infants in each group completed eight weeks of the study. Prothrombin times did not differ between the two groups. Mean (SD) plasma vitamin K1 values (in ng/ml) decreased in both groups over time, but were higher in the oral group at 14 and 56 days: 2.0 (1.6) v 1.3 (1.1) at 14 days; 0.5 (0.3) v 0.5 (0.7) at 30 days; and 0.5 (0.8) v 0.2 (0.2) at 56 days of life. PIVKA II was raised (> or = 0.1 AU/ml) in cord blood in 47% of the infants. By 14 days, only one infant in each group had a raised PIVKA II value and both of these initially had high concentrations of PIVKA II in cord blood. At 30 days, there were no raised PIVKA II values. At 56 days, there were no raised PIVKA II values in the oral group, although three infants in the IM group had raised values. CONCLUSIONS: Plasma vitamin K concentrations were at least equal or significantly higher in babies given oral vitamin K supplements compared with IM treated babies at the time points measured. Through the first 8 weeks of life, multiple doses of the new oral preparation maintain haemostasis and vitamin K status in breast fed infants at least equal to that of the intramuscular preparation.