bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlation of Confined Placental Mosaicism with Fetal Intrauterine Growth Retardation
Authors: Stipoljev F. , Latin V. , Kos M. , Mišković B. , Kurjak A.
Published: April 2003

Objective: Our purpose was to determine if the frequency of confined placental mosaicism in newborns with unexplained intrauterine growth retardation (IUGR) was higher compared with infants with appropriate growth in utero and the outcome of these pregnancies. Study Design: A total of 20 cases with unexplained IUGR and 20 cases with appropriate growth for gestational age has been studied. Amnion, chorion and villi biopsy specimens were obtained from growth-retarded cases and controls at delivery. Cord blood specimens for 48-hour lymphocyte cultures were obtained from all infants with IUGR. Results: Karyotype analysis revealed confined placental mosaicism in two of 20 (10%) cases with IUGR. In one growth retarded case and one appropriate growth for gestational age case, mosaicism was also confirmed in the amnion. Cytogenetic analysis from peripheral blood of newborns showed normal karyotype in all cases. Three pregnancies in the group of fetuses with IUGR (15%) ended with fetal death compared with normal fetal surveillance of all cases from the control group. Conclusion: Confined placental mosaicism was detected two times more frequently from placentas of growth- retarded infants compared with those of newborns with appropriate growth. The fetal loss was significantly higher in the group of cases with IUGR compared with the control group.
Late Onset of Discordant Growth in a Monochorionic Twin Pregnancy: Vascular Anastomoses Determine Fetal Growth Pattern and Not Placental Sharing
Authors: Nikkels Peter G.J. , van Gemert Martin J.C. , Briët Jan Willem
Published: April 2003

Twin-twin transfusion syndrome in monochorionic twin pregnancies has a complex and variable clinical presentation. We present the first documented case where two unidirectional arteriovenous anastomoses connecting the donor twin’s larger with the recipient’s smaller placental part produce late onset of discordant growth and subsequent twin-twin transfusion syndrome. We conclude that the haemodynamic effects of the anastomoses caused the observed discordant fetal development and not the unequally shared placenta.
Recombinant Parvovirus B19 Empty Capsids Inhibit Fetal Hematopoietic Colony Formation in vitro
Authors: Lindton Bim , Tolfvenstam Thomas , Norbeck Oscar , Markling Lola , Ringdén Olle , Westgren Magnus , Broliden Kristina
Published: April 2003

Erythroid lineage cells are target cells for human parvovirus B19, and a natural infection often results in transient anemia. To determine whether recombinant B19 capsid proteins (VP1/VP2) also inhibit human hematopoietic progenitor growth, a model system was set up. The B19 capsids were inoculated into primary cultures of hematopoietic stem cells derived from human fetal liver, resulting in a 70–95% reduction of BFU-E (burst-forming unit erythroid cells) as compared with the medium control. A similar effect was seen in human hematopoietic stem cell cultures derived from cord blood and adult bone marrow. Preincubation of the B19 capsids with either a monoclonal antibody to the virus or with B19 IgG positive human sera reduced the inhibitory effect. Furthermore, the inhibitory effect could be reduced by preincubating the target cells with a monoclonal antibody to the cellular receptor for the virus, the P antigen. These findings thus show that the inhibition of colony formation of human hematopoietic stem cells can occur in the absence of parvovirus B19 nonstructural proteins. We speculate that B19 capsid could provide a possible strategy to downregulate indigenous hematopoiesis in fetal stem cell transplantations.
Neonatal Leukemoid Reaction and Early Development of Bronchopulmonary Dysplasia in a Very Low-Birth-Weight Infant
Authors: Zanardo Vincenzo , Magarotto Mariella , Rosolen Angelo
Published: April 2003

The factors controlling the recruitment of inflammatory cells and the activation of the cytokine cascade in low-birth-weight premature infants have been implicated in the sequence of multiorgan inflammatory diseases, including the chronic lung disease of prematurity, bronchopulmonary dysplasia. This article describes a 982-gram, 25 (+2 days) weeks’ gestation male infant, who had a leukemoid reaction throughout the first week of life, followed by early development of bronchopulmonary dysplasia.
Prenatal Sonographic Features of Congenital Lobar Emphysema
Authors: Babu R. , Kyle P. , Spicer R.D.
Published: April 2003

The prenatal sonographic features of congenital lobar emphysema (CLE) have not been well characterised. Five cases have been reported in the literature and on all these occasions either an echogenic (3) or a cystic (2) lung lesion was detected prenatally and the diagnosis was confirmed after the operation. This is the sixth case of CLE in the literature with prenatal sonographic features documented. The prenatal scans of a 23-year-old lady performed at 22 weeks of gestation revealed cystic lesions and increased echogenicity of the right fetal lung. There were no other anomalies and the karyotype was normal. The lesion decreased in size at 28 weeks and the baby was born by a normal vaginal delivery at 41 weeks. CT scan performed on day 6 confirmed cystic changes on the right lung with compression of the right lower lobe. A repeat CT scan performed at 4 months revealed extensive cystic changes in a hyper-inflated right lung and mediastinal shift to the left. At operation, abnormally inflated right upper and middle lobes were found suggesting a CLE. There were no subsequent complications after removal and histology confirmed CLE. The reported cases are reviewed and the prenatal sonographic features of CLE are discussed.
A Rare Case of Non-Immune Hydrops fetalis: Double-Chambered Right Ventricle
Authors: Marton Tamás , Hajdú Júlia , Papp Zoltán
Published: April 2003

A double-chambered right ventricle was diagnosed prenatally by ultrasound examination in a case of fetal hydrops and polyhydramnios. Delivery was induced at the 28th week. 2D echocardiography of the newborn confirmed the intrauterine diagnosis. To our knowledge, this is the first reported case of prenatally diagnosed divided right ventricle, causing hydrops.
Towards Standardization of the Human Cytomegalovirus Antigenemia Assay
Authors: Verschuuren Erik A.M. , Harmsen Martin C. , Limburg Pieter C. , van der Bij Wim , van den Berg Arie P. , Kas-Deelen Adri ana M. , Meedendorp Boelo , van Son Willem J. , The T. Hauw
Published: April 2003

The Human Cytomegalovirus antigenemia (HCMV-Agemia) test has been accepted worldwide as a clinical tool in the diagnosis and management of HCMV-associated syndromes in immunocompromised patients. The many modifications proposed since the first description by our laboratory make standardisation of the HCMV-Agemia assay necessary to enable multicenter clinical trials. We report the initial work for standardization of the HCMV-Agemia assay. A standard protocol is proposed, the optimal distribution conditions are investigated and the results of the shipment of positive and negative test slides as well as of two sets of coded internal standard slides are discussed. The main conclusions are that standard slides can be distributed at room temperature and that the results of participating laboratories with the coded internal standard slides were strikingly similar in spite of differences in HCMV-Agemia protocols used by participating laboratories.
Significance of Qualitative Polymerase Chain Reaction Combined with Quantitation of Viral Load in the Diagnosis and Follow-Up of Cytomegalovirus Infection after Solid-Organ Transplantation
Authors: Vanpoucke H. , van Vlem B. , Vanholder R. , van Renterghem L.
Published: April 2003

Quantitative PCR was evaluated in the monitoring of patients with ongoing posttransplantation cytomegalovirus (CMV) infection and antiviral therapy, compared to leukoDNAemia and serology. From January 1998 until May 1999, 61 patients were followed up weekly during 3 months after transplantation by a qualitative PCR. The quantitative PCR was performed on plasma samples from 21 selected patients, of whom 12 had a primary infection and 9 a reactivation or reinfection. Analysis of the viral load differences showed that the viral loads in patients with a primary infection were significantly higher than viral loads in patients with a reactivation (p < 0.01). Based on the results of our study, we can state that qualitative PCR is a good marker for initiating preemptive therapy. In addition, viral quantitation is clinically useful for accurate diagnosis of established CMV disease, and monitoring of antiviral therapy.
Plasmid DNA Vaccines: Investigation of Integration into Host Cellular DNA following Intramuscular Injection in Mice
Authors: Ledwith Brian J. , Manam Sujata , Troilo Philip J. , Barnum Amy B. , Pauley Cindy J. , Griffiths II Thomas G. , Harper Laural B. , Beare Carolann M. , Bagdon Walter J. , Nichols Warren W.
Published: April 2003

The primary safety concern for DNA vaccines is their potential to integrate into the host cell genome. We describe an integration assay based on purification of high-molecular-weight genomic DNA away from free plasmid using gel electrophoresis, such that the genomic DNA can then be assayed for integrated plasmid using a sensitive PCR method. The assay sensitivity was approximately 1 plasmid copy/µg DNA (representing ∼150,000 diploid cells). Using this assay, we carried out integration studies of three different plasmid DNA vaccines, containing either the influenza hemagglutinin, influenza matrix or HIV gag gene. Six weeks after intramuscular injection, free plasmid was detected in treated muscle at levels ranging from approximately 1,000 to 4,000 copies/µg DNA. At 6 months, the plasmid levels ranged between 200 and 800 copies/µg DNA. Gel purification of genomic DNA revealed that essentially all of the detectable plasmid in treated quadriceps was extrachromosomal. If integration had occurred, the frequency was ≤1–8 integrations per 150,000 diploid cells, which would be at least three orders of magnitude below the spontaneous mutation rate. Our results suggest that the risk of mutation due to integration of plasmid DNA vaccines following intramuscular injection is negligible.
DNA Vaccines: Development, Standardization and Regulation
Authors: Cichutek Klaus
Published: April 2003

Nucleic acid vaccines contain nonvectored nucleic acids intended to be used as prophylactic vaccines in humans or animals. In addition to the Guidelines Assuring the Quality of DNA Vaccines published by the WHO, further standards for the manufacture and preclinical testing are being developed. Theoretical risks have been taken into account and assessed before human use has been considered. Legal requirements for clinical trials and licensing of nucleic vaccines are in place in Germany and other European member states which allow further testing and development of proprietary medicinal products based on nucleic acids and intended for prophylactic vaccination.
Characterization and Regulation of the Receptor Tyrosine Kinase Tie-1 in Platelets
Authors: Tsiamis Achilleas C. , Hayes Paul , Box Helen , Goodall Alison H. , Bell Peter R.F. , Brindle Nicholas P.J.
Published: April 2003

The receptor tyrosine kinase Tie-1 is expressed predominantly on endothelial cells where it has an essential role in blood vessel formation. Targeted disruption of the Tie-1 gene results in a lethal phenotype with severe disruption to the normal integrity of the vasculature. In an examination of Tie-1 in vivo, we observed a significant pool of the receptor present in the circulation associated with the platelet fraction. Western blotting reveals the platelet form of Tie-1 to be a protein of approximately 110 kDa, this contrasts with the 135/125-kDa doublet found in endothelial cells. Platelet activation results in increased surface expression of Tie-1. The closely related receptor tyrosine kinase Tie-2/Tek is not present in platelets. Endothelial Tie-1 undergoes metalloprotease-mediated ectodomain cleavage in response to phorbol ester and other agonists. Tie-1 cleavage leads to release of the extracellular domain and generation of a cell-associated intracellular domain with signalling capacity. The potential for cleavage was investigated in platelets. In contrast to endothelial Tie-1, phorbol ester does not stimulate truncation of the platelet receptor, suggesting these cells lack one or more components of the regulated metalloprotease system controlling Tie-1. These data demonstrate the Tie-1 receptor tyrosine kinase is present on platelets and its surface expression is regulated. Furthermore, platelet Tie-1 differs significantly from the endothelial receptor. Platelet Tie-1 has the potential to modulate endothelial function by competing for any Tie ligands and may have signalling roles important in controlling aspects of platelet behaviour.
Signalling Pathways Activated by 5-HT1B/5-HT1D Receptors in Native Smooth Muscle and Primary Cultures of Rabbit Renal Artery Smooth Muscle Cells
Authors: Hinton J.M. , Hill P.B. , Jeremy J.Y. , Garland C.J.
Published: April 2003

The potential of primary cultures of rabbit renal artery vascular smooth muscle cells (VSMCs) was assessed as a means to investigate the signalling pathways linked to 5-hydroxytryptamine (5-HT) 5-HT1B/5-HT1D receptors in native arteries. In renal artery segments denuded of endothelium, incubated with ketanserin and prazosin (each 1 µM), and prestimulated with 20 mM K+ Krebs buffer, 5-HT and CP 93,129, a 5-HT1B receptor agonist, evoked concentration-dependent contractions. GR 127935, a 5-HT1B/5-HT1D receptor antagonist, significantly antagonised 5-HT-evoked contractions at nanomolar concentrations. Reverse transcription polymerase chain reaction (RT-PCR) of mRNA from smooth muscle cells from the isolated renal artery and from primary cultures of VSMCs from the same artery expressed mRNA transcripts for the 5-HT1B receptor and the 5-HT1D receptor in both preparations. The sequence of the PCR fragments corresponded to the known sequence for these receptors. Application of 5-HT evoked a concentration-dependent, pertussis toxin (PTx)-sensitive reduction in cyclic AMP in both cultured cells and intact artery (cyclic AMP concentration reduced by 65.53 ± 3.33 and 52.65 ± 5.34% from basal with 10 µM 5-HT, respectively). The effect of 10 µM 5-HT on cAMP was increased in the presence of 20 mM K+ (reduced by 82.50 ± 2.50 and 87.54 ± 3.97%, respectively). In intact arteries, contraction through 5-HT1B/5-HT1D receptors was significantly attenuated by inhibitors of phosphatidylinositol 3-kinase (wortmannin) and activated mitogen-activated protein kinase (MAPK), MEK (U01296). In the cultured VSMCs, activated MAPK was identified by immunocytochemistry and immunoblotting after stimulation with 5-HT, but only if 20 mM K+ was present at the onset of stimulation. These data provide the first direct evidence that 5-HT1B/5-HT1B receptors are linked to the activation of MAPK and indicate that primary cultures of renal VSMCs could provide a model system to study further the signalling pathways linked to these receptors.
Effects of Assessment and Processing Techniques on the Shape of Arterial Pressure-Distension Loops
Authors: Hoeks Arnold P.G. , Willigers Jean M. , Reneman Robert S.
Published: April 2003

The dynamic mechanical characteristics of the arterial wall, especially its alinearity and viscoelastic behaviour, are revealed by pressure-distension loops, where the change in diameter (distension) of a lumen is plotted as a function of the change in transmural pressure. For linearity, the pressure-distension loop should be closed and straight, while viscous behavior results in an open loop. For a correct interpretation of the loops, it is essential that both distension and pressure are recorded simultaneously at the same location and processed by circuitries having the same frequency characteristics. The present paper aims at quantifying the effect of misalignment in recording position and of mismatch in frequency characteristics by analyzing the distension and pressure waveforms obtained at known locations in a phantom rig under pulsatile conditions with equipment with a validated frequency response. It is concluded that misalignment of only a few millimeters or mismatch in frequency response, even if the cutoff frequencies are far beyond the signal frequencies of interest, strongly affects the shape of the pressure-distension loop. If the forward and reflected pressure wave partially coincide, position misalignment cannot adquately be corrected for. The frequency mismatch, however, can be corrected for by crosswise preprocessing a waveform by a filter with the frequency response of the registration equipment used for the recording of the other waveform. It is important to consider these artifacts in interpreting pressure-distension loops in the clinical situation.
Human Umbilical Vein Endothelial Cells Differ from Other Endothelial Cells in Failing to Express ABO Blood Group Antigens
Authors: O’Donnell James , Mille-Baker Blandine , Laffan Michael
Published: April 2003

Most in vitro investigations of endothelial cell function have been based on the behaviour of human umbilical vein endothelial cells (HUVEC) in primary tissue culture. However, it is becoming apparent that there is a marked degree of heterogeneity among endothelial cells derived from different vascular beds. We have studied primary HUVEC from 45 umbilical cords. Contrary to previously published reports, we were unable to detect ABO antigens on the surface of cultured HUVEC from group A or B cords. This was not due to an absence of precursor H substance which was uniformly expressed on HUVEC in primary tissue culture. Further investigation revealed an absence of A enzyme activity in A type HUVEC and a level of mRNA only just detectable by RT-PCR. The absence of functional A enzyme activity was confirmed by demonstrating the absence of A substance on von Willebrand factor secreted by A type cells. HUVEC appear to be the only vascular endothelial cells that do not express ABO blood group antigens. We speculate that this may help protect the cord from maternal antibodies during gestation. The lack of ABO blood group antigens on HUVEC may significantly affect their surface function, and therefore care should be taken when extrapolating conclusions from results obtained with these cells (the most widely used human endothelial cell model in vitro) to the properties of adult endothelial cells.
Diadenosine Polyphosphates Are Largely Ineffective as Agonists at Natively Expressed P2Y1 and P2Y2 Receptors on Cultured Human Saphenous Vein Endothelial Cells
Authors: Conant Alan R. , Fisher Michael J. , McLennan Alexander G. , Simpson Alec W.M.
Published: April 2003

The diadenosine polyphosphates are a group of long-lasting compounds, released into the bloodstream by platelet degranulation. They mediate endothelium-dependent vasodilatation in several animal vascular systems via P2Y receptors coupled to increases in cytoplasmic calcium ([Ca2+]c). However, there is little evidence of diadenosine-mediated vasodilatation in the human vasculature, and a direct interaction with natively expressed P2Y receptors on human endothelium has not been demonstrated. We have therefore studied the effects of diadenosines on primary cultures of human saphenous vein endothelial cells (HSVECs) and related this to the expression of P2Y receptors. HSVECs were loaded with the calcium-sensitive dye fura-2, and nucleotide-stimulated [Ca2+]c responses were recorded. HSVECs responded to 10 µM UTP, ATP and 2-methylthio-ATP but not to UDP. Consistent with the recorded [Ca2+]c responses, RT-PCR analysis of HSVEC RNA amplified specific products for the P2Y1 and P2Y2 receptors but not the P2Y4 and P2Y6 receptors. HSVECs responded to Ap3A with a rise in [Ca2+]c, but none of the other diadenosines tested elicited a response. Therefore natively expressed human P2Y1 and P2Y2 receptors are insensitive to diadenosine polyphosphates with the exception of Ap3A. We would therefore predict that the diadenosine polyphosphates have only a limited vasodilatory role in human saphenous veins.
Evaluation of β-Galactosidase Activity in Tissue in the Presence of Blood
Authors: Pelisek Jaroslav , Armeanu Sorin , Nikol Sigrid
Published: April 2003

The reporter gene for β-galactosidase is frequently used to determine the efficiency of gene transfer in arteries. However, blood is often present in arterial explants and may compromise the results by the presence of hemoglobin. The light absorption of hemoglobin is similar to the absorption of several colorimetric products of the commonly used β-galactosidase substrates, including o-nitrophenyl-β-D-galactopyranoside (ONPG) and chlorophenol red galactopyranoside (CPRG). This may result in false-positive measurements of β-galactosidase enzyme activity. The aim of this investigation was to determine the most appropriate method for quantification of β-galactosidase activity in the presence of blood. Colorimetric substrates (ONPG, CPRG) or the chemiluminescent Galacton-Plus substrate were used, and light absorption was measured at different concentrations of erythrocyte extract. Among the β-galactosidase substrates tested, CPRG was the most appropriate, allowing detection of enzyme activity at concentrations as low as 0.05 mU, independent of blood contamination. Addition of reducer stabilized enzyme activity for at least 5 h. Endogenous β-galactosidase activity was evaluated and used to correct results. CPRG substrate, in combination with the reducer agent mercaptoethanol, was found to be the optimal reagent for quantifying β-galactosidase activity in the presence of blood after nonviral in vivo reporter gene transfection, even with a relatively low transfer efficiency.
Causes of Thrombophilia Yet to Be Discovered: A Personal View
Authors: Griffin John H. , Deguchi Hiroshi , Fernandez Jose A.
Published: April 2003

Prediction of genetic risk factors for venous thrombosis might best be left for only wise soothsayers. Nonetheless, based on the principle that hypercoagulability, either systemic or vascular-bed-specific, predisposes to thrombosis, we venture some speculations. Hyperactivity of platelets could be caused by elevated numbers of surface glycoproteins or defective signal transduction pathways. The reported efficacy of aspirin for prevention of venous thrombosis is consistent with the prediction that certain platelet defects may increase the risk of venous thrombosis. Hyperlipidaemia is associated with hypercoagulability, and lipoproteins exhibit procoagulant (e.g. triglyceride-rich particles and oxidized low-density lipoprotein) or anticoagulant (e.g. high-density-lipoprotein’s cofactor activity for activated protein C/protein S) activities. This leads to the prediction that defects in lipids and/or lipoproteins may increase the risk for venous thrombosis. Interestingly, statins were recently reported to prevent the occurrence of venous thrombosis in the HERS (Heart and Estrogen/Progestin Replacement) trial. We also predict that new defects in the protein C pathway (e.g. defective endothelial protein C receptor or novel cofactors for activated protein C/protein S) will be discovered. Risk factors affecting the majority of patients will likely in- volve new single nucleotide polymorphisms (SNPs) like the factor V nt G1691A or prothrombin nt G20210A SNPs. The Human Genome Project will soon accelerate discovery of new SNPs that are risk factors for venous thrombosis.
A Prospective Randomized Study Comparing Nifedipine versus Ritodrine for the Suppression of Preterm Labour
Authors: Al-Qattan Fawzia , Omu Alexander E. , Labeeb Najwa
Published: April 2003

Nifedipine is a dihydropyridine and a calcium channel blocker during the second phase of the action potential of uterine smooth muscle cells, and ritodrine is a β-sympathomimetic. Objective of Study: To compare the efficacy and side-effects of oral nifedipine to ritodrine in the inhibition of preterm labour. Methodology: Sixty parturients admitted to the Maternity Hospital with preterm labour who fulfilled the inclusion criteria were randomized into two equal therapy groups: (a) oral nifedipine (n = 30) and (b) intravenous ritodrine (n = 30). During the period, the parturients were under continuous monitoring of fetal well-being, maternal uterine contractions, blood pressure, and pulse and respiratory rates. Both groups were given dexamethasone and followed up through delivery and the early neonatal period. Results: The incidence of preterm deliveries during the study period was 6.5%. Ritodrine had a quicker onset of inhibition of uterine contractions, especially between 20 and 40 min after initiation of tocolytic therapy (p < 0.04). Labour was delayed on the average for 40 h in the nifedipine group compared to 24 h in the ritodrine group (p < 0.05). Eighteen patients (60%) in the nifedipine group had cessation for more than 48 h compared to 7 (30.4%) in the ritodrine group (p < 0.05). Nifedipine inhibited uterine contractions for more than 7 days in more patients than ritodrine (13 versus 5, p < 0.05). Ten patients in the nifedipine group went beyond 36 weeks of gestation compared to 4 in the ritodrine group (p < 0.03). In 5 (17.9%) of the ritodrine group compared to none in the nifedipine group, treatment was abandoned because of severe side-effects of nausea (11 versus 2, p < 0.01) and palpitations (16 versus 3, p < 0.004). There were no significant differences in the Apgar scores and neonatal morbidity. More infants in the ritodrine group (17, 73.9%) than in the nifedipine group (14, 46.1%, p < 0.05) were admitted to the neonatal unit. Conclusion: Nifedipine is recommended for aborting preterm contractions because it has fewer side-effects, superior efficacy and greater ease of administration than intravenous ritodrine.
Effects of Aminoguanidine and Meloxicam on Nitric Oxide and Prostaglandin E Production Induced by Lipopolysaccharide in the Hypothalamus and Anterior Pituitary of the Rat
Authors: Mohn Claudia , Lomniczi Alejandro , Faletti Alicia , Scorticati Camila , Elverdin Juan C. , McCann Samuel M. , Rettori Valeria
Published: April 2003

Background/Objective: Injection of bacterial lipopolysaccharide (LPS) into male rats activates genes that in turn induce many enzymes that participate in the animals’ response to LPS. There is induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in many tissues. This induction could result from combination with cell surface LPS receptors that directly induce both genes, or the nitric oxide (NO) released as a result of iNOS induction could induce COX-2. Methods: To distinguish between these two possibilities, specific inhibitors of iNOS and COX-2 activity, aminoguanidine (AG) and meloxicam (MLX), respectively, were injected either peripherally or intracerebroventricularly (i.c.v.), and their effect on NO and prostaglandin E (PGE) production induced by LPS in the medial basal hypothalamus (MBH) and anterior pituitary gland (AP) were determined. Results: Peripheral injection of AG blocked iNOS-derived NO production in the AP but not in the MBH. When AG was injected i.c.v., iNOS-derived NO production in the MBH was blocked. MLX injected peripherally blocked COX-2-derived PGE2 production in the MBH and AP, whereas AG injected peripherally or i.c.v. was ineffective. Since AG was only effective in blocking iNOS-derived NO production in the MBH when injected i.c.v., AG apparently does not effectively cross the blood brain barrier, whereas MLX injected peripherally inhibited PGE production, probably by inhibiting COX-2 activity in both the MBH and AP. AG was ineffective in preventing the increase in PGE derived from COX-2 in either the MBH or AP. Conclusion: LPS directly induces both enzymes, iNOS and COX-2, in the hypothalamus and AP.
Corticosteroids Influence the Action Potential Firing Pattern of Hippocampal Subfield CA3 Pyramidal Cells
Authors: Okuhara Dayne Y. , Beck Sheryl G.
Published: April 2003

Corticosteroids regulate gene expression through the activation of mineralocorticoid and glucocorticoid receptors. The hippocampus contains the highest density of mineralocorticoid and glucocorticoid receptors in the central nervous system. The modulation of neuron excitability by corticosteroids in hippocampal subfield CA1 is well documented. However, it is not known whether corticosteroids produce different effects across the various hippocampal subfields. Therefore, we used intracellular recording techniques to examine the actions of chronic corticosteroid treatment (2 weeks) on the electrophysiological properties of rat hippocampal subfield CA3 pyramidal cells. The treatment groups used in this investigation were: adrenalectomy (ADX), selective mineralocorticoid receptor activation with aldosterone (ALD), mineralocorticoid and glucocorticoid receptor activation with high levels of corticosterone (HCT), and SHAM. Corticosteroid treatment altered the percentage of nonburst and burst firing neurons. The percentages of nonbursting cells were 74 and 62% in tissue from ADX and HCT animals compared to 42 and 41% in ALD and SHAM animals, respectively. The corticosteroid-induced effect on the ratio of nonbursting to bursting cells does not appear to be secondary to changes in the cell’s membrane input resistance, resting potential, time constant, action potential, slow-or fast-afterhyperpolarizing potential properties. Based on these results we conclude that corticosteroids are important for maintaining the ratio of nonburst and burst firing pyramidal neurons in subfield CA3. These novel results are distinct from those previously reported for subfield CA1, suggesting that corticosteroids have different effects across hippocampal subfields.
Identification of Gap Junctional Connexin-32 mRNA and Protein in Gonadotropin-Releasing Hormone Neurons of the Female Rat
Authors: Hosny Somaya , Jennes Lothar
Published: April 2003

Pulsatile gonadotropin-releasing hormone (GnRH) release from the median eminence is critical for the appropriate function of the pituitary gonadotropes and for the generation of a preovulatory gonadotropin surge. The mechanisms by which many GnRH axon terminals are synchronized to release GnRH in a coordinated fashion into the capillaries of the primary plexus are unknown as are the anatomical sites at which the regulation of GnRH neurons takes place. While many neurotransmitters have been shown to influence GnRH release, it is not clear if such neurotransmitters regulate GnRH neurons directly through synaptic interactions or through intermediate neurons. An alternative mechanism of interneuronal communication is provided by gap junctions which allow a rapid, bidirectional exchange of signals. In order to explore if GnRH neurons synthesize the appropriate proteins to form gap junctions with adjacent cells we used double immunohistochemistry for GnRH and connexins-26, -32 or -43 as well as dual in situ hybridization to identify GnRH mRNA and connexin-32 mRNA. The results show that all GnRH neurons contain connexin-32 immunoreactive puncta at their perikarya and, occasionally, at their axon terminals in the median eminence while connexin-26 and -43 immunoreactivity was absent in GnRH neurons. In addition, connexin-32 mRNA was detected in GnRH mRNA containing neurons. However, gap junctional connections between adjacent GnRH neurons were not observed. The data suggest that gap junctional coupling of GnRH neurons with neighboring non-GnRH containing cells may occur and may represent a mechanism by which GnRH neurons can be synchronized or by which hormonal or neurotransmitter signals can be conveyed to the GnRH neurons.
Quantitation and Regulation of Pyroglutamyl Peptidase II Messenger RNA Levels in Rat Tissues and GH3 Cells
Authors: Lin Jun , Wilk Sherwin
Published: April 2003

The distribution of the mRNA of the specific thyrotropin-releasing-hormone (TRH)-degrading enzyme pyroglutamyl peptidase II (EC 3.4.19.6) in rat tissues and brain regions and its regulation in rat tissues and in GH3 cells was studied by a reliable and quantitative solution hybridization ribonuclease protection assay. The distribution of pyroglutamyl peptidase II mRNA levels was uneven with the highest level of mRNA found in brain. Within brain the distribution of pyroglutamyl peptidase II mRNA was heterogeneous. A single dose of T3 markedly increased the level of pyroglutamyl peptidase II mRNA in the pituitary (p < 0.01) and in the liver (p < 0.05). In GH3 cells, exposure to T3 at concentrations from 10–10 to 10–6M for time periods of 2–24 h, did not change pyroglutamyl peptidase II mRNA levels. Acute administration of TRH to rats had no effect on pyroglutamyl peptidase II mRNA levels. By contrast, TRH down-regulated pyroglutamyl peptidase II mRNA in GH3 cells. A similar effect was produced in GH3 cells by activators of protein kinase C. These studies reveal fundamental differences in the mechanism of regulation of pyroglutamyl peptidase II mRNA in pituitary and in GH3 cells. Elevation of pyroglutamyl peptidase II mRNA in liver by T3 suggests that this organ is the source of the enzyme in serum.
Absence of an Inhibitory Vasopressinergic Tone on LH Release in Pubertal Male Rhesus Macaques
Authors: Lado-Abeal Joaquin , Norman Reid L.
Published: April 2003

During puberty, primates begin to secrete LH, and presumably GnRH, at night and then eventually throughout the day as they mature. We examined the role of vasopressin, a putative inhibitor of the GnRH pulse generator, on LH secretion in pubertal male macaques both during the day when the GnRH pulse generator was not active and during the night when pulsatile LH secretion was observed. As has been found in other primates, LH and testosterone levels were low during the day and elevated at night. A potent vasopressin receptor antagonist (VPa) was administered during the middle 5 h of a 15-hour daytime or nighttime blood collection period to determine the effects on LH, testosterone and cortisol secretion. Both during the day and night, cortisol secretion was elevated during VPa infusion, suggesting that this V1a receptor antagonist has agonist activity on the V1b receptor in the pituitary involved in vasopressin-stimulated corticotropin release. Mean LH levels during VPa infusion were not different from the control period for that group during the day when LH secretion was absent. Mean LH levels were significantly higher (p < 0.05) than pre-VPa LH levels at night when pulsatile LH secretion was observed. However, LH levels at night during VPa were not higher than levels in untreated animals at the same time. The results of these studies demonstrate that LH and testosterone profiles are similar to those observed in human males during the pubertal transition and suggest that the absence of daytime gonadotropin release during puberty is not due to inhibitory vasopressinergic tone on the GnRH pulse generator.
Evidence for a Leptin-Neuropeptide Y Axis for the Regulation of Growth Hormone Secretion in the Rat
Authors: Vuagnat Béatrice A.M. , Pierroz Dominique D. , Lalaoui Meriem , Englaro Piera , Pralong François P. , Blum Werner F. , Aubert Michel L.
Published: April 2003

The obese gene (OB) product, leptin, has been shown to exert control on metabolic processes such as food intake and body weight homeostasis, possibly through a neuropeptide Y (NPY) neurotransmission. More recently, leptin has been shown to control several neuroendocrine axes, modulating pituitary hormone secretions in function of metabolic conditions. Since in the rat growth hormone (GH) secretion is dependent upon prevailing metabolic conditions, and NPY has been shown to be implicated in the feedback mechanisms of this hormone, we reasoned that leptin could also exert control over GH secretion and we examined this hypothesis in male rats submitted to a 3-day fast. Circulating leptin concentrations measured by RIA abruptly fell to low values after 24 h of fasting and remained low thereafter. Upon refeeding, leptin secretion regularly increased. As shown by others, pulsatile GH secretion had disappeared after 3 days of fasting. Centrally administered leptin (10 µg/day, i.c.v. infusion initiated at the beginning of the fasting period) totally prevented the disappearance of pulsatile GH secretion. No leak of centrally administered leptin to the general circulation was observed. Infusing the same amount of leptin intracerebroventricularly to rats receiving ad libitum feeding produced a severe reduction in food intake but maintained a normal GH secretory pattern. In contrast, pair-fed rats, submitted to the same food restriction, exhibited a marked reduction in GH secretion. Hypothalamic NPY gene expression, estimated by Northern blot analysis, was significantly increased in fasting rats, and decreased in leptin-treated, fasting rats. In rats receiving ad libitum feeding, leptin treatment reduced NPY gene expression, consistent with the observed reduction in food intake, whereas pair-fed animals logically exhibited increased NPY gene expression. In both situations with reduced feeding, normal GH secretion was seen in leptin-treated animals exhibiting low NPY gene expression, whereas decreased or abolished GH secretion was seen in animals not receiving leptin and exhibiting increased NPY mRNA levels. Interestingly, despite maintenance of normal GH secretion in leptin-treated, fasting rats, plasma IGF-I levels were low, as in vehicle-treated rats. Indeed, hepatic gene expression for both GH receptor and IGF-I was markedly reduced by fasting, and no correction was seen with leptin treatment. In summary, the regulation of GH secretion, at least the changes linked with malnutrition, appears to be dependent upon a leptin signal, perceived centrally, possibly related to circulating levels of this new hormone. The present data suggest that leptin can rescue normal pulsatile GH secretion by preventing the documented inhibitory action of NPY on GH secretion.
Role of Glucocorticoids in the Modulation of Corticotropin-Releasing Hormone mRNA Level by the Endogenous Benzodiazepine Receptor Ligand Octadecaneuropeptide in Rat Brain
Authors: Givalois Laurent , Li Songyun , Pelletier Georges
Published: April 2003

We have recently demonstrated that the endozepine octadecaneuropeptide (ODN) exerts an inhibitory influence on corticotropin-releasing hormone (CRH) mRNA expression. The effect is mediated by GABAA receptors and is reversed by adrenalectomy. In order to investigate the involvement of peripheral steroids and more particularly of glucocorticoids in the ODN modulation of CRH mRNA expression, we have evaluated, in adrenalectomized and castrated male rats (ADX/CX), the effect of dexamethasone (DEX) pretreatment on CRH mRNA expression induced by central injection of ODN. Variations in the CRH mRNA expression in the hypothalamic paraventricular nucleus have been studied using quantitative in situ hybridization. The intracerebroventricular injection of ODN (4 µg/kg), as previously reported, induced a significant inhibition of CRH mRNA expression in sham-operated rats (–33%). This inhibition was reversed in ADX/CX male rats (+65% vs. sham vehicle-injected rats and +20% vs. ADX/CX vehicle-injected rats). Pretreatment with DEX (5 mg/kg) during 4 days induced in ADX/CX rats a decrease of 22% (vs. ADX/CX vehicle-injected rats) in the CRH mRNA signal, which became comparable to that observed in sham vehicle-injected rats. Pretreatment of ADX/CX animals with DEX prevented the ODN-induced increase in CRH mRNA expression, inducing rather a 16 and 30% inhibition when compared to vehicle- and ODN-injected ADX/CX rats, respectively. Moreover the CRH mRNA levels observed in ODN-injected ADC/CX rats were higher than those observed in sham vehicle- and sham ODN-injected rats (+16% vs. sham vehicle-injected rats and +63% vs. sham ODN-injected rats). These results indicate that dexamethasone treatment in ADX/CX rats can restore mRNA levels to those observed in sham-operated animals but not the inhibiting effect induced by ODN. Together with previous findings, these results suggest that adrenal and/or gonadal factor(s) other than glucocorticoids are involved in ODN modulation of the HPA axis.
Growth Hormone Releasing Hormone Expression During Postnatal Development in Growth Hormone-Deficient Ames Dwarf Mice:mRNA in situ Hybridization
Authors: Hurley David L. , Wee Beth E.F. , Phelps Carol J.
Published: April 2003

Several genetic mutations in mice and rats that produce lifelong growth hormone (GH) deficiency result in overexpression of GH-releasing hormone (GHRH) mRNA in hypothalamic arcuate nucleus neurons. In order to examine the development of this condition, GHRH mRNA expression was quantified in Ames dwarf (df/df) and normal (DF/?) mice at 1 (day of birth), 3, 7, 14, 21 and 60 postnatal days (d) following in situ hybridization. Total mRNA was assessed using computer-assisted densitometry after X-ray film autoradiography, and mRNA expression per neuron was quantified by counts of grains per cell after emulsion autoradiography. Total GHRH mRNA was the same in dwarf and normal mice at 1, 3 and 7d. GHRH mRNA in dwarfs increased at 14d to 240% of that in DF/? (p < 0.005); the percentage overexpression in dwarf mice remained ≧200% through 60d, although total GHRH mRNA increased in both dwarfs and normals during this period. GHRH mRNA per neuron was the same in normal and dwarf mice at 1d, then increased in dwarfs to 190% of that in normals at 3d (p < 0.05), and rose to 300% of normal levels by 7d and beyond (p < 0.005). There was no sexual dimorphism in expression by either measure in normal or dwarf mice. These results indicate that an increase in GHRH mRNA in Ames dwarf mice is first detectable at 3d, a period of approximately 7d after the failure to initiate GH production, which occurs normally at embryonic day 17.5. The onset of GHRH overexpression occurs earlier than the decline of either hypophysiotropic somatostatin or dopamine in Ames dwarf mice. This difference may be due to the stimulatory action of GHRH, as opposed to the inhibitory effects of factors examined previously.
Effects of Amylin and Salmon Calcitonin on β-Endorphin-Induced Growth Hormone and Prolactin Secretion in the Rat
Authors: Pagani Francesca , Netti Carmela , Guidobono Francesca , Lattuada Norma , Ticozzi Chiara , Sibilia Valeria
Published: April 2003

In this study we examined the possible interplay of amylin (AMY) and salmon calcitonin (sCT) in the central control of growth hormone (GH) and prolactin (PRL) secretion in male rats. For this purpose we first compared effects of central intracerebroventricular (i.c.v.) admininstration of various doses of AMY (2.5–2,500 ng/rat) and sCT (2.2–220 ng/rat) on β-endorphin (β-END, 0.5 µg/rat)-induced GH and PRL secretion. AMY and sCT dose-dependently inhibited β-END-induced GH secretion, whereas only sCT was able to inhibit β-END-induced PRL secretion. To examine whether the GH inhibitory effect of AMY was due to the possible cross-reactivity of AMY and sCT on the same receptors in the CNS, we pretreated some rats with the AMY antagonist (AMY8–37, 2.5 µg/rat, i.c.v.). AMY8–37 significantly enhanced the GH-stimulatory action of β-END. AMY8–37, administered prior to AMY and sCT, significantly removed the inhibitory effect of both AMY and sCT on β-END-induced GH release, suggesting that both peptides mediate their response on GH through a common receptor. In vitro competition binding studies on rat hypothalamic membranes have shown that both AMY and sCT compete with [125I]rAMY binding with half inhibition (IC50) values of 3.6 × 10–11 and 1.6 × 10–10 M, respectively. Binding of [125I]sCT was inhibited by sCT with an IC50 of 1.09 × 10–10M and to a lesser extent by AMY with an IC50 of 1.3 × 10–6 M. Thus it is possible that the two peptides recognize a common hypothalamic receptor but with different affinities (sCT > AMY). Overall these data indicate that AMY behaves as a mimic of sCT in the central control of GH secretion. The failure of AMY, at variance with sCT, to modify the PRL-releasing activity of β-END indicates that different receptor subtypes for sCT are involved in the endocrine effects of sCT and only those mediating the modulatory action of GH respond to AMY.
Growth Hormone-Releasing Activity of Thymulin on Pituitary Somatotropes Is Age Dependent
Authors: Brown OscarA. , Sosa YolandaE. , Dardenne Mireille , Pléau Jean-Marie , Goya RodolfoG.
Published: April 2003

Thymulin is a Zn-bound nonapeptide produced by the thymic epithelial cells (TEC) whose secretion is modulated by growth hormone (GH), among others. We assessed the ability of thymulin to influence the release of GH from dispersed anterior pituitary (AP) cells from young, middle-aged and senescent Sprague-Dawley female rats. Perifused and incubated AP cells were used in different sets of experiments and GH release was measured by RIA. Perifusion of young and senescent AP cells with thymulin doses, ranging from 10–8 to 10–5 M, gave a logarithmic dose-response pattern of GH. Supernatants from TEC lines also showed GH secretagogue activity. The GH release was always lower in the senescent cells. AP cells incubated with 10–8–10–3 M thymulin showed a time- and dose-dependent response, the latter being bell-shaped with a maximum at 10–7 M thymulin. Preincubation of thymulin with an antithymulin serum completely quenched the secretagogue activity of the hormone. Coincubation of thymulin with GHRH revealed a semiadditive release of GH in young and middle-aged AP cells and an additive effect in senescent cells. In middle-aged AP cells, the synthetic GH secretagogue GHRP-6 showed a synergistic effect with thymulin on GH release. The calcium chelator EGTA, but not the calcium ionophore A23187, blocked the GH-releasing activity of thymulin in AP cells. The cAMP enhancers, caffeine, NaF and forskolin significantly increased the thymulin-stimulated release of GH while trifluoperazine, a protein kinase C inhibitor, had no effect. The inositol phosphate enhancer LiCl potentiated the action of thymulin on GH release. The data suggest that the GH-releasing activity of thymulin is receptor-mediated and involves calcium, cAMP and inositol phosphates. In addition, senescence appears to impair somatotrope responsiveness to thymulin.
Leukemia Inhibitory Factor as an Intrapituitary Mediator of ACTH Secretion
Authors: Schwartz Jeffrey , Ray DavidW. , Perez FrankM.
Published: April 2003

In the normal anterior pituitary, intercellular interactions are important for the expression of hormones including adrenocorticotropin (ACTH). Leukemia inhibitory factor (LIF) is secreted by anterior pituitary cells and stimulates both basal and corticotropin-releasing hormone (CRH)-stimulated ACTH secretion in AtT20 cells. To determine the effects of LIF on normal pituitary cells, we measured the effects of LIF and an immunoneutralizing antiserum against LIF on ACTH secretion by cultured sheep anterior pituitary cells. In intact populations of anterior pituitary cells, LIF (10 nM) stimulated ACTH secretion (from 0.30 ± 0.06 to 0.77 ± 0.01 ng/well per 3 h) and antiserum to LIF (by itself) had no effect (0.29 ± 0.05 ng/well per 3 h). In marked contrast, following pharmacological elimination of CRH-target cells, a condition known to disinhibit ACTH secretion, basal ACTH secretion was elevated (0.74 ± 0.13 ng/well per 3 h); LIF produced no further stimulation (0.73 ± 0.22 ng/well per 3 h) but immunoneutralization of LIF significantly reduced secretion to 0.50 ± 0.10 ng/well per 3 h. Medium, conditioned by exposure to CRH-target-depleted cultures of anterior pituitary cells, increased net ACTH secretion (from 0.29 ± 0.03 to 6.54 ± 0.71 ng/well per 3 h), when added as a challenge to naive, cultured anterior pituitary cells. Inclusion of antiserum to LIF significantly attenuated (5.29 ± 0.62 ng/well per 3 h) this response. The presence in and secretion of LIF by normal individual pituitary cells was detected using immunocytochemical methods. Seven to 8% of all cells stained positively for LIF, with 66 ± 11% of those secreting detectable amounts of LIF under unstimulated conditions. LIF colocalized with TSH and LH in pituitary cells. Taken together, these data suggest that LIF can stimulate ACTH secretion by normal anterior pituitary cells, and potentially plays a role as an intrapituitary stimulator of ACTH secretion under certain conditions.