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Human disease caused by highly pathogenic avian influenza (HPAI) H5N1 can lead to a rapidly progressive viral pneumonia leading to acute respiratory distress syndrome. There is increasing evidence from clinical, animal models and in vitro data, which suggests a role for virus-induced cytokine dysregulation in contributing to the pathogenesis of human H5N1 disease. The key target cells for the virus in the lung are the alveolar epithelium and alveolar macrophages, and we have shown that, compared to seasonal human influenza viruses, equivalent infecting doses of H5N1 viruses markedly up-regulate pro-inflammatory cytokines in both primary cell types in vitro. Whether this H5N1-induced dysregulation of host responses is driven by qualitative (i.e activation of unique host pathways in response to H5N1) or quantitative differences between seasonal influenza viruses is unclear. Here we used microarrays to analyze and compare the gene expression profiles in primary human macrophages at 1, 3, and 6 h after infection with H5N1 virus or low-pathogenic seasonal influenza A (H1N1) virus. We found that host responses to both viruses are qualitatively similar with the activation of nearly identical biological processes and pathways. However, in comparison to seasonal H1N1 virus, H5N1 infection elicits a quantitatively stronger host inflammatory response including type I interferon (IFN) and tumor necrosis factor (TNF)-alpha genes. A network-based analysis suggests that the synergy between IFN-beta and TNF-alpha results in an enhanced and sustained IFN and pro-inflammatory cytokine response at the early stage of viral infection that may contribute to the viral pathogenesis and this is of relevance to the design of novel therapeutic strategies for H5N1 induced respiratory disease.

Background/aims: To compare the optic disc parameters between patients with non-arteritic anterior ischaemic optic neuropathy (NAION) and normal controls, using optical coherence tomography (OCT) and Heidelberg Retinal Tomograph III (HRT), and to evaluate the structure-function relationship in NAION eyes., Methods: Both eyes of 22 patients with typical unilateral NAION of > or =6 months' duration and 52 eyes from 52 randomly selected normal subjects underwent Humphrey visual field (HVF) examination and measurement of optic disc and retinal nerve fibre layer thickness (RNFLT)., Results: For the NAION-affected eyes, NAION fellow eyes and normal controls, the ocular magnification-corrected OCT disc areas were respectively 1.849 (SD 0.343) mm(2), 1.809 (0.285) mm(2) and 1.964 (0.386) mm(2); the cup areas were 0.246 (0.187) mm(2), 0.172 (0.180) mm(2) and 0.469 (0.332) mm(2). On HRT, the disc areas were 2.11 (0.38) mm(2), 2.06 (0.40) mm(2) and 2.16 (0.42) mm(2); and the cup areas were 0.28 (0.34) mm(2), 0.25 (0.18) mm(2) and 0.48 (0.32) mm(2). On both OCT and HRT, the cup areas and cup-disc area ratios (CDAR) of both eyes of NAION patients were significantly smaller than controls (p< or =0.01), but the disc areas were not (p> or =0.21). There was a significant correlation between HVF mean deviation and OCT RNFLT (r = 0.44, p = 0.04) but not with HRT RNFLT (p = 0.30) in NAION-affected eyes., Conclusion: NAION patients have smaller optic cups and CDARs in both eyes compared with controls. A larger sample size is necessary to demonstrate if disc size affects the risk of developing NAION. The NAION-affected eyes' OCT RNFLT correlated with HVF mean deviation but the HRT RNFLT did not.

Lipoprotein glomerulopathy is a rare kidney disease in which lipoprotein thrombi are seen in the glomerular capillaries. Most of these patients are found in Japan and East Asian countries. The presenting symptoms include proteinuria, an abnormal plasma lipoprotein profile that resembles type III hyperlipoproteinaemia, and a marked increase in serum apolipoprotein E concentration. Previous studies have suggested that lipoprotein glomerulopathy might be related to APOE gene mutation. No effective therapeutic regimen has been established for lipoprotein glomerulopathy. We report the first case of biopsy-proven lipoprotein glomerulopathy in Hong Kong in a patient who presented with nephrotic syndrome and dyslipidaemia. DNA analysis revealed apolipoprotein E Kyoto together with a novel apolipoprotein E mutation, apolipoprotein E (Asp230Tyr) Hong Kong. There was significant improvement in the clinical parameters and resolution of symptoms after the introduction of statins. Further studies will be needed to clarify the role of apolipoprotein E Hong Kong and its interaction with apolipoprotein E Kyoto in the pathogenesis of lipoprotein glomerulopathy.

Aim: To evaluate the reliability of lens density measurement with anterior segment optical coherence tomography (OCT) and its association with the Lens Opacity Classification System Version III (LOCS III) grading., Methods: Fifty-five eyes from 55 age-related cataract patients were included. One eye from each subject was selected at random for lens evaluation. After dilation, lens photographs were taken with a slit lamp and graded against the LOCS III standardised condition. Anterior segment OCT imaging was performed on the same eyes with a high-resolution scan. The association between the anterior segment OCT nucleus density measurement and LOCS III nuclear opalescence (NO) and nuclear colour (NC) scores was evaluated with the Spearman correlation coefficient. Anterior segment OCT measurement precision, coefficient of variation (CVw), and intraclass correlation coefficient (ICC) were calculated., Results: The mean NO and NC scores were 3.39 (SD 1.10) and 3.37 (SD 1.27), respectively. Significant correlations were found between anterior segment OCT nuclear density measurements and the LOCS III NO and NC scores (r = 0.77 and 0.60, respectively, both with p<0.001). The precision, CVw and ICC of anterior segment OCT measurement were 2.05 units, 4.55% and 0.98, respectively., Conclusion: Anterior segment OCT nucleus density measurement is reliable and correlates with the LOCS III NO and NC scores.

Objective: To investigate the prevalence of metabolic syndrome in Chinese renal transplant recipients, using two different sets of diagnostic criteria., Design: Cross-sectional study., Setting: Regional hospital, Hong Kong., Patients: All Chinese patients who received solitary living-related or cadaveric kidney transplantation from 1 July 1997 to 31 December 2005 in our hospital with follow-up of more than 6 months were recruited. The diagnosis of metabolic syndrome was made according to the National Cholesterol Education Program-Adult Treatment Panel III (NCEP-ATPIII) criteria and the International Diabetes Federation criteria., Results: Using the modified (Asian) NCEP-ATPIII criteria, a total of 39 (32%) of 121 patients had metabolic syndrome, which included 20/69 (29%) of the males and 19/52 (37%) of the females. Using the International Diabetes Federation criteria, metabolic syndrome was diagnosed in 26% of the patients, 22% in males and 31% in females. In our patients, the most common component of metabolic syndrome was hypertension and the least common was low high-density-lipoprotein-cholesterol level. Low high-density-lipoprotein-cholesterol levels were significantly more common in female patients., Conclusion: This study shows that there is a high prevalence of metabolic syndrome in our Chinese renal transplant recipients.

Poor human-to-human transmission of influenza A H5N1 virus has been attributed to the paucity of putative sialic acid alpha2-3 virus receptors in the epithelium of the human upper respiratory tract, and thus to the presumed inability of the virus to replicate efficiently at this site. We now demonstrate that ex vivo cultures of human nasopharyngeal, adenoid and tonsillar tissues can be infected with H5N1 viruses in spite of an apparent lack of these receptors.

Background: Rapid and simple methods for diagnosing human influenza A (H5N1) disease urgently needed. The limited data so far suggest that the currently available rapid antigen detection kits have poor clinical sensitivity for diagnosis of human H5N1 disease., Objectives: To compare the analytical sensitivity of six commercially available rapid antigen detection kits for the detection of "human" (subtypes H1N1, H3N2) and "avian" (subtype H5N1) influenza A viruses., Study Design: Six commercially available test kits for the detection of influenza A were investigated. Analytic sensitivity for the detection of two contemporary H1N1, two H3N2 and three H5N1 viruses was determined using virus culture as a reference method., Results and Conclusions: Each test kit detected the H5N1 virus subtypes as efficiently as they detected conventional human viruses of subtypes H1N1 or H3N2. However, limits of detection of influenza viruses of all subtypes by antigen detection kits were >1000-fold lower than virus isolation. Thus, the reportedly poor clinical sensitivity of these antigen detection kits for diagnosis of patients with H5N1 disease is not due to a difference of sensitivity for detecting avian influenza H5N1 compared to human influenza viruses.

An otherwise well 70-year-old man presented with a non-specific complaint of epistaxis caused by an underlying necrotic natural killer-cell lymphoma complicated by a maggot infestation. He failed to attend for treatment after discharge but re-presented 3 weeks later with an acute exacerbation of his chronic pulmonary obstructive disease. During those 3 weeks his nasal condition had advanced rapidly with extensive tumour infiltration and necrosis affecting his nose and face. The natural clinical course, overall prognosis, and available treatment modalities are briefly discussed.

By yeast two-hybrid screening using the calcium-binding protein ALG-2 as bait a new target of ALG-2 was identified, the RNA-binding protein RBM22. In order to confirm these interactions in vivo we prepared fluorescent constructs by using the monomeric red fluorescent protein to label ALG-2 and the enhanced green fluorescent protein to label RBM22. Confocal microscopy of NIH 3T3 cells transfected with either ALG-2 or RBM22 expression constructs encoding fluorescent fusion proteins alone revealed that the majority of ALG-2 was localized in the cytoplasm whereas RBM22 was located in the nucleus. When cells were co-transfected with expression vectors encoding both fusion proteins ALG-2 was found in the nucleus indicating that RBM22 which can shuttle between the cytoplasm and the nucleus may play a role in nuclear translocation of ALG-2. Using zebrafish as a model mRNA homologues of ALG-2 and RBM22 were microinjected into the blastodisc-yolk margin of zebrafish embryos at the 1-cell stage followed by monitoring the fusion proteins during development of the zebrafish. Hereby, we observed that ALG-2 alone evenly distributed within the cell, whereas in the presence of RBM22 the two proteins co-localized within the nucleus. More than 95% of the two proteins co-localized within the same area in the nucleus suggesting a functional interaction between the Ca(2+)-signaling protein ALG-2 and the RNA-binding protein RBM22.

A 52-year-old man with 6 years' history of human immunodeficiency virus infection who was receiving highly active antiretroviral therapy presented with acute renal failure and nephrotic syndrome. Renal biopsy revealed features consistent with nephropathy associated with human immunodeficiency virus infection. Treatment consisted of intravenous methylprednisolone followed by oral prednisolone. The patient's renal function improved, although proteinuria persisted. Human immunodeficiency virus-associated nephropathy is very rare in Asian populations and is more common among blacks. To the best of our knowledge, this is the first documented case of nephropathy associated with human immunodeficiency virus infection occurring in Hong Kong.

Infection with avian influenza A virus of the H5N1 subtype (isolates A/HK/212/03 and A/HK/213/03) was fatal to one of two members of a family in southern China in 2003. This incident was preceded by lethal outbreaks of H5N1 influenza in waterfowl, which are the natural hosts of these viruses and, therefore, normally have asymptomatic infection. The hemagglutinin genes of the A/HK/212/03-like viruses isolated from humans and waterfowl share the lineage of the H5N1 viruses that caused the first known cases of human disease in Hong Kong in 1997, but their internal protein genes originated elsewhere. The hemagglutinin of the recent human isolates has undergone significant antigenic drift. Like the 1997 human H5N1 isolates, the 2003 human H5N1 isolates induced the overproduction of proinflammatory cytokines by primary human macrophages in vitro, whereas the precursor H5N1 viruses and other H5N1 reassortants isolated in 2001 did not. The acquisition by the viruses of characteristics that enhance virulence in humans and waterfowl and their potential for wider distribution by infected migrating birds are causes for renewed pandemic concern.

Ultrahigh molecular weight polyethylene (UHMWPE)/quartz composites were compression molded in the presence of organosiloxane, and then hydrolyzed. The used organosiloxane is vinyl tri-ethyloxyl silane. The gelation, the melting behavior, the crystallinity, the mechanical properties and the wear resistance of UHMWPE/quartz composites were investigated. The results showed that organosiloxane can act as a cross-linking agent for UHMWPE matrix and serve as a coupling agent for improving the bonding between the quartz particles and the UHMWPE matrix. The correlation between the various properties and the morphology of the composites has been discussed. At about 0.5phr organsiloxane while the degree of crystallinity of the composite is at the peak value of 57%, the mechanical properties and the wear resistance of UHMWPE/quartz composites reaches their maximum.

Erythropoietin and its receptor have been identified in human, murine and ovine placentas. Based on the common embryonic origin of the placenta and fetal membranes, we postulated that erythropoietin is similarly expressed in the fetal membranes. Using in situ hybridization and immunohistochemistry, we tested the hypothesis that ovine fetal membranes are sites of erythropoietin production and action. At 86, 103 and 138 days gestation, erythropoietin mRNA and protein were present in the amnion localized to the cell layer consisting largely of amniotic epithelium and in the chorion localized to the chorionic columnar cells consisting of cytotrophoblasts. Binucleate cells, differentiated cytotrophoblasts known to produce hormones, were identified in the chorion in the region of erythropoietin expression but were not observed in amniotic tissue. The erythropoietin receptor protein was present in the amnion and chorion at 103 and 138 days gestation but was not observed in either tissue at 86 days. In summary, erythropoietin appears to be produced as well as utilized within the ovine amnion and chorion. Within the amnion, the amniotic epithelial cells express the erythropoietin gene whereas, within the chorion, either the cytotrophoblasts or the binuclear cells may be the source. Due to the presence of the receptor, we speculate that the erythropoietin produced in the membranes may mediate fetal membrane function and/or growth through an autocrine and/or paracrine mechanism. Further, the fetal membranes may be the source of erythropoietin in the amniotic fluid., (Copyright 2001 Harcourt Publishers Ltd.)

Long-term loss of fetal blood can occur with fetomaternal hemorrhage, vasoprevia, or placental previa. Our objective was to determine the effects of progressive fetal blood loss over 10 days on fetal plasma erythropoietin (EPO) concentration and its relationship to arterial PO(2), hematocrit, and the volume of blood loss. Late-gestation fetal sheep (n = 8) were hemorrhaged daily at a rate of 1 ml/min over 10 days. The extent of hemorrhage differed in each fetus and ranged from 30 to 80 ml/day, with the cumulative volume removed ranging from 78 to 236 ml/kg estimated fetal weight. Four fetuses served as time controls. EPO concentration measurements were by radioimmunoassay. Statistical analyses included regression, correlation, and analysis of variance. We found that EPO and arterial PO(2) were unchanged until the cumulative hemorrhage volume exceeded 20-40 ml/kg. Once this threshold was exceeded, plasma EPO concentration increased progressively throughout the study and averaged 14.3 +/- 3.2 times basal values on day 10. EPO concentration, arterial PO(2), and hematocrit changes were related curvilinearly to cumulative hemorrhage volume (P < 0.01), whereas the relationship between plasma EPO and arterial PO(2) was log linear (P < 0.001). We conclude that 1) fetal plasma EPO concentration and arterial PO(2) are insensitive to a slow, mild-to-moderate blood loss over several days; 2) unlike the rapid return of EPO to normal within 48 h after acute hemorrhage, fetal EPO concentration undergoes a progressive increase with moderate-to-severe blood loss over several days; 3) the long-term hemorrhage-induced changes in EPO are best correlated with arterial PO(2); and 4) the fetal EPO response to hemorrhage does not appear to be limited by the fetus's ability to produce EPO.

This report describes a patient with mixed normal anion gap hyperchloremic metabolic and respiratory acidosis associated with hypokalemia attributed to cough mixture abuse. Metabolic acidosis was likely related to an overdose of ammonium chloride, whereas respiratory acidosis was probably related to the effect of hypokalemia on respiratory muscles, causing hypoventilation. Hypokalemia was caused by a transcellular shift of potassium induced by ephedrine and pseudoephedrine. Both ammonium chloride and ephedrine were probably present in the cough mixture obtained by our patient as an over-the-counter medication. Physicians should be aware of the potential for cough mixture abuse to cause major electrolyte disturbances that may carry the risk for major cardiac arrhythmias, particularly in youth.

Cefepime is a cephalosporin with a broad spectrum of activity against most gram-positive and gram-negative pathogens. In this study, we attempted to compare the safety and efficacy of cefepime monotherapy against the potentially more toxic combination of vancomycin and netilmicin in the treatment of continuous ambulatory peritoneal dialysis (CAPD)-associated bacterial peritonitis. Eighty-one consecutive CAPD patients who presented with peritonitis from January 1, 1998, to June 30, 2000, were recruited for study. Patients were randomized to be administered either intraperitoneal (IP) cefepime, 1 g once daily (group A), or intravenous vancomycin and netilmicin at conventional doses (group B) for 10 days. Bacterial growth was obtained in 52 episodes (66%), and pathogens identified included gram-positive organisms (30 episodes; 38%), gram-negative organisms (14 episodes; 18%), mixed organisms (2 episodes; 2.5%), and fungus (6 episodes; 8%). Eight patients were excluded after randomization for various reasons (6 patients, fungal peritonitis; 2 patients, wrong diagnoses). Because of the relatively low peritonitis rate after the use of a disconnect system, the sample size of this study was relatively small, giving a power of 0.45. There were no significant differences in primary response rates and cure rates (no relapse >28 days after completion of antibiotic therapy) between both groups of patients (group A versus group B, 82% [32 of 39 patients] versus 85% [29 of 34 patients] and 72% [28 of 39 patients] versus 76% [26 of 34 patients], respectively; P = not significant). No significant side effect was encountered in either group. Total peritonitis-related hospitalizations were 84 patient-days (1, 7, 8, 11, 20, and 37 patient-days) and 115 patient-days (3, 6, 9, 14, 21, 21, and 41 patient-days), whereas total costs per patient cure were estimated to be US $1,039 and US $1,371 in groups A and B, respectively. We conclude that once-daily 1-g IP cefepime monotherapy is a simple, safe, and cost-effective alternative to vancomycin and netilmicin therapy in the treatment of CAPD-associated bacterial peritonitis.

In ovine placentae at 100 days gestation, we localized the expression of erythropoietin (EPO) mRNA by in situ hybridization and determined the cellular localization of EPO protein and EPO receptor protein by fluorescence immunohistochemistry. Erythropoietin mRNA was observed in maternal tissue at the apical tips of the fetal cytotrophoblastic villi at their interface with the maternal caruncle and was absent from both the centrally located fetal-maternal tissue and the more peripherally located maternal caruncle. An EPO-protein-associated fluorescent signal was observed in the same interface region as the EPO mRNA hybridization signal. An intense fluorescent signal associated with EPO receptor protein was observed in the apical fetal-maternal interface region with a lower density signal dispersed throughout the remainder of the interdigitating fetal-maternal tissue. The predominant cells in the apical fetal-maternal interface were identified as binucleate cells by immunohistochemistry. Thus the localization of the binucleate cells was the same as that for the EPO mRNA and the EPO protein, whereas the EPO receptor had a more generalized distribution. Since the binucleate cells are hormone producing cells, we speculate that the binucleate cells are the source of the EPO that is present in ovine placenta., (Copyright 2001 Harcourt Publishers Ltd.)

The receptor tyrosine kinases, kinase-insert domain-containing receptor (KDR) and fms-like tyrosine kinase (Flt-1), and their ligand vascular endothelial growth factor (VEGF) are essential for the development and maintenance of placental vascular function during pregnancy. To further understand the role of VEGF in mediating angiogenesis and vascular permeability during development, the cellular localization of KDR and Flt-1 mRNA and protein, and the distribution of(125)I-VEGF binding sites in placenta, chorion and amnion of ovine fetuses were examined at three different gestational ages. In placentae at 62, 103 and 142 days, the predominant site of KDR mRNA and protein, and VEGF binding was the maternal vascular endothelium. In addition, a specific, although weak, signal for KDR mRNA was found in the maternal epithelium. At 103 and 142 days but not 62 days gestation, KDR mRNA and protein as well as VEGF binding sites were abundantly present in the endothelium of villous blood vessels. In the fetal membranes at 62, 103 and 142 days gestation, KDR mRNA and protein were expressed in the amniotic epithelium and intramembranous blood vessel endothelium, where binding of(125)I-VEGF was strong. There was no KDR mRNA or VEGF binding in the chorionic cytotrophoblast. Flt-1 expression was not detectable in placentae or fetal membranes at the three ages studied. In summary, the results demonstrated that VEGF receptors are present in the maternal and fetal vasculatures of the ovine placenta. This expression is consistent with a capillary growth-promoting function of KDR and its ligand VEGF. Further, the presence of KDR and VEGF binding sites in ovine fetal membranes suggests a role for VEGF in promoting intramembranous vascularity and permeability throughout gestation., (Copyright 2001 Harcourt Publishers Ltd.)

Objective: In the ovine fetus subjected to 24 hours of hypoxia, urinary flow is normal within a few hours from the onset of hypoxia and there is a maintained inhibition of swallowing. We hypothesized that 4 days of fetal hypoxia would lead to polyhydramnios., Study Design: Five late-gestation fetal sheep were subjected to hypoxia for 4 days and 7 other late-gestation fetal sheep served as time control animals. Fetal hypoxia was produced on postsurgical days 5 through 9 by continuous intratracheal nitrogen insufflation to the ewe. On days 3, 5, 7, and 9 after surgery, amniotic fluid volume, fetal urinary flow rate, and the compositions of maternal and fetal blood, amniotic fluid, and fetal urine were measured. A 3-factor analysis of variance was used for statistical analysis., Results: During the period of experimental hypoxia the mean (+/-SE) fetal PaO(2) was 16.0 +/- 0.6 mm Hg, versus 21.2 +/- 0.7 mm Hg in control sheep (P <.001). Fetal hypoxia was associated with increased urinary flow on days 7 and 9, averaging 1410 +/- 310 and 2101 +/- 345 mL/d, respectively, versus 585 +/- 92 and 699 +/- 78 mL/d, respectively, in control animals (P <.001). Amniotic fluid volume was unchanged with time and averaged 960 +/- 159 mL in hypoxic fetuses on postsurgical days 7 through 9 and 851 +/- 130 mL in control animals (P =.60). Fetal blood lactate increased in the hypoxic animals, averaging 3.4 +/- 2.1 mmol/L versus 1.6 +/- 0.3 mmol/L in control animals (P =.02). Fetal urinary excretions of sodium, potassium, chloride, and lactate increased significantly during hypoxia, by 170% to 400%., Conclusion: Four days of nitrogen-induced hypoxia in the ovine fetus resulted in excess fetal urinary flow approximating 1000 mL/d greater than normal without the development of polyhydramnios. Because amniotic fluid volume did not change and hypoxia is a known inhibitor of fetal swallowing, we speculate that intramembranous absorption of amniotic water, electrolytes, and lactate increased.

Background: Parvovirus B19 (B19) infection is known to cause chronic infection leading to anemia in immunocompromised patients. Although nosocomial B19 infections in immunocompetent patients have been documented, no outbreaks in immunocompromised patients have been previously reported. Whether transmission can occur from a patient with chronic infection is also unknown., Methods: An outbreak of B19 infection in a renal transplant unit was investigated by molecular analysis of the virus strains and a case-control study., Results: Three patients had genetically identical virus strains suggesting the occurrence of nosocomial transmission. The index case transmitted infection many weeks after the onset of her clinical symptoms. Other patients at risk of acquiring infection were those most intensively immunosuppressed. Viral load in the serum correlated with the hematological response. A rebound in the viral load was associated with clinical relapse and the failure of i.v. immunoglobulin therapy., Conclusion: Nosocomial transmission of B19 can occur from immunocompromised patients even when they are in the chronic stage of the infection. The clinical and virological response to i.v. immunoglobulin therapy is variable and depends on the overall level of immunosuppression of the patient.

Objective: Obstruction of the fetal esophagus does not always produce the expected polyhydramnios. This is because of increased intramembranous absorption of amniotic fluid into the fetal circulation. A possible mediator for this increased absorption is vascular endothelial growth factor (VEGF). The present objective was to explore whether VEGF gene expression and action would be induced in fetal membranes and placentas of ovine fetuses after esophageal ligation., Study Design: Five late-gestation fetal sheep underwent esophageal ligation and 5 served as control animals. On postoperative day 9, amnion, chorion, and placenta were collected for cellular localization and quantitation of VEGF messenger ribonucleic acid by in situ hybridization and Northern blot analysis. Reverse-transcription polymerase chain reaction was used to identify the VEGF molecular forms. Immunostaining with Ki-67 antibody was used to determine the proliferation of vascular endothelium in the fetal membranes and placentas., Results: VEGF messenger ribonucleic acid was localized in amniotic epithelium, chorionic cytotrophoblast, and cytotrophoblast of the placenta. VEGF164 was the major transcript expressed in these tissues. The abundance of VEGF messenger ribonucleic acid in the amnion and chorion, but not in the placenta, was significantly increased in the ligated fetuses in comparison with the control fetuses. The proliferation of the intramembranous blood vessel endothelium was greater in the ligated fetuses than in the control fetuses., Conclusion: The levels of VEGF messenger ribonucleic acid and the proliferation of vascular endothelium in the amnion and chorion increased after fetal esophageal ligation. This provides a possible mechanism for the enhanced intramembranous absorption of amniotic fluid through increased vascularity and permeability of the fetal membranes, thus ameliorating the development of polyhydramnios. We speculate that the signal(s) that mediate the increase in VEGF expression is present in either the fetal urine or the fetal lung secretions, or both.

To further understand the role of vascular endothelial growth factor (VEGF) in mediating angiogenesis and vascular permeability during development in the sheep placenta and fetal membranes, we examined the localization of VEGF mRNA and protein in placental, chorionic and amniotic tissues by in situ hybridization and immunohistochemistry in ovine fetuses at 62, 102 and 141 days gestation (term=150 days). In the placenta, VEGF mRNA expression and VEGF protein immunostaining were strong in cytotrophoblasts surrounding the villi. In addition, VEGF protein was localized in smooth muscle cells around fetal and maternal blood vessels and in the maternal epithelium. There was no apparent difference in placental VEGF mRNA or protein levels associated with advancing gestation. In the fetal membranes, VEGF mRNA was detected in the amniotic epithelium and the chorionic cytotrophoblastic cell layer. The intensity of the hybridization signals in both amnion and chorion appeared low at 62 days, moderate at 102 days and high at 141 days gestation. VEGF protein was detected in amniotic epithelium and chorionic cytotrophoblasts at all gestational ages studied. The increase in VEGF gene expression in fetal membranes as term approaches suggests that during fetal development VEGF may promote the vascularity and permeability of the microvessels which perfuse the fetal membranes, as well as permeability of the amniotic membrane itself. Thus VEGF may participate in the regulation of amniotic fluid volume., (Copyright 2000 Harcourt Publishers Ltd.)

Objectives: Vascularity of the surface of the placenta in humans and of the placenta and fetal membranes in several species including sheep is an important determinant of intramembranous absorption of amniotic fluid. Our previous studies have shown that the total blood vessel surface area in ovine amnion and chorion increases with advancing gestation. Vascular endothelial growth factor (VEGF) is a potent angiogenic and permeability factor and is found to be expressed in the ovine placenta and fetal membranes. To investigate the role of VEGF in maintaining the absorptive function of the intramembranous microvessels, the present study was undertaken to determine the gestational change in gene expression of VEGF and its receptors, kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase (Flt-1), in ovine placenta, chorion, and amnion., Methods: Total RNA was extracted from placental cotyledon, chorion, and amnion of ovine fetuses at 60-140 days of gestation. The relative abundance of VEGF, KDR, and Flt-1 mRNA was determined by Northern blot analysis, and VEGF molecular forms expressed were identified by reverse transcriptase polymerase chain reaction. The gestational changes in mRNA levels of VEGF and its receptors were analyzed by regression analysis., Results: In ovine placenta, chorion, and amnion, VEGF mRNA levels increased significantly from 60 to 140 days. The major VEGF molecular form expressed in these tissues was VEGF164, whereas VEGF120, VEGF144, and VEGF188 were present at lower levels. In the placenta, KDR was the primary VEGF receptor expressed, although Flt-1 was also detected at very low levels. In the amnion and chorion, KDR was the only receptor expressed. A gestational-dependent change in VEGF receptor expression was not observed in the placenta and membranes., Conclusions: The increase in VEGF gene expression with advancing gestation in the amnion and chorion where KDR is expressed suggests that VEGF and its receptor are important determinants of vascularity and permeability, and thus exchange capacity, of the intramembranous pathway.

A novel apparatus in which fluids may be injected and sampled at high pressure is described. Bioseparation applications of the apparatus were demonstrated in three model systems: (1) lambdaDNA was eluted under pressure from an anion exchange column into a low-salt (0.25 M) buffer, thereby eliminating conventional time-consuming desalting procedures required for downstream analysis of the DNA; (2) RNA was separated under pressure from a RNA/DNA mixture, thereby enabling rapid differential preparation of nucleic acids; and (3) an antibody was purified from a protein mixture by affinity capture at one pressure and dissociation from the antigen binding partner at a second pressure, thereby enabling the immunoreactivities of both antibody and antigen to be preserved during the separation process., (Copyright 1999 Academic Press.)

The highest sensitivity nucleic acid gel stains developed to date are optimally excited using short-wavelength ultraviolet or visible light. This is a disadvantage for laboratories equipped only with 306- or 312-nm UV transilluminators. We have developed a new unsymmetrical cyanine dye that overcomes this problem. This new dye, SYBR Gold nucleic acid gel stain, has two fluorescence excitation maxima when bound to DNA, one centered at approximately 300 nm and one at approximately 495 nm. We found that when used with 300-nm transillumination and Polaroid black-and-white photography, SYBR Gold stain is more sensitive than ethidium bromide, SYBR Green I stain, and SYBR Green II stain for detecting double-stranded DNA, single-stranded DNA, and RNA. SYBR Gold stain's superior sensitivity is due to the high fluorescence quantum yield of the dye-nucleic acid complexes ( approximately 0.7), the dye's large fluorescence enhancement upon binding to nucleic acids ( approximately 1000-fold), and its capacity to more fully penetrate gels than do the SYBR Green gel stains. We found that SYBR Gold stain is as sensitive as silver staining for detecting DNA-with a single-step staining procedure. Finally, we found that staining nucleic acids with SYBR Gold stain does not interfere with subsequent molecular biology protocols., (Copyright 1999 Academic Press.)

Streptozotocin-induced diabetes for 4 weeks resulted in a decrease in proopiomelanocortin (POMC) mRNA in both the anterior lobe (AL) and the neuro-intermediate lobe (NIL) of the rat pituitary. The beta-endorphin levels decreased in the NIL but not in the AL. It is concluded that the synthesis of POMC in the pituitary is inhibited in diabetic rats and that there is a decrease in beta-endorphin release from the anterior pituitary.

We describe the development of a sensitive fluorescence-based solution assay for RNA using a new dye, RiboGreen RNA quantitation reagent. RiboGreen reagent exhibits >1000-fold fluorescence enhancement and high quantum yield (0.65) upon binding nucleic acids, with excitation and emission maxima near those of fluorescein. Unbound dye is essentially nonfluorescent and has a large extinction coefficient (67,000 cm-1 M-1). The RiboGreen assay allows detection of as little as 1.0 ng/ml RNA in a standard fluorometer, filter fluorometer, or fluorescence microplate reader-surpassing the sensitivity achieved with ethidium bromide by 200-fold. The linear quantitation range for RiboGreen reagent extends over three orders of magnitude in RNA concentration. Using 750 nM RiboGreen reagent, we quantitated 20 ng/ml to 1.0 microg/ml RNA. By diluting the reagent to 75 nM, we could quantitate 1.0 to 50 ng/ml RNA. Both assay ranges exhibited linear fluorescence increases versus RNA concentration (r2 = 0.999). Assay linearity was maintained in the presence of salts, protein, urea, ethanol, chloroform, agarose, and some detergents. Several different RNA types yielded similar signal intensities and detection sensitivities. The assay is easy to use, rapid, and readily adaptable for automation., (Copyright 1998 Academic Press.)

Previous studies have demonstrated that extracellular ATP is able to activate Ca2+-dependent Cl- channels which may be important in circumventing the defective cAMP-dependent pancreatic ductal HCO3- secretion in cystic fibrosis (CF). The present study further investigated the effect of extracellular ATP on the stimulation of HCO3- secretion across CF pancreatic duct cells (CFPAC-1). Cells were grown in culture plate inserts which enabled the access of a pH microelectrode to the apical compartment. Changes in apical pH upon stimulation by extracellular ATP, as an indication of HCO3- secretion, were measured. ATP induced a rapid increase in apical pH, which reached a plateau with an averaged pH unit of 0.3 higher than that measured in unstimulated cells. The effect of ATP was concentration dependent. The ATP-induced change in pH could be blocked by apical addition of Cl- channel blockers, indicating that activation of apical Cl- channel is vital for HCO3- secretion by the pancreatic duct cells. Together with the previous finding, the present study suggests that HCO3- secretion may be stimulated in CF pancreatic duct cells by extracellular ATP via a cAMP-independent pathway.

Qualitative and quantitative polymerase chain reaction (PCR) for human herpesvirus 6 (HHV-6) DNA in whole blood and plasma was correlated with serology and clinical assessment in 143 children hospitalized for undifferentiated febrile illness to evaluate options for diagnosis of primary HHV-6 infection on the acute blood specimen. PCR and serology for HHV-7 were done in parallel to define serologic cross-reactions. Using HHV-6 seroconversion as the reference standard, detection of HHV-6 DNA in whole blood in the absence of antibody in the plasma was the most reliable evidence of primary HHV-6 infection. Detection of HHV-6 DNA in plasma and a high virus load in whole blood (>3.3 log10 copies/5 microL) had a sensitivity of 90% and 100%, respectively, in diagnosing primary HHV-6 infection. However, both were occasionally found in patients with other infections, possibly associated with HHV-6 reactivation. Maternal antibody may confound interpretation of serology in patients under 3 months of age.

The use of pressure to disrupt immune complexes was demonstrated in two model systems: prostate-specific antigen (PSA) and anti-PSA antibody; and epiglycanin, a mucin glycoprotein, and an antibody specific to that protein. Dissociation of the anti-PSA antibody from the immobilized PSA antigen was observed when pressures of 415 MPa and 550 MPa (1 MPa approximately 144 psi) were applied at room temperature (approximately 21 degrees C). Application of pressures ranging from 140 MPa to 550 MPa resulted in dissociation of antibody from epiglycanin. In both cases, the rebinding of dissociated antibody to immobilized antigen indicated that the effect of high pressure on the binding of the immune complexes was reversible. These findings suggest that application of high hydrostatic pressure has the potential to be used to significantly improve the sensitivity and specificity of clinical assays.

To examine the role of vascular endothelial growth factor (VEGF) in mediating angiogenesis and vascular permeability during fetal development, we determined the gene expression of VEGF in ovine fetal tissues. Further, we cloned and sequenced the ovine VEGF cDNA encoding VEGF164 from sheep placenta. VEGF protein was localized in epithelial cells of the placenta and fetal kidney, and in hepatocytes of the fetal liver. By Northern analysis, a major VEGF mRNA species of 3.7 kb was identified in all tissues examined, with abundance highest in the lung and lowest in the liver. The most prominent molecular form expressed in ovine fetal tissues appeared to be VEGF164 with low levels of expression of VEGF120 and VEGF188. Cloning and sequence analysis of the most abundant form of ovine VEGF cDNA in the placenta confirmed the prediction of a 164-amino acid peptide, with a putative N-terminal signal sequence of 26 amino acids. Comparison of the VEGF cDNA sequence among different species revealed that VEGF is highly conserved suggesting an important role in development.

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen with potent permeability properties. This growth factor exists in several isoforms; the most abundant form present in most tissues is VEGF165. The different isoforms exhibit differences in biologic function. During development, VEGF is expressed in multiple embryonic and fetal tissues, with the highest levels found in the lung, kidney, and heart. Vascular endothelial growth factor is also expressed in placental tissues and fetal membranes, and this expression increases with advancing gestation. In the fetal heart and placenta, VEGF expression is inducible by hypoxia. Two receptors, KDR and Flt-1, have been identified for VEGF. They are widely expressed in vascular endothelial cells and are also found in placental tissues where VEGF is localized. In humans, Flt-1 appears to be the predominant receptor, whereas in the cow and sheep, KDR is the major receptor expressed. The presence of VEGF and its receptors in placental tissues throughout gestation strongly suggests that VEGF plays an important role in the development and maintenance of placental vascular function during pregnancy. The localization of VEGF in fetal membranes and the fetal surface of the placenta raises the possibility that VEGF may be involved in the regulation of amniotic fluid volume and composition.

Objective: Insulin-like growth factors (IGF)-I and -II have been implicated in growth and differentiation during embryonic and fetal development. To examine the role of the IGFs in growth of the fetal heart, we determined the gene expression of IGF-I and IGF-II in the four cardiac chambers of the ovine fetus from 58 to 146 days' gestation (term = 147 days)., Methods: Total RNA was obtained from the cardiac chambers, analyzed by Northern blot, and hybridized to ovine specific cDNA probes for IGF-I and IGF-II. The resulting autoradiograms were subjected to light densitometry, and the intensity of the IGF signals was normalized to the respective 28S ribosomal RNA signals., Results: In the atria and ventricles, IGF-I mRNA abundance was very low throughout the gestational period studied, whereas IGF-II mRNA levels were higher and readily detectable. In the atria, IGF-I mRNA was very low at 60 days' gestation and appeared to increase gradually toward term. Abundance of IGF-II mRNA was high at 60 days, increased further until 120 days, and decreased slightly toward term. In contrast, in the ventricles, IGF-I mRNA increased from 60 to 100 days and then declined moderately at term. Levels of IGF-II mRNA in the ventricles were high at 60 days and decreased progressively to low levels at term. No difference in IGF-I or IGF-II mRNA levels was noted between the right and left atria or right and left ventricles., Conclusion: These results suggest that developmental patterns for IGF-I and IGF-II gene expression exist in the ovine fetal heart, and the patterns differ between the atria and ventricles. Further, these gestational trends differ from those for atrial natriuretic factor (ANF) found in our previous studies, indicating that expression of the ANF gene in the fetal heart may not be associated with cardiac growth and differentiation.

The effect of pituitary adenylate cyclase-activating polypeptide (PACAP-27) on tyrosine hydroxylase activity has been studied in intact, cultured, bovine adrenal chromaffin cells. Tyrosine hydroxylase activity was determined in situ by measuring the production of 14CO2 following the hydroxylation and rapid decarboxylation of [14C]tyr offered to the cells. PACAP-27 increased tyrosine hydroxylase activity 3-fold over 10 min. With an EC50 of 10-20 nM. PACAP-38 was approximately 2-fold less potent. Removing extracellular Ca2+ reduced basal tyrosine hydroxylase activity and the activation produced by both PACAP-27 and forskolin by about 20%. In the absence of extracellular Ca2+, chelation of intracellular Ca2+ by treating cells with BAPTA-AM (50 microM) caused a consistent 40-50% reduction in basal tyrosine hydroxylase activity and in the responses to forskolin and PACAP-27. The tyrosine hydroxylase activation produced by PACAP-27 was unaffected by the protein kinase C inhibitor Ro 3l-8220 (3 microM), but was reduced by 85% by the protein kinase A inhibitor H89 (10 microM). PACAP-27 increased cellular cyclic AMP levels 3-fold at 100 nM. The results suggest that PACAP-27 activates tyrosine hydroxylase in bovine chromaffin cells through cyclic AMP formation and protein kinase A activation, and that both extracellular and intracellular Ca2+ modulate the effect of the adenylate cyclase/cyclic AMP/protein kinase A signalling pathway on tyrosine hydroxylase activity.

Objective: The purpose of this study was to explore the gene expression of vascular endothelial growth factor (VEGF) in placental cotyledon, chorion, and amnion of the ovine fetus., Study Design: Time-dated pregnant sheep with singleton or twin fetuses at a gestational age ranging from 100 to 140 days were used for the study. Placental cotyledonary, chorionic, and amniotic tissues were collected and processed for messenger ribonucleic acid analysis by Northern blotting and reverse transcription-polymerase chain reaction., Results: By use of a phosphorus 32-labeled human VEGF complementary deoxyribonucleic acid probe, a prominent VEGF messenger ribonucleic acid transcript of 3.7 kb was detected in the cotyledon, chorion, and amnion. A minor band of 1.7 kb was also found but only in the cotyledon and chorion. The abundance of messenger ribonucleic acid encoding VEGF was highest (p < 0.001) in the cotyledon and lowest in the amnion. In these tissues polymerase chain reaction-amplified products corresponding to VEGF121, VEGF165, VEGF189, and VEGF206 were identified by ethidium bromide. In addition, a polymerase chain reaction fragment corresponding to VEGF145 was observed. These fragments produced specific hybridization signals with the human VEGF radioactive probe where the intensity of the signal was strongest for VEGF165 and weakest for VEGF189., Conclusions: VEGF gene expression was detected in the cotyledon, chorion, and amnion of the near-term ovine fetus. These findings suggest that vascular endothelial growth factor may play a role in the induction of angiogenesis and promotion of permeability in the microvessels that perfuse the placental and fetal membranes.

Objective: The present study quantified the abundance of atrial natriuretic factor (ANF) messenger RNA (mRNA) and determined the developmental pattern of ANF gene expression in the four cardiac chambers of the ovine fetus during the last two-thirds of gestation., Methods: Twenty-one fetuses from 13 time-dated pregnant ewes at gestational ages of 60-145 days were used for this study. Total RNA from fetal atria and ventricles was extracted and ANF mRNA was analyzed by Northern blotting. The ANF mRNA signal was quantified by light densitometry. The abundance of ANF mRNA in the cardiac chambers across gestational ages was analyzed by linear regression analysis and one-way analysis of variance., Results: Atrial natriuretic factor mRNA was much more abundant in the atria than in the ventricles of all fetuses at each gestational age studied. Atrial ANF mRNA levels were lowest in the younger fetuses at 60 days and increased with advancing gestation. Ventricular ANF mRNA levels were highest in fetuses at 60 days and decreased to almost nondetectable levels near term. No difference in ANF mRNA abundance was noted between the right and left atria or the right and left ventricles at each gestational age., Conclusion: A developmental pattern of ANF gene expression is demonstrated in the ovine fetal heart during the last two-thirds of gestation. This pattern shows that atrial ANF mRNA abundance increases while ventricular abundance decreases as the fetus matures. Expression of the ANF gene in the fetal period may be regulated developmentally or induced by cardiovascular changes in utero.

Objectives: The current study investigated the developmental changes in atrial natriuretic factor peptide content and messenger ribonucleic acid localization in the atria and ventricles of the ovine fetus throughout the second half of gestation., Study Design: Ovine fetuses from 67 to 146 days' gestation (term 147 days) and newborn lambs were used for the study. Tissue atrial natriuretic factor contents were determined by radioimmunoassay, and atrial natriuretic factor messenger ribonucleic acid distribution was determined by in situ hybridization., Results: In fetal atria, atrial natriuretic factor peptide levels were much greater than those in the ventricles. The levels in the atria increased with advancing gestation from 70 to 140 days, reflecting an increase in weight of the atrial chambers. A similar trend was not observed in the ventricles. In the atria, atrial natriuretic factor peptide content (per unit protein) reached high levels at 100 to 110 days' gestation; this was associated with an increase in level of atrial natriuretic factor gene expression. In the ventricles, atrial natriuretic factor peptide content and gene expression were very low throughout the second half of gestation, except for a peak in content that occurred at 100 days. Atrial natriuretic factor messenger ribonucleic acid abundance was much greater in the atria than in the ventricles in fetuses from 90 to 130 days' gestation. The distribution of atrial natriuretic factor messenger ribonucleic acid was homogeneous throughout the thickness of the atria and ventricles of the fetal heart., Conclusion: During the second half of gestation in the ovine fetus, the expression of atrial natriuretic factor messenger ribonucleic acid in the atria and ventricles paralleled the appearance of the peptide.

Objective: The purpose of our study was to explore the urinary responses of the ovine fetus to indomethacin levels comparable with those used therapeutically in the human fetus., Study Design: After a 1-hour control period, chronically catheterized ovine fetuses between 125 and 139 days of gestation were given an intravenous bolus of indomethacin (0.05 mg/kg estimated fetal weight) followed by a 0.0025 mg/kg/min continuous infusion for 5 hours. The experimental group (n = 9) was compared with a vehicle-only infusion group (n = 10)., Results: There was a sustained 55.7% +/- 9.5% (mean +/- SEM) decrease in urinary output by 2 hours of indomethacin infusion (p < 0.00001, analysis of variance). Urinary osmolality, potassium, and chloride concentrations underwent sustained increases during the infusion period (p < 0.005). Free water clearance decreased by 67.5% +/- 12.0% (p < 0.001). Fetal arterial pressure increased only transiently (p < 0.05), and increases in venous pressure (p = 0.013) and heart rate (p < 0.0001) were sustained. Fetal plasma arginine vasopressin concentration increased during indomethacin infusion (p < 0.05) and was correlated with the fall in urinary flow rate and free water clearance (p = 0.002). During vehicle infusion no significant changes were observed in any of the variables., Conclusions: Our data indicate that the fetus undergoes antidiuresis when exposed to low levels of indomethacin and that the observed antidiuresis is mediated by a decrease in free water clearance. The reduction in free water clearance may be mediated by increases in plasma arginine vasopressin concentrations.

Objective: This study investigated the maturational change in the atrial natriuretic factor response to hypoxia in ovine fetuses and the role of the autonomic nervous system and arginine vasopressin in modulating this response., Study Design: Chronically catheterized ovine fetuses from 110 to 135 days' gestation were subjected to 30 minutes of hypoxia. The fetuses were either intact, treated with hexamethonium to block the autonomic nervous system, or treated with a pressor antagonist of arginine vasopressin., Results: Hypoxia elevated plasma atrial natriuretic factor levels by 1635 +/- 265 pg/ml in immature fetuses; this response was greater than the increase of 748 +/- 189 pg/ml in mature fetuses (p < 0.0001). Blockade of the autonomic nervous system reduced the atrial natriuretic factor response and suppressed the vascular pressure changes to hypoxia in immature but not in mature fetuses. A vasopressin pressor antagonist suppressed the atrial natriuretic factor but not vascular pressure responses to hypoxia in mature fetuses., Conclusions: This study demonstrates that the immature fetus manifested a greater atrial natriuretic factor response to hypoxia than did the mature fetus. This enhanced response appeared to be dependent on the modulatory effects of the autonomic nervous system on vascular pressures during hypoxia. In addition, arginine vasopressin appeared to augment the atrial natriuretic factor response to hypoxia in the mature fetus independent of vascular pressure changes.

Intraamniotically injected substances such as arginine vasopressin and digoxin have been found to rapidly appear in the ovine fetal circulation, irrespective of whether the fetal esophagus has been ligated or occluded. To determine if the ovine fetal oral-nasal membrane plays a significant role in this fetal absorption of amniotic substances, we used two groups of chronically catheterized fetal sheep with a surgical glove sewed over the fetal head to prevent access of the head to amniotic fluid. In the first group 22.5 micrograms of arginine vasopressin was injected into the amniotic cavity; in the second group 22.5 micrograms was injected into the glove over the fetal head. We found that, after injection into the amniotic cavity, there were rapid and highly significant increases in amniotic fluid arginine vasopressin concentrations, from 6.1 +/- 1.3 to 51,249 +/- 18,182 pg/ml (mean +/- SE) (p less than 0.00001). Concurrently there was a rapid increase in fetal plasma arginine vasopressin concentrations from 4.5 +/- 1.3 to 93.8 +/- 18.9 pg/ml (p less than 0.00001). The increase was significant within 15 minutes and reached a maximum at 60 minutes after the injection. Fetal arterial pressure increased by 10 +/- 2 mm Hg, whereas heart rate decreased by 30 +/- 5 beats/min (p less than 0.00001). In contrast, after the injection into the glove covering the fetal head, there were no significant changes in any of the measured parameters. This suggests that the ovine fetal oral-nasal membrane is not a significant route of absorption of amniotic fluid arginine vasopressin and that the most likely route of absorption is the vascularized fetal surface of the placenta and vascularized fetal membranes, i.e., the intramembranous pathway.

The purpose of the present study was to determine whether endogenous atrial natriuretic factor participates in the maintenance of normal vascular pressure and renal function in ovine fetuses at 128 to 130 days' gestation. Circulating atrial natriuretic factor in the fetus was immunoneutralized by an intravenous bolus injection of an atrial natriuretic factor antiserum at a dilution of 1:2000 (low dose, n = 7) or 1:400 (high dose, n = 6). In the high-dose group, plasma atrial natriuretic factor concentration was significantly reduced by 65 +/- 14 pg/ml from basal levels of 165 +/- 12 pg/ml within 10 minutes and remained reduced for the 90-minute period after the injection. Fetal arterial pressure acutely and transiently decreased, but at 50 minutes arterial pressure increased and was elevated for the remainder of the experiment. Urine flow and urinary excretion rates of sodium, potassium, and chloride were reduced within 10 minutes after the injection. Urine flow rate was suppressed for as long as plasma atrial natriuretic factor concentrations were reduced. Fetuses in the low-dose and control groups showed no significant change in cardiovascular or renal function. In response to atrial natriuretic factor antiserum injection, plasma angiotensin II concentrations were increased, whereas plasma arginine vasopressin concentrations were unchanged. These results suggest that endogenous atrial natriuretic factor is involved in the maintenance of arterial pressure and urinary excretion in the ovine fetus.

The purpose of this experiment was to establish dose-response relationships for the effects of angiotensin II on arterial pressure, venous pressure, heart rate, and blood volume in the ovine fetus. Chronically catheterized fetal sheep at 132 +/- 1 (SE) days' gestational age were infused with angiotensin II at 4.8 +/- 1.1 (n = 7), 27.7 +/- 4.6 (n = 7), 102.2 +/- 16.7 (n = 6), or 239.0 +/- 30.9 (n = 4) ng/min/kg fetal body weight for 30 minutes. Fetal arterial pressure increased at the three highest doses. Fetal venous pressure, heart rate, and blood volume responded only at 102.2 and 239.0 ng/min/kg. At 239.0 ng/min/kg, arterial pressure increased by 17.7 +/- 1.6 mm Hg (p less than 0.00001), venous pressure increased by 1.5 +/- 0.3 mm Hg (p less than 0.0005), blood volume decreased by 7.8 +/- 2.2% (p less than 0.0001), and heart rate initially decreased by 14 +/- 4 beats/min, followed by an increase of 52 +/- 17 beats/min from control (p less than 0.0005) at the end of angiotensin II infusion. Thus this study shows that angiotensin II affected multiple fetal cardiovascular variables in a dose-dependent manner, suggesting that it is an important regulatory hormone for the entire fetal cardiovascular system.

To investigate the effects of blood volume reduction on fetal plasma atrial natriuretic factor concentrations, chronically catheterized ovine fetuses at 109 to 138 days' gestation were subjected either to withdrawal of two consecutive blood samples or to a moderate hemorrhage. In fetuses from which two blood samples of 3.5 ml each (approximately 1% of fetal blood volume) were withdrawn under basal conditions at 15- to 30-minute intervals, plasma atrial natriuretic factor concentrations in the second sample decreased by 17 +/- 7 pg/ml from 178 +/- 8 pg/ml in the first sample (p less than 0.02). If the fetal blood removed during the first sample was replaced with an equal volume of maternal blood, plasma atrial natriuretic factor concentrations did not change significantly. In these same samples, plasma arginine vasopressin and angiotensin II concentrations were unchanged between the two samples regardless of whether volume was replaced. In fetuses subjected to hemorrhages of 21% +/- 2% over 10 minutes without blood replacement, plasma atrial natriuretic factor concentration at 1.5 hours after hemorrhage was suppressed by 42 +/- 10 pg/ml from basal level of 139 +/- 9 pg/ml (p less than 0.05). Plasma atrial natriuretic factor returned to control levels by 5.5 hours after hemorrhage as blood volume returned to normal. Thus in the ovine fetus a reduction in blood volume results in a decrease in plasma atrial natriuretic factor concentrations. Also, atrial natriuretic factor appears to be more sensitive to changes in blood volume than other vasoactive hormones studied.

Recently an intramembranous pathway was reported in the ovine fetus as a route for the movement of a significant volume of water from the amniotic cavity directly into the fetal blood, which perfuses the fetal membranes and fetal surface of the placenta. To test whether this pathway could be an avenue for the movement of arginine vasopressin from the amniotic cavity into the fetal circulation, we injected 1 to 25 micrograms of arginine vasopressin into the amniotic cavity of two groups of chronically catheterized fetal sheep: a control group of seven animals and a group of seven animals with surgical ligation of the fetal esophagus. We found similar and highly significant increases of arginine vasopressin concentrations in both control and surgically ligated fetuses in amniotic fluid (p less than 0.00001), fetal plasma (p less than 0.0001), and fetal urine (p less than 0.0001). Both groups had similar increases in arterial (p less than 0.0001) and venous (p less than 0.003) pressures with simultaneous decreases in urine flow (p less than 0.001) and heart rate (p less than 0.0001) after the intraamniotic injection of arginine vasopressin. We conclude that amniotic arginine vasopressin can be rapidly absorbed in its biologically active form directly into the fetal circulation through the intramembranous pathway. Furthermore, the observation that esophageal ligation did not alter this absorption suggests that the intramembranous pathway may be important in the regulation of amniotic fluid volume and composition.

The present study was designed to test the hypothesis that arginine vasopressin mediates the fetal cardiovascular response to acute hypoxia. Chronically catheterized sheep fetuses at 126 to 138 days' gestation were infused with either an arginine vasopressin pressor antagonist (n = 8) or saline solution as control (n = 8). A 30-minute hypoxia was induced by infusion of nitrogen containing 5% carbon dioxide into the maternal trachea. Fetal arterial PO2 decreased 13.1 +/- 1.3 (SE) mm Hg from a basal value of 23.8 +/- 1.5 mm Hg and there was no significant difference in the degree of hypoxia between the two groups. Fetal arterial and venous pressures increased significantly, whereas the blood volume decreased, but these changes were similar between the control and arginine vasopressin-blocked fetuses. Heart rate fell similarly in both groups during hypoxia by an average of 35 beats/min. At the termination of hypoxia, heart rate in the blocked group rebounded to levels significantly above baseline and remained elevated for 30 minutes, whereas heart rate in the control group returned slowly to basal values. Recovery of arterial pressure, venous pressure, and blood volume were similar in the two groups. Thus it appears that arginine vasopressin may mediate in part the fetal heart rate response to acute hypoxia. However, the blood volume, arterial pressure, and venous pressure responses to hypoxia appear to be induced by factors other than arginine vasopressin.

Vasoactive intestinal peptide (VIP) was found in the adrenal gland of ovine fetuses at 130-135 days gestation and was shown to stimulate catecholamine secretion. VIP was demonstrated by immunocytochemistry using the indirect antibody-enzyme method. VIP-immunoreactive nerve fibers were observed in the capsule, zona glomerulosa and inner layer of the cortex as well as in the medulla; furthermore small clusters of VIP-containing cell bodies were found at the corticomedullary border. To study the direct effect of VIP on catecholamine release, fetal adrenal medulla was dispersed into single cells and incubated in vitro with VIP for 6 hours. Catecholamine release into the medium was measured at 1, 3 and 6 hours. At 6 hours of incubation, VIP stimulated total catecholamine release from fetal adrenomedullary cells in a dose-dependent manner at concentrations ranging from 10(-8) to 10(-4) M. The release of norepinephrine and epinephrine, but not dopamine, was significantly enhanced. The presence of VIP in the fetal adrenal cortex and medulla, and the ability of VIP to stimulate catecholamine release from fetal adrenomedullary cells in vitro suggest that VIP may be an important modulator of medullary catecholamine secretion during fetal life.

Long term destruction of the tuberoinfundibular dopaminergic neurons results in increased responsiveness of the anterior pituitary to the suppression of PRL release by dopamine. In the present study we tested whether long term destruction of these neurons by medial basal hypothalamic lesions would also increase the potency of dopamine in suppressing the release and apparent synthesis of PRL. Anterior pituitaries from ovariectomized control rats and ovariectomized rats lesioned for 2 weeks were incubated for 3 h in medium 199 containing [3H]leucine in the presence or absence of various concentrations of dopamine. Labeled PRL was separated by polyacrylamide gel electrophoresis and quantitated by liquid scintillation spectroscopy. Concentrations of dopamine ranging from 10(-8)-10(-6) M caused a dose-dependent suppression of labeled newly synthesized PRL released into the medium from anterior pituitaries of medial basal hypothalamus-lesioned animals. With pituitaries from the nonlesioned rats, newly synthesized PRL release was progressively inhibited by 10(-7) and 10(-6) M dopamine, while 10(-8) M dopamine actually significantly stimulated PRL release. Total labeled PRL (that released and that remaining in the gland) was equally suppressed by all three concentrations of dopamine in pituitaries from lesioned animals, but only a minor effect was observed at the highest concentration of dopamine with the control pituitaries. Therefore, the potency of dopamine to suppress the release and apparent synthesis (total labeled PRL) of newly synthesized PRL from pituitaries of long term lesioned animals was increased.