Human rhinovirus-induced inflammatory responses are inhibited by phosphatidylserine containing liposomes.

Human rhinovirus (HRV) infections are major contributors to the healthcare burden associated with acute exacerbations of chronic airway disease, such as chronic obstructive pulmonary disease and asthma. Cellular responses to HRV are mediated through pattern recognition receptors that may in part signal from membrane microdomains. We previously found Toll-like receptor signaling is reduced, by targeting membrane microdomains with a specific liposomal phosphatidylserine species, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine (SAPS). Here we explored the ability of this approach to target a clinically important pathogen. We determined the biochemical and biophysical properties and stability of SAPS liposomes and studied their ability to modulate rhinovirus-induced inflammation, measured by cytokine production, and rhinovirus replication in both immortalized and normal primary bronchial epithelial cells. SAPS liposomes rapidly partitioned throughout the plasma membrane and internal cellular membranes of epithelial cells. Uptake of liposomes did not cause cell death, but was associated with markedly reduced inflammatory responses to rhinovirus, at the expense of only modest non-significant increases in viral replication, and without impairment of interferon receptor signaling. Thus using liposomes of phosphatidylserine to target membrane microdomains is a feasible mechanism for modulating rhinovirus-induced signaling, and potentially a prototypic new therapy for viral-mediated inflammation.

Genetic variation in hepatic glucose-6-phosphatase system genes in cases of sudden infant death syndrome.

Genetic deficiencies of the hepatic glucose-6-phosphatase system, either of the enzyme (G6PC1) or of the glucose-6-phosphate transporter (G6PT1), result in fasting hypoglycaemia. Low hepatic G6PC1 activities were previously reported in a few term sudden infant death syndrome (SIDS) infants and assumed to be due to G6PC1 genetic deficiencies. In preterm infants, failures of postnatal activation of G6PC1 expression suggest disordered development as a novel cause of decreased G6PC1 activity in SIDS. G6PC1 and G6PT1 functional and mutational analysis was investigated in SIDS and non-SIDS infants. G6PC1 hepatic activity was abnormally low in 98 SIDS (preterm, n=13; term, n=85), and non-SIDS preterm infants (n=35) compared to term non-SIDS infants (n=29) and adults (n=9). Mean glycogen levels were elevated, except in term non-SIDS infants. A novel G6PT1 promoter polymorphism, 259C --> T was found; the - 259*T allele frequency was greater in term SIDS infants (n=140) than in term control infants (n=119) and preterm SIDS infants (n=30). Heterozygous and homozygous prevalence of 259C --> T was 38.6% and 7.1%, respectively, in term SIDS infants. In cell-based expression systems, the presence of - 259T in the promoter decreased basal luciferase activity by 3.2-fold compared to - 259C. Glucose-6-phosphatase latency in hepatic microsomes was elevated (indicating decreased G6PT1 function) in heterozygous and homozygous - 259T states. Delayed postnatal appearance of hepatic glucose-6-phosphatase in infants makes them vulnerable to hypoglycaemic episodes and this may occur in some SIDS infants. However, SIDS may be an association of more complex phenotypes in which several genes interact with multiple environmental factors. A UK-wide DNA Biobank of samples from all infant deaths, with an accompanying epidemiological database, should be established by pathologists to allow cumulative data to be collected from multiple genetic investigations on the same large cohort of samples, with the aim of selection of the best combination of genetic markers to predict unexpected infant death.

Alcohol abuse--a persistent preventable risk for congenital anomalies.

OBJECTIVE: To examine whether alcohol abuse (ALC) continued to be a health hazard to pregnant women in the 1990s. STUDY DESIGN: Analysis of a perinatal data base comprising 170,258 women with singleton pregnancies. Univariate cross table analysis and logistic regression were conducted to examine the association between alcohol abuse and congenital malformations coded according to the International Classification of Diseases (ICD). RESULTS: 14,727/170,258 mothers (8.6%) admitted to ALC during pregnancy and 36,705/170,258 (21.6%) to smoking. Anomaly rates for ALC (365/14,092, 4.3%) vs. Non-ALC (6187/149,344, 4.0%) differed significantly (p<0.001). The rates of specific anomalies varied between <0.1% and 1.1%. Odds ratios for 16 ICD 9 anomaly categories were >1 in 14 instances overall (Sign test, p=0.004), in 12 instances in women <30 years (p=0.08), and in 13 instances in women over 30 years (p=0.02). Congenital anomalies of the "respiratory system" (ICD9 748), of "genital organs" (ICD9 752.1), of the "integument" (ICD9 757), and "other anomalies of limbs/other musculoskeletal anomalies" (ICD 755/756) were statistically significantly associated with ALC, especially in women>30 years. CONCLUSION: ALC in pregnancy continued to be an important factor independently associated with an increased incidence of a broader range of congenital anomalies than previously recognized. Risk for anatomic anomalies was increased in offspring of ALC women over age 30, consistent with previous reports of increased risk of neurobehavioral abnormality in offspring of women over 30.

Expression level dependent changes in the properties of P2X2 receptors.

The currents of P2X(2) receptors expressed in Xenopus oocytes or HEK293 cells show significant cell-to-cell variation in many properties including the rate of desensitization and the magnitude of potentiation by zinc or acidic pH. In this study, we examined whether differences in expression levels underlie this variability. We injected Xenopus oocytes with different concentrations of RNA encoding rat P2X(2) to give a wide range of maximum current amplitudes, and then measured the potentiation of responses to 10 micro M adenosine 5'-triphosphate (ATP) by zinc or acidic pH. Individual oocytes showed potentiation ratios that ranged from 1.4- to 25-fold. Oocytes with small amplitude responses to a saturating concentration of ATP tended to have larger potentiation ratios than oocytes with large amplitude responses. This phenomenon was explained by an inverse correlation between the EC(50) for ATP and the maximum current amplitude, with the EC(50) decreasing from about 37 to 7 micro M as expression level increased. In contrast, the Hill coefficient was not correlated with the maximum current amplitude. Truncated receptors lacking the last 76 amino acids also showed an inverse correlation between the EC(50) and the maximum current amplitude. Thus, the interactions that cause expression-dependent changes in P2X(2) receptor properties must involve domains proximal to position H397.

Iodine deficiency associated with parenteral nutrition in extreme preterm infants.

Infants are in negative iodine balance on current standard regimens of total parenteral nutrition, with a mean iodine intake of 3 micro g/kg/day (150 ml/kg/day). The recommended enteral intake of iodine for preterm infants is 30 micro g/kg/day. Gastrointestinal absorption of iodine is high, suggesting that parenteral intakes should approach enteral recommendations.

An inadequate glycaemic response to glucagon is linked to insulin resistance in preterm infants?

AIMS: To define clinical, metabolic, and hormonal characteristics of preterm infants relative to glucagon responsiveness. METHODS: Two phase study of 78 preterm infants (25-36 weeks gestation) on regular four hourly feeds anticipating discharge home at 36 weeks mean corrected gestation. In phase 1 infants were fasted until hypoglycaemic, or maximally for eight hours. Endocrine and metabolic profiles were obtained at completion. Phase 2 was performed the following day. A feed was omitted and replaced by a bolus dose of intravenous glucagon (100 micro g/kg). Main outcome measures were measurements of blood glucose and lactate concentrations, taken immediately pre-glucagon, and thereafter every 15 minutes for 60 minutes. A rise in glucose concentration of >1 mmol/l (55 infants) was defined as an adequate response to glucagon. An inadequate glycaemic response was <1 mmol/l (23 infants). RESULTS: Several differences in fasting blood glucose and hormone concentrations were identified in infants with an inadequate glycaemic response to glucagon compared to those with an adequate response: relative fasting hyperglycaemia (mean 3.7 v 3.3 mmol/l, p = 0.008); fasting hyperinsulinaemia (mean 4.3 v 2.6 mU/l, p = 0.014); an increased insulin:glucagon ratio (0.19 v 0.11, p = 0.014), and a lower insulin sensitivity QUICKI index (0.19 v 0.22, p = 0.04). There was no distinctive phenotype to reliably predict response to glucagon. CONCLUSION: Some preterm infants show an inadequate glycaemic response to glucagon and have features suggestive of insulin resistance. The potential long term implications of such insulin resistance may have appreciable public health consequences.

Polymorphisms in genes involved in glucose metabolism in cases of sudden infant death syndrome.

BACKGROUND: Infants with intra-uterine growth retardation have an increased risk of Sudden Infant Death Syndrome (SIDS). Hypoglycaemia is also significantly correlated with retardation of intra-uterine growth. A number of mutations in key proteins involved in regulation of blood glucose (e.g. glucokinase) have been found to result in reduced birth weight. Heterozygous mutations in the coding region of the glucokinase gene have been shown to cause MODY (a form of early onset Type II diabetes mellitus). The aim was to screen a cohort of SIDS and control infants who were either growth retarded or appropriately grown for gestational age to determine if any mutations and/or polymorphisms were present in the glucokinase gene. METHODS: PCR, denaturing high performance liquid chromatography on an automated Transgenomic WAVE DNA fragment analysis system and DNA sequencing. RESULTS: Genomic DNA was isolated from 129 infants who were either growth retarded or appropriately grown for gestational age. We found several rare novel polymorphisms in the glucokinase gene in the infant samples. However, none of the samples contained any of the mutations in the glucokinase gene previously reported in cases of MODY. CONCLUSIONS: We have found rare novel polymorphisms in the glucokinase gene in the infant samples. In contrast in these samples, we have not found any examples of the previously reported mutations in the coding region of the glucokinase gene found in MODY. This clearly shows that while MODY babies are often small, MODY is not a common cause of either intra-uterine growth retardation or of SIDS.

Developmental disorders of glucose metabolism in infants.

BACKGROUND: Developmental failures to adequately control postnatal blood glucose levels are common in the transition from fetal to infant life and can persist for many months. The standard method of functionally measuring hepatic glucose production and/or disordered glucose production is the response to a glucagon tolerance test. METHOD: We adapted the standard glucagon tolerance test used for children and adults for use in preterm infants. 79 consecutive preterm infants gestational age range 25-36 weeks (mean 32.2 weeks), mean birth weight 1.66 kg admitted to the Neonatal Intensive Care Unit, Ninewells Hospital, Dundee and who survived to discharge home were recruited into the study. At the time of discharge home the characteristics of the group were as follows: adjusted mean gestational age 36.7 weeks, mean discharge weight 2.23 kg. RESULTS: In this study of preterm infants the maximal increase in plasma glucose following administration of a glucagon tolerance test is 1.39 +/- 07 mmol/L, n = 78 (range 0-3.98 mmol/L). CONCLUSIONS: An increase in plasma glucose of less than 4 mmol/L is considered abnormal in adults following administration of a fasting glucagon tolerance test. The responses of preterm infants and adults to glucagon are clearly different. The attenuated response to glucagon in the preterm infants is consistent with the low levels of hepatic glucose-6-phosphatase activity in premature infants as glucose-6-phosphatase is the terminal step of the two main pathways of liver glucose production.

Differential expression of sulfotransferase enzymes involved in thyroid hormone metabolism during human placental development.

Thyroid hormone is essential for normal human development, and disruption of thyroid hormone homeostasis at critical developmental stages can result in severe and often long-term effects on crucial organs such as the brain and lungs. Numerous factors control the bioavailability of receptor active thyroid hormone T(3). Sulfation, catalyzed by sulfotransferase enzymes (SULTs), is an important pathway of thyroid hormone metabolism by which T(4) is irreversibly converted to inactive reverse T(3) rather than active T(3). The human fetus and neonate have high levels of circulating sulfated iodothyronines, although the source of these is not clear. The placenta forms the link between the fetus and its mother and is involved in transfer of thyroid hormone early in pregnancy, although its capacity for sulfation is unknown. We therefore examined expression of the SULTs involved in iodothyronine metabolism during human placental development. SULT activity was measured in human placental cotyledon and membranes (amnion, chorion, and decidua basalis) from 13-42 wk of gestation, and Western blot analysis was employed to verify enzyme activity data. Phenol and catecholamine sulfotransferases were expressed at the highest levels and were generally higher in the villous than membranous tissues. SULT1A1 activity showed significant correlation with sulfation of 3,3'-T(2), suggesting that this enzyme is primarily responsible for placental T(2) sulfation. Estrogen sulfotransferase was present at extremely low levels during early pregnancy, although in mid- and late gestation increased expression in the (predominantly maternal-derived) decidual component of the placenta was observed. Hydroxysteroid sulfotransferase, T(3), reverse T(3), and T(4) SULT activities were also low in all tissues examined, and expression of SULTs 1B1 and 1C2 were essentially undetectable by Western blot analysis. The results highlight a tissue-specific regulation of SULT expression during placental development, demonstrate very low sulfation of iodothyronines suggesting that the placenta is not a major source of circulating sulfated iodothyronines in the fetus.

The effects of hypoxia and hyperoxia on fetal-placental vascular tone and inflammatory cytokine production.

OBJECTIVE: To determine the effects of fetal hypoxia and hyperoxia on placental vascular tone and production of interleukin-6 and tumor necrosis factor-alpha. STUDY DESIGN: The maternal and fetal circulation of 2 cotyledons from 5 human placentas were perfused for 4 hours. The fetal circulation of 1 cotyledon was perfused with hypoxic Hanks' balanced salt solution; the other was perfused with hyperoxic Hanks' balanced salt solution. Fetal vascular pressures were recorded every 10 minutes, and fetal vein effluents were collected hourly. RESULTS: Fetal-placental vascular perfusion pressure was reduced from baseline during hypoxic conditions. Cytokine concentrations were elevated during hyperoxic conditions compared with hypoxic conditions, with significant differences achieved at 2, 3, and 4 hours for interleukin-6 and at 4 hours for tumor necrosis factor-alpha. CONCLUSION: Fetal-placental vasodilation may be a compensatory mechanism to improve hypoxic conditions. Supraphysiologic oxygenation may contribute to the fetal inflammatory response syndrome and to the development of cerebral palsy.

Regulation of prostaglandin availability in human fetal lung by differential localisation of prostaglandin H synthase-1 and prostaglandin dehydrogenase.

Specialisation of the respiratory portion of human fetal lung commences around 20-24 weeks gestation. In contrast, human fetal lung in vitro has the capacity to self-differentiate from 12 weeks gestation when grown in media devoid of growth factors or hormones, suggesting activation of autocrine or paracrine factors in vitro, or removal of the fetus from in utero inhibitory mechanisms. Prostaglandins play a key role during in vitro human fetal lung development and are synthesised by prostaglandin H synthase-1 (PGHS-1) and inactivated by 15-hydroxyprostaglandin dehydrogenase (PGDH) with formation of inactive 13,14-dihydro-15-keto-prostaglandins. We have used quantitative immunohistochemistry to determine expression and localisation of PGHS-1, PGDH, PGE2 and 13,14-dihydro-15-keto-PGE2 (PGEM) in human fetal lung with in situ hybridisation to localise PGHS-1 and PGDH mRNA. For the catabolic enzyme PGDH, amounts of mRNA, protein and enzyme product PGEM are increased within epithelium of distal as compared to more proximal airways. For PGHS-1, comparable amounts of mRNA, protein and enzyme product PGE2 are found in proximal and distal lung epithelium. Catabolism by PGDH is a sensitive mechanism for regulating bioavailability of prostaglandins and we propose that active catabolism of prostaglandins within human fetal lung epithelium is an inhibitory mechanism retarding epithelial differentiation in utero.

Thioredoxin reductase and cytoplasmic glutathione peroxidase activity in human foetal and neonatal liver.

Cytosolic thioredoxin reductase (TR) is an FAD-containing homodimeric selenoenzyme which, together with thioredoxin (Trx) and NADPH, forms a powerful oxidoreductase system. Cytoplasmic glutathione peroxidase (GPX-1) is a selenoprotein with antioxidant activity. The TR/Trx system has been associated with cellular processes including regulation of cell growth, and modification of activity of transcription factors. TR may also act as an antioxidant. We have measured TR activity, TR concentration, and GPX-1 activity in human hepatic cytosols from foetuses and neonates. The concentration of TR was significantly greater (P<0.05) in foetal (43.6, 37.9-50.8 microg/g protein, median, interquartile range) than in neonatal liver (11.6, 8.70-15.0 microg/g). This was also true of TR activity which was 2.1, 1.8-2.5 U/g protein in foetal, and 0.65, 0.44-0.74 U/g protein in neonatal liver (P<0.0005). Similarly, GPX-1 activity was significantly higher (P<0.005) in the foetal (199.7, 144.0-227.9 U/g protein) than in neonatal (77.0, 58.4-110.3 U/g protein) hepatic cytosol. Overall, foetal liver expressed approx. 3-fold higher activities of TR and GPX-1 than neonatal liver.

Sulfation of thyroid hormone and dopamine during human development: ontogeny of phenol sulfotransferases and arylsulfatase in liver, lung, and brain.

Sulfation is an important mechanism for regulating the biological activity of numerous hormones and neurotransmitters in man. Here we have investigated the ontogeny of sulfotransferases (SULT) and sulfatase (ARS) involved in the metabolism of thyroid hormone and dopamine. SULT1A1 enzyme activity was lower in postnatal liver and lung than in fetal tissues. Hepatic SULT1A3 (dopamine) was expressed at high levels early in development, but decreased substantially in late fetal/early neonatal liver and was essentially absent from the adult liver. In lung, significant SULT1A3 activity was observed in the fetus, but neonatal levels were considerably lower. In brain, the highest activity was observed in the choroid plexus for SULT1A1, with low and widespread activity for both SULT1A1 and SULT1A3 in other brain regions. SULT activity with 3,3'-diiodothyronine (3,3'-T(2)) as substrate was measured in all tissues and correlated significantly with SULT1A1 activity (4-nitrophenol), suggesting that SULT1A1 is primarily responsible for the sulfation of this iodothyronine. The developmental expression of SULT1A3 and SULT1A1 in liver and brain was confirmed by immunoblot, and immunohistochemistry of developing liver showed substantial expression of these proteins in hemopoietic cells in fetal liver. We also detected low activity for the hydrolysis of 3,3'-T(2) sulfate by ARS, although there was less distinction between fetal and neonatal samples than with SULT activities. We have therefore shown that the developing fetus has substantial sulfation capacity. Sulfation may therefore play a major role in the homeostasis of hormones and other endogenous compounds as well as in detoxification in the fetus, particularly as other conjugating enzyme systems, such as the UDP-glucuronosyltransferases, are not expressed at significant levels until the neonatal period.

Consent to autopsy for neonates.

OBJECTIVES: To determine parents' views on autopsy after treatment withdrawal. DESIGN: Face to face interviews with 59 sets of bereaved parents (108 individual parents) for whose 62 babies there had been discussion of treatment withdrawal. RESULTS: All except one couple were asked for permission for postmortem examination; 38% refused. The main reasons for declining were concerns about disfigurement, a wish to have the child left in peace, and a feeling that an autopsy was unnecessary because the parents had no unanswered questions. The diagnosis, the age of the child, and the approach of the consultant appeared to influence consent rates. Of those who agreed to autopsies, 92% were given the results by the neonatologist concerned. Whether or not they had agreed to the procedure, at 13 months no parent expressed regrets about their decision. CONCLUSIONS: Autopsy rates in the East of Scotland stand at 62%. Parents' perceptions are an important element in consent to postmortem examination.

Deciding on treatment limitation for neonates: the parents' perspective.

UNLABELLED: The reported study aimed to explore parents' perceptions of treatment withdrawal/withholding; their experience and their opinions. In-depth face-to-face interviews at 3 and 13 months after the baby's death were conducted with 108 parents of 62 babies from the East of Scotland. Of the parents, 56% (60/180) believed they had decided to stop treatment. A further 4/59 sets of parents subsequently wished they had taken responsibility. Only one parent who did decide felt guilt 3 months after the event. Parents identified two essential factors which minimise doubt: full and honest information and concrete evidence of a poor prognosis. CONCLUSION: The majority of parents wish to be active in decision making on behalf of their baby. Doing so does not appear to have adverse consequences. The pacing of events in the process of deciding and managing the dying is critical. Dissatisfaction is reduced if parents are given time and evidence to help them assimilate the reality at each stage.

Influence of gestational age and maternal height on fetal femur length calculations.

OBJECTIVE: To determine whether current methods of detecting Down syndrome based on fetal femur length calculations are influenced by gestational age or maternal height. METHODS: Four formulas were used to calculate expected femur length (FL) based on the fetal biparietal diameter (BPD) between 15 0/7 weeks' gestation and 19 6/7 weeks' gestation. For each gestational age, the BPD:FL ratio for women shorter than one standard deviation (SD) below the mean height was compared with the ratio for women taller than one SD above the mean height. A measured:expected FL ratio of 0.91 or less and a BPD:FL ratio greater than 1.5 SD above the mean was considered abnormal. RESULTS: The four formulas used to calculate measured:expected FL ratios were significantly more likely to be abnormal at 15--16 weeks' gestation, compared with 18-19 weeks' gestation (P <.05). Maternal height correlated with femur lengths at 18 and 19 weeks' gestation (P <.05) but not at earlier gestational ages. At 18 and 19 weeks' gestation, women shorter than one SD below the mean were twice as likely to have an abnormal BPD:FL ratio compared with women taller than one SD above the mean (relative risk 2.38; 95% confidence interval 1.21, 4.69). CONCLUSION: Early gestational age increases a woman's risk of having an abnormal measured:expected FL ratio, whereas short stature increases a woman's risk of having an abnormal BPD:FL ratio at later gestational ages. These findings indicate that risk assessment for fetal Down syndrome for such patients might be inaccurate. (Obstet Gynecol 2001;97:742-6.

Thyroid hormone metabolism and the developing human lung.

Thyroid hormones are involved in the regulation of fetal lung development, and maturation is accelerated in animal models by antepartum exposure to raised concentrations of the receptor-active thyroid hormone triiodothyronine and glucocorticoids. It is essential that the nature of the regulation of the spatial and temporal metabolism of iodothyronines in the human fetus and infant is known before effective therapies can be developed to modify human lung maturation. Thyroid hormone bioavailability to the human fetus is regulated in part by enzymatic deiodination and reversible sulfation of iodothyronines, with contributions from other factors such as fetomaternal and fetoamniotic hormone transfers, fetal thyroid gland production, and the activities of plasma membrane transporters mediating uptake of iodothyronines from plasma into tissues.