Expanding the BP1-BP2 15q11.2 Microdeletion Phenotype: Tracheoesophageal Fistula and Congenital Cataracts.

The proximal q arm of chromosome 15 contains breakpoint regions BP1-BP5 with the classic deletion of BP1-BP3 best known to be associated with Prader-Willi and Angelman syndromes. The region is approximately 500 kb and microdeletions within the BP1-BP2 region have been reported in patients with developmental delay, behavioral abnormalities, and motor apraxia as well as dysmorphic features including hypertelorism, cleft or narrow palate, ear abnormalities, and recurrent upper airway infections. We report two patients with unique, never-before-reported 15q11.2 BP1-2 microdeletion syndrome findings, one with proximal esophageal atresia and distal tracheoesophageal fistula (type C) and one with congenital cataracts. Cataracts have been described in Prader-Willi syndrome but we could not find any description of cataracts in Angelman syndrome. Esophageal atresia and tracheoesophageal fistula have not been reported to our knowledge in either syndrome. A chance exists that both cases are sporadic birth defects; however, the findings of the concomitant microdeletion cannot be overlooked as a possible cause. Based on our review of the literature and the presentation of our patients, we recommend that esophageal atresia and distal tracheoesophageal fistula as well as congenital cataracts be included in the phenotypic spectrum of 15q11.2 BP1-2 microdeletion syndrome.

Extremely low birth weight infants are at high risk for auditory neuropathy.

UNLABELLED: Auditory neuropathy (AN) is a condition in which transmission of sound to the brain is abnormal. This is reflected as an electrophysiologic profile of normal otoacoustic emissions (OAE), with abnormal auditory brainstem evoked responses (ABR). Functionally speech perception is impaired and management strategies remain controversial. AN can be missed if high-risk newborns are screened for hearing loss with only OAE testing. The rate of sensorineural hearing loss (SNHL) in high-risk nursery infants is 10 times greater compared with normal term newborns. Therefore, we hypothesize that infants from the neonatal intensive care unit (NICU) are at significantly higher risk for AN than normal term infants. OBJECTIVE: The objective of this study is to establish a prevalence rate and characterize risk factors for NICU graduates who demonstrate the AN electrophysiologic profile. STUDY DESIGN: This retrospective study examined infants admitted to the NICU at Kapi'olani Medical Center for Women and Children in Honolulu, HI from 1999 through 2003. Infants were screened with automated ABR. Diagnostic testing and OAE were performed before discharge if the ABR was abnormal. Hospital courses of 24 AN, 71 SNHL and 95 gestational age (GA)-matched control infants with normal hearing were reviewed. RESULT: With a SNHL prevalence of 16.7/1000, the rate for AN was 5.6/1000 NICU infants. Compared to infants with SNHL, infants with AN were significantly younger (GA 28.3+/-4.8 AN vs 32.9+/-5.2 weeks SNHL, P<0.0001) and smaller (BW 1318+/-894 AN vs 1968+/-1006 g SNHL). Nearly two-thirds of the AN infants were ELBW and had significantly longer hospital stays compared to SNHL infants of the same birth weight group. Exposure to furosemide, aminoglycosides, vancomycin or dexamethasone was associated with increased AN but not SNHL. Peak bilirubin level correlated with SNHL but not AN. CONCLUSION: Low birth weight NICU infants are at significant risk for AN. ELBW infants are at significantly higher risk for both AN and SNHL. Infants admitted to the NICU should be routinely screened by automated ABR and if abnormal, further evaluation should be started before hospital discharge. Early identification of AN will result in better understanding of this disorder and lead to the development of appropriate intervention strategies.

Loop diuretics and in vitro relaxation of human fetal and newborn mouse airways.

This study was designed to test the hypotheses that furosemide directly causes relaxation in human fetal airway and that delivery of loop diuretics to either the adventitial or epithelial surface of newborn mouse airway results in equivalent relaxation. Isometric tension changes were measured in human fetal (11-16 wk) trachea and mainstem bronchus rings exposed to furosemide (300 microM) or saline after acetylcholine or leukotriene D(4) constriction. Significant decreases in isometric tension to furosemide were demonstrated after constriction with acetylcholine or leukotriene D(4). To examine the site of effect and mimic aerosolized and systemic administration, furosemide (3-300 microM) and bumetanide (0.3-30 microM) were applied separately to epithelial and adventitial surfaces of newborn mouse airways. No differences in airway diameter changes to epithelial or adventitial furosemide or bumetanide were observed, but a 10-fold difference in potency was found. In summary, human fetal airway relaxed to furosemide when constricted with either neurotransmitter or inflammatory mediator in vitro. Further, no differences in relaxation to equimolar epithelial and adventitial furosemide were observed in isolated newborn mouse airway. Taken together, this provides evidence that furosemide has a direct, nonepithelial-dependent effect on airway smooth muscle tone.

Differential effects of inhaled nitric oxide and hyperoxia on pulmonary dysfunction in newborn guinea pigs.

This study tested the hypothesis that inhaled nitric oxide (NO) and combined NO and hyperoxia will result in less pulmonary dysfunction and delay onset of respiratory signs compared with hyperoxia-exposed newborn guinea pigs (GPs). GPs were exposed to room air (n = 14), 95% O(2) (n = 36), 20 parts per million (ppm) NO (n = 14), or combined 20 ppm NO and 95% O(2) (NO/O(2), n = 13) for up to 5 days. Data evaluated included latency interval for onset of respiratory distress, pressure volume curves, lung histology, and bronchoalveolar lavage (BAL) polymorphonuclear cells (PMNs), proteolytic activity, and total protein. NO-exposed GPs did not develop respiratory distress and had no evidence of pulmonary dysfunction. O(2)-exposed GPs developed respiratory distress after 1-5 days (median 4.0) vs. 3-5 days (median 5.0) for NO/O(2) exposure (P < 0.05). BAL from O(2)-exposed GPs showed increased PMNs compared with NO/O(2)-exposed GPs. O(2)- and NO/O(2)-exposed GPs had comparable reduced lung volumes, lung histology, and increased BAL proteinase activity and total protein. In summary 1) O(2) exposure resulted in multiple measures of pulmonary dysfunction in newborn GPs, 2) 5-day exposure to NO produced no noticeable respiratory effects and pulmonary dysfunction, and 3) short-term exposure (

m-hydroxy benzoylecgonine recovery in fetal guinea pigs.

Recently, meta-hydroxybenzoylecgonine (m-OH BE) was identified by gas chromatography-mass spectroscopy during quantitative analysis for cocaine. Identification of m-OH BE in addition to the routinely identified benzoylecgonine by gas chromatography-mass spectroscopy confirmatory assays may increase detection of cocaine-exposed infants and decrease false negative results. However, it is not known whether m-OH BE is derived directly from benzoylecgonine or from hydroxylated cocaine, or whether this metabolite is produced in the fetus or transferred across the placenta from the maternal circulation. We quantitated the recovery of cocaine, benzoylecgonine, and m-OH BE from amniotic fluid, fetal meconium, fetal intestine, and maternal urine for up to 4 days after single dose administration of either cocaine or benzoylecgonine to pregnant time-bred guinea pigs. m-OH BE was recovered from meconium after maternal injections of cocaine and benzoylecgonine. There was no significant detection of m-OH BE from amniotic fluid or intestine and minimal recovery from maternal urine after either cocaine or benzoylecgonine administration. Detection of m-OH BE in meconium increased the identification of in utero exposed guinea pigs, and the greatest yield of m-OH BE from meconium occurred later than that observed for cocaine or benzoylecgonine.

Cross-desensitization to furosemide and salbutamol in isolated neonatal guinea pig airways.

Airway hyperresponsiveness in neonatal chronic lung disease is treated with both furosemide, a diurectic that inhibits the Na-K-2Cl cotrasporter, and salbutamol, a beta2-adrenoceptor agonist. Tachyphylaxis to both drugs in vitro has been described. This study was conducted to determine if the relaxation response in newborn guinea pig airways to furosemide and salbutamol can be cross-desensitized in vitro. Tracheal ring segments from 4- to 7-day-old guinea pigs were suspended in HEPES buffer for measurement of isometric tension. Segments were pre-treated with either furosemide (300 microM, 1 h) or salbutamol (10 microM, 30 min). After constriction with 3 microM acetylcholine, relaxation response to salbutamol or furosemide, respectively, was measured. Pretreatment with furosemide diminished relaxation response to salbutamol [87 +/- 3% (n = 11) vs. 117 +/- 8% (n = 10), p < 0.05], as compared to saline-treated controls. In addition, pretreatment with salbutamol diminished relaxation response to furosemide [53 +/- 2% (n = 11) vs. saline-treated (83 +/- 7%, n = 7, p < 0.05) and DMSO-treated controls (69 +/- 5%, n = 5, p < 0.05)]. Measurements of 86Rb uptake, cyclic AMP levels and responses in the presence of charybdotoxin make it unlikely that Na-K-2Cl cotransporter activity, stimulation of cAMP, or opening of maxi-K+ channels are mechanisms involved in the cross-desensitization to furosemide and salbutamol in vitro.

Amiloride-induced contraction of isolated guinea pig, mouse, and human fetal airways.

Nebulized amiloride has been proposed as therapy in cystic fibrosis to block Na+ hyperabsorption in airway epithelium and prevent dehydration of secretions. Patients with cystic fibrosis often have reaction airways. Bovine and canine trachea relax to amiloride in vitro, suggesting another benefit as a bronchodilator, whereas guinea pig trachea, a useful model of human airways, does not. We hypothesized that human airways would respond like guinea pig airways. Airway ring segments from guinea pigs, mice, and human fetuses were constricted with the concentration of acetylcholine producing 50-75% maximum contraction. Subsequent changes in isometric tension to cumulative additions of amiloride (10(-8)-10(-4) M) were measured. Guinea pig airways contracted 29 +/- 5%, mouse airways contracted 23 +/- 6%, and human fetal airways contracted 30 +/- 8%. Contraction to amiloride was mimicked by dimethylamiloride, a more selective inhibitor of the Na+/H+ antiporter, and was attenuated by protein kinase C (PKC) inhibition with GF109203X and staurosporine. The present study indicates that amiloride-induced airway contraction in guinea pigs and mice closely parallels the response in isolated human airways and that the mechanism may involve the Na+/H+ antiporter and PKC.

Furosemide-induced airway relaxation in guinea pigs: relation to Na-K-2Cl cotransporter function.

This study tested the hypothesis that airway relaxation to furosemide is mediated via the Na-K-2Cl cotransporter. If this mechanism exists in airway smooth muscle like in vascular smooth muscle, changes in airway relaxation should be associated with changes in Na-K-2Cl cotransporter function, and both should be substrate dependent. Tracheal rings from newborn guinea pigs were bathed in standard (STD) or varying low Cl- concentration ([Cl-]) N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Isometric relaxation to 300 microM furosemide or 10(-8) to 10(-5) M salbutamol was measured. Airway segments were incubated with rubidium-86 (86Rb) in STD or varying low [Cl-] HEPES, with and without 300 microM furosemide or 25 microM salbutamol. Furosemide was unable to reduce 86Rb uptake at 10 mM [Cl-], although relaxation was still observed in 10 mM [Cl-]. Salbutamol did not affect 86Rb uptake. This study demonstrated that there is a furosemide-sensitive Na-K-2Cl cotransporter in newborn guinea pig trachea. However, the effect of furosemide on cotransporter function did not always directly correspond to differences in relaxation, suggesting that the Na-K-2Cl cotransporter may play a major, but not exclusive, role in furosemide-induced airway relaxation.

Tachyphylaxis to furosemide in isolated airways of guinea pigs.

This in vitro study was conducted to determine whether tachyphylaxis of guinea pig airway to furosemide occurs under conditions that produce tachyphylaxis to the beta 2-adrenoceptor agonist, salbutamol. Isometric tension was measured in tracheal rings bathed in HEPES buffer from 4-6 d newborn guinea pigs of either sex, and 6 wk old males. Paired rings were first incubated with furosemide, 30 or 300 microM, or control for 60 min, washed, then constricted with 3 microM acetylcholine. At stable contraction, relaxation to furosemide (30 microM-1 mM) was measured. For comparison, similar experiments were performed with 10 microM salbutamol incubation for 30 min. 86Rb uptake, a marker for K+ transport and Na-K-Cl cotransport activity, was also measured in these airway segments. Pre-exposure to these airway relaxants did not affect contractile force generation by acetylcholine. Tracheal desensitization to both salbutamol and furosemide was observed. Partial recovery of furosemide induced relaxation was seen one hour after desensitization. Pre-exposure to 300 microM furosemide did not inhibit the decrease in 86Rb uptake normally observed with furosemide. In summary, we found that: 1) tachyphylaxis of guinea pig airway relaxation occurred with both salbutamol and furosemide under similar experimental conditions; however 2) inhibition of 86Rb uptake by furosemide was not affected by prior exposure. Taken together, these results suggest that furosemide induced airway relaxation could be affected by repeated or prolonged exposure, but this response may not be associated with changes in furosemide-sensitive Na-K-Cl cotransporter activity.

Acute hyperoxic injury attenuates the relaxing effects of "loop" diuretics and salbutamol on large airways of newborn guinea pigs.

We have previously found an age-dependent relaxing effect of furosemide in normal fetal, newborn, and adult guinea pig airways with fetal trachea exhibiting the greatest relaxation and adult tissue the least. This study was designed to expand upon this finding by determining if in vivo hyperoxic exposure would influence in vitro airway relaxation mediated by the loop diuretics, furosemide and ethacrynic acid, and the beta 2-adrenoceptor agonist, salbutamol. Newborn guinea pigs were raised in > 95% FiO2 until ill; controls in room air. Isometric relaxation to 3 x 10(-5) M furosemide, 3 x 10(-6) M ethacrynic acid, or 10(-8)-10(-6) M salbutamol was recorded in 3 x 10(-6) M histamine-constricted airway rings. Ethacrynic acid, like furosemide, relaxed newborn guinea pig airways. Hyperoxia did not alter the contractile effect of 3 x 10(-6) M histamine but did significantly decrease the relaxing effect of furosemide, ethacrynic acid, and salbutamol. Loop diuretic mediated airway relaxation was accentuated in HEPES buffer when compared with Krebs, whereas salbutamol-mediated relaxation was unaffected. These results suggest that hyperoxia nonspecifically decreases airway responsiveness to the relaxing agents studied.

Effect of environmental conditions on emergency department use by wheezing children.

STUDY OBJECTIVE: To examine in children the relationship of wheezing to measurable environmental factors. STUDY DESIGN: Multiple regression analysis was used to measure correlation with air quality, weather, and seasonal and infection-related variables. RESULTS: Daily wheezing census was significantly correlated with weather and seasonal variables and the daily infection census. We are not certain which weather variable is the dominant factor in the weather association because all of the weather variables have some degree of colinearity. Air quality as measured by carbon monoxide and airborne particles was not shown to be associated with wheezing. CONCLUSION: A high incidence of pediatric emergency department presentations for wheezing are associated with weather, infections, and months of the year.

Methamphetamine detection from meconium and amniotic fluid in guinea pigs depends on gestational age and metabolism.

Significant adverse perinatal effects of maternal methamphetamine use have been reported, but little is known about factors influencing methamphetamine screening test results during the perinatal period. We tested the hypothesis that gestational age would affect quantitative recovery of methamphetamine in meconium and amniotic fluid. Time-bred guinea pigs received an intraperitoneal (i.p.) injection of 1 mg/kg methamphetamine at either 44 days (0.65 of term, n = 5), 50 days (0.74, n = 8), 56 days (0.82, n = 9) or 63 days (0.93, n = 4) gestation. At 1 or 7 days after i.p. methamphetamine, meconium and amniotic fluid were collected for quantitative methamphetamine assay by gas chromatography-mass spectrometry. Recovery from amniotic fluid and meconium 1 day after injection was influenced by gestational age. Greater values in amniotic fluid and meconium and a higher percentage of positive samples were seen in older fetuses. Collectively at all gestational ages, combined testing of amniotic fluid and meconium yielded detectable methamphetamine or its metabolites in 87% of guinea pigs 1 day after injection. However, methamphetamine was not detectable 1 week after injection in any sample (n = 63) at either 0.74 or 0.82 of term except for one positive amniotic fluid sample. Finally, demethylation of methamphetamine to amphetamine was higher in older fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)

Transient enhancement of multidrug resistance by the bile acid deoxycholate in murine fibrosarcoma cells in vitro.

Recent studies have implicated protein kinase C (PKC) activation in drug resistance in vitro. PKC can be activated directly by phorbol-ester tumor promoters as well as by the bile acid deoxycholate. In this report, we demonstrate that deoxycholate, at concentrations that are chronically present in the lumen of the colon in vivo, mimicked phorbol-ester tumor promoters by protecting Adriamycin (ADR)-sensitive and multidrug-resistant (MDR) murine fibrosarcoma UV-2237M cells from ADR cytotoxicity. Deoxycholate also enhanced the resistance of the MDR cell line UV-2237M-ADRR to the cytotoxic effects of vincristine and vinblastine. In contrast to cytotoxic drug-selected MDR phenotypes, deoxycholate-induced drug resistance was transient and required continuous exposure to the bile acid. The protein kinase inhibitor H7 completely reversed the protection against ADR cytotoxicity conferred on UV-2237M-ADRR cells by deoxycholate, providing evidence that deoxycholate exerts its protective effects by a mechanism that involves stimulation of protein phosphorylation and not merely by detergent effects on membrane permeability. PKC consists of a family of at least seven isozymes with distinct modes of activation and substrate specificities. We previously reported that MDR UV-2237M cell lines contain higher levels of PKC activity than the parental ADR-sensitive UV-2237M cell line (O'Brian et al., FEBS Lett 246: 78-82, 1989). The present report shows that PKC-III is a major PKC isozyme in ADR-sensitive and MDR UV-2237M cell lines. Thus, the resistance to ADR induced by the phorbol esters in UV-2237M cell lines provides strong evidence that PKC-III activation confers protection against ADR on ADR-sensitive and MDR UV-2237M cell lines. Furthermore, since deoxycholate is an endogenous molecule in the colonic epithelium, our finding that physiological concentrations of deoxycholate can render cells more resistant to chemotherapeutic drugs in vitro may have implications for the biology and therapy of intestinal cancers.

Significant enhanced superoxide anion (O2-) production in vitro by peripheral blood monocytes of lepromatous leprosy patients stimulated with liposome and suppression by C-reactive protein (CRP).

Peripheral blood monocytes (PBM) from normal individuals, infectious mononucleosis (IM) and leprosy patients were stimulated with liposome. The mean and standard error of superoxide anion (O2-) generated in nm/1.5 X 10(5) PBM/well for 5 normal subjects and 3 IM patients was 2.9 +/- 0.5 and 3.1 +/- 0.2, respectively. Monocytes stimulated with 100 ng C-reactive protein (CRP) incorporated into liposome gave values of 3.3 +/- 0.3 and 2.7 +/- 0.1 nm O-2/1.5 X 10(5) PBM/well for normals and IM, respectively. No significant differences in O2- production between liposome and liposome with incorporated CRP were shown. PBM from lepromatous patients demonstrated a significant (p less than 0.01) increase in O2- production with liposome alone compared with tuberculoid patients (3.5 +/- 0.4 vs 1.8 +/- 0.3). The most dramatic suppression of O2- shown when purified CRP was added to the mixtures in all groups examined [0.4 +/- 0.1 (500 ng), 0.3 +/- 0.0 (500 ng), 1.5 +/- 0.1 (100 ng), and 1.3 +/- 0.6 (100 ng) nm O2-/1.5 X 10(5) PBM/well for normals, IM, lepromatous, and tuberculoid, respectively]. Results of O2- formation with incorporation of CRP into liposome as compared with liposome alone had no significant effect on PBM of lepromatous or tuberculoid patients. It is suggested that CRP may play a significant role in regulation of oxygen free radicals formed during acute and chronic inflammatory episides.