Mature and immature platelets during the first week after birth and incidence of patent ductus arteriosus.

BACKGROUND: Thrombocytopenia is a risk factor for patent ductus arteriosus. Immature and mature platelets exhibit distinct haemostatic properties; however, whether platelet maturity plays a role in postnatal, ductus arteriosus closure is unknown. METHODS: In this observational study, counts of immature and mature platelets (=total platelet count - immature platelet count) were assessed on days 1, 3, and 7 of life in very low birth weight infants (<1500 g birth weight). We performed echocardiographic screening for haemodynamically significant patent ductus arteriosus on day 7. RESULTS: Counts of mature platelets did not differ on day 1 in infants with (n = 24) and without (n = 45) haemodynamically significant patent ductus arteriosus, while infants with significant patent ductus arteriosus exhibited lower counts of mature platelet on postnatal days 3 and 7. Relative counts of immature platelets (fraction, in %) were higher in infants with patent ductus arteriosus on day 7 but not on days 1 and 3. Receiver operating characteristic curve analysis unraveled associations between both lower mature platelet counts and higher immature platelet fraction (percentage) values on days 3 and 7, with haemodynamically significant ductus arteriosus. Logistic regression analysis revealed that mature platelet counts, but not immature platelet fraction values, were independent predictors of haemodynamically significant patent ductus arteriosus. CONCLUSION: During the first week of postnatal life, lower counts of mature platelets and higher immature platelet fraction values are associated with haemodynamically significant patent ductus arteriosus. Lower counts of mature platelet were found to be independent predictors of haemodynamically significant patent ductus arteriosus.

Vascular endothelial growth factor polymorphism rs2010963 status does not affect patent ductus arteriosus incidence or cyclooxygenase inhibitor treatment success in preterm infants.

BACKGROUND: Vascular endothelial growth factor is critically involved in ductus arteriosus closure. Polymorphisms in the vascular endothelial growth factor gene have been associated with several diseases in neonates and adults. AIM: Herein, we investigated if vascular endothelial growth factor polymorphism rs2010963 status is associated with patent ductus arteriosus incidence and/or pharmacological treatment success. METHODS: We assessed rs2010963 status in 814 preterm infants (<1500 g birth weight) by means of restriction fragment length polymorphism analysis. DNA samples were obtained from dry-spot cards used for the German national newborn screening program. Clinical data were obtained by retrospective chart review. RESULTS: We could not find any statistically significant difference in the incidence of patent ductus arteriosus depending on vascular endothelial growth factor rs2010963 polymorphism status. Furthermore, no statistically significant associations between vascular endothelial growth factor polymorphism rs2010963 status and cyclooxygenase inhibitor treatment success were observed. CONCLUSION: Our results indicate that there is no association between vascular endothelial growth factor polymorphism rs2010963 status and the occurrence of patent ductus arteriosus or the response to cyclooxygenase inhibitor treatment in a large cohort of preterm infants. Additional studies are needed to determine the role of genetic factors on patent ductus arteriosus incidence and treatment response.

Ibuprofen and indomethacin differentially regulate vascular endothelial growth factor and its receptors in ductus arteriosus endothelial cells.

BACKGROUND: Cyclooxygenase inhibitors are widely applied to facilitate ductal closure in preterm infants. The mechanisms that lead to patent ductus arteriosus closure are incompletely understood. Vascular endothelial growth factor plays pivotal roles during ductal closure and remodelling. Aim The aim of this study was to investigate the effects of ibuprofen and indomethacin on the expression of vascular endothelial growth factor and its receptors in a primary rat ductus arteriosus endothelial cell culture. METHODS: Protein expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 1 and 2 was confirmed in rat ductus arteriosus and aorta by immunofluorescence staining. Fetal rat endothelial cells were isolated from ductus arteriosus and aorta using immunomagnetic cell sorting and treated with ibuprofen or indomethacin. mRNA expression levels were assessed by quantitative polymerase chain reaction analysis. RESULTS: In ductal endothelial cells, ibuprofen significantly induced vascular endothelial growth factor and its receptor 2, but not receptor 1, whereas indomethacin did not alter the expression levels of the vascular endothelial growth factor system. In contrast, ibuprofen significantly induced vascular endothelial growth factor and its receptors 1 and 2 in aortic endothelial cells, whereas indomethacin only induced vascular endothelial growth factor receptor 2. CONCLUSION: Our results indicate differential effects of ibuprofen and indomethacin on the expression levels of the vascular endothelial growth factor system in ductus arteriosus endothelial cells. In addition, vessel-specific differences between ductal and aortic endothelial cells were found. Further in vivo studies are needed to elucidate the biological significance of these findings.

Lung function in very low birth weight infants after pharmacological and surgical treatment of patent ductus arteriosus - a retrospective analysis.

BACKGROUND: The indications and strategies for treatment of patent ductus arteriosus (PDA) are controversial, and the safety and long-term benefits of surgical PDA closure remain uncertain. The aim of this study was to compare the lung function of very low birth weight (VLBW) infants after successful PDA treatment with a cyclooxygenase inhibitor or secondary surgical ligation. METHODS: A total of 114 VLBW infants (birth weight < 1500 g), including 94 infants (82%) with a birth weight < 1000 g, who received treatment for hemodynamically significant PDA (hsPDA), were examined at a median postmenstrual age of 48 weeks. All infants were initially given pharmacological treatment, and 40 infants (35%) required PDA ligation. Lung function testing (LFT) included tidal breathing measurements, measurement of respiratory mechanics assessed by the occlusion test, whole-body plethysmography, SF6 multiple breath washout, forced expiratory flow (V'maxFRC) by the rapid thoracoabdominal compression technique, exhaled NO (FeNO), and arterialized capillary blood gas analysis. RESULTS: On the day of the LFT, the 2 groups had similar postconceptional age and body weight. However, the PDA ligation group was more immature at birth (p < 0.001) and had reduced respiratory compliance (p < 0.001), lower V'maxFRC (p = 0.006), increased airway resistance (Raw) (p < 0.001), and impaired blood gases (p < 0.001). Multivariate analysis showed that PDA surgery was an independent risk factor for increased Raw. CONCLUSION: PDA ligation after failed pharmacological treatment is associated with impaired lung function as compared to successful pharmacological closure in infants at a postmenstrual age of 48 weeks. However, only Raw was independently affected by PDA ligation, while all other differences were merely explained by patient characteristics.

Clinical, Laboratory, and Placental Findings in Perinatal Listeriosis.

Clinical, laboratory, and placental manifestations of perinatal listeriosis are highly variable. Herein, we retrospectively analyzed all patients treated for neonatal listeriosis at the Charité University Medical Center in Berlin, Germany, 1999-2013. A total of 16 cases were identified. In 14 patients listeriosis was confirmed in neonatal specimens, while in two only the placenta tested positive. Elevated C-reactive protein and/or interleukin-6 levels were only inconsistently found, while a marked white blood cell left shift was present in all infants, if available. All but one infant manifested symptoms on the first day of life. Most patients required respiratory support, while none developed meningoencephalitis as evidenced by clinical or cerebrospinal fluid findings. Two patients died, all other patients survived without sequelae. In conclusion, perinatal listeriosis is still associated with significant morbidity and mortality. Clinical and laboratory findings are highly heterogeneous, but extreme leukocyte left shift seems to be a common feature.

Natural History of Patent Ductus Arteriosus in Very Low Birth Weight Infants after Discharge.

Data on the natural history of infants discharged with patent ductus arteriosus is sparse. We report on the 36-months follow-up after hospitalization in 68 infants discharged with an open ductus arteriosus. Notwithstanding a high spontaneous closure rate, catheter intervention in 5 infants illustrates a critical need for cardiologic follow-up.

Isolation and culture of fibroblasts, vascular smooth muscle, and endothelial cells from the fetal rat ductus arteriosus.

The ductus arteriosus (DA), a fetal arterial shunt vessel between the proximal descending aorta and the pulmonary artery, closes shortly after birth. Initial functional closure as a result of the DA's smooth muscle contraction is followed by definite anatomical closure. The latter involves several complex mechanisms like endothelial cushion formation and smooth muscle cell migration resulting in fibrosis and sealing of the vessel. These complex steps indicate highly specialized functions of the DA vascular smooth muscle cells (VSMCs), endothelial cells, and fibroblasts. Herein, we describe a new reproducible method for isolating VSMCs, endothelial cells, and fibroblasts of high viability from fetal rat DA using immunomagnetic cell sorting. Purity of the different cell cultures was assessed by immunohistochemistry and flow cytometry and ranged between 85 and 94%. The capability of the VSMCs to react to hypoxic stimuli was assessed by intracellular calcium and ATP measurements and by VEGF mRNA expression analysis. VSMCs respond to hypoxia with decreases in intracellular calcium concentrations and ATP levels, whereas VEGF mRNA expression increased 3.2-fold. The purified vessel-specific different cell types are suitable for subsequent gene expression profiling and functional studies and provide important tools for improving our understanding of the complex processes involved in the closure of the DA.

Vascular endothelial growth factor and its soluble receptor in infants with congenital cardiac disease.

Patients with cyanotic congenital cardiac disease often develop major aortopulmonary collaterals. Vascular endothelial growth factor is a key promoter of angiogenesis. Its soluble receptor-1 acts as a potent antagonist. We studied 30 infants with cyanotic congenital cardiac disease and 27 infants with acyanotic congenital cardiac disease. Central venous plasma vascular endothelial growth factor and soluble vascular endothelial growth factor receptor-1 levels were measured before, and 24 and 96 hours after surgery. There was no difference between plasma vascular endothelial growth factor levels in infants with cyanotic and those with acyanotic congenital cardiac disease. In cyanotic infants, the soluble vascular endothelial growth factor receptor-1 levels tended to be higher than in the acyanotic infants. In conclusion, there is no significant difference in the plasma levels of vascular endothelial growth factor and its soluble receptor-1 between infants with cyanotic and those with acyanotic congenital cardiac disease.

Ibuprofen augments bilirubin toxicity in rat cortical neuronal culture.

Premature infants are at risk for bilirubin-associated brain damage. In cell cultures bilirubin causes neuronal apoptosis and necrosis. Ibuprofen is used to close the ductus arteriosus, and is often given when hyperbilirubinemia is at its maximum. Ibuprofen is known to interfere with bilirubin-albumin binding. We hypothesized that bilirubin toxicity to cultured rat embryonic cortical neurons is augmented by coincubation with ibuprofen. Incubation with ibuprofen above a concentration of 125 microg/mL reduced cell viability, measured by methylthiazole tetrazolium reduction, to 68% of controls (p < 0.05). Lactate dehydrogenase (LDH) release increased from 29 to 38% (p < 0.01). The vehicle solution did not affect cell viability. Coincubation with 10 microM unconjugated bilirubin (UCB)/human serum albumin in a molar ratio of 3:1 and 250 microg/mL ibuprofen caused additional loss of cell viability and increased LDH release (p < 0.01), DNA fragmentation, and activated caspase-3. Preincubation with the pan-caspase inhibitor z-val-ala-asp-fluoromethyl ketone abolished ibuprofen- and UCB-induced DNA fragmentation. The study demonstrates that bilirubin in low concentration of 10 microM reduces neuron viability and ibuprofen increases this effect. Apoptosis is the underlying cell death mechanism.

Population pharmacokinetic analysis of Ibuprofen enantiomers in preterm newborn infants.

The aim of this pharmacokinetic analysis was to develop and validate a population pharmacokinetic model for R- and S-ibuprofen from samples obtained after 3 successive administrations of ibuprofen (10-5-5 mg/kg) at 24-hour intervals to preterm newborn infants aged from <6 hours to 8 days of life. A model including unilateral bioconversion of R-ibuprofen into S-ibuprofen was developed using the software NONMEM. R- and S-ibuprofen plasma concentrations were adequately fitted by this model. Estimated clearance and volume of distribution were 3.5 mL/h/kg and 173 mL/kg, respectively, with a calculated half-life (t((1/2))) of 34.3 hours for S-ibuprofen. Estimated clearance at birth and volume of distribution were 25.5 mL/h/kg and 306 mL/kg with a t((1/2)) at birth of 8.3 hours for R-ibuprofen. R-ibuprofen elimination increased during the first week of life, whereas S-ibuprofen pharmacokinetics were weakly modified. Therefore, because the activity of the 2 enantiomers differs, it is important that subsequent studies consider R- and S-enantiomers separately. Mean simulated ibuprofen concentrations at various dose regimens were in agreement with observed concentrations. The present analysis allows a more accurate estimation of the ibuprofen pharmacokinetics as parameters could be estimated separately for each enantiomer and the effect of postnatal age on the elimination of R-ibuprofen was elicited.

B-type natriuretic peptide to predict ductus intervention in infants <28 weeks.

Patent ductus arteriosus (PDA) is frequent in neonates with gestational age of less than 28 wk. Clinical and echocardiographic signs define hemodynamic significance of PDA, but do not reveal the need for PDA intervention in the first days of life. B-type natriuretic peptide (BNP) has been proposed as a screening tool for PDA in preterm infants. To determine whether BNP can predict the need for PDA intervention, plasma BNP was measured by chemiluminescence immunoassay in 67 preterm infants <28 wk (median 26) on the second day of life in a prospective blinded study. PDA intervention was based on specified clinical and echocardiographic findings. Twenty-four patients (intervention group) received treatment for PDA and 43 patients (controls) remained without intervention. BNP concentrations were higher in the intervention (median 1069 pg/mL) than in the control group (247 pg/mL, p < 0.001). BNP correlated positively with ductal size (R = 0.46, p < 0.001) and atrial/aortic root ratio (R = 0.54, p < 0.001). In conclusion, plasma BNP proved to be a good predictor for ductus intervention (area under the curve: 0.86) with the best cutoff at 550 pg/mL on the second day of life in ventilated infants less than 28 wk gestation (sensitivity: 83%; specificity: 86%).

The expression of VEGF and its receptors in the human ductus arteriosus.

Programmed proliferative degeneration of the human fetal ductus arteriosus (DA) preceding definite postnatal closure has a large developmental variability and is controlled by several signaling pathways. Among these vascular endothelial growth factor (VEGF) and its receptors (VEGF-Rs) play an important role. Until now, gestational age dependent expression of VEGF and its receptors has not been investigated in a large number of human DA tissue specimens. We examined protein expression of VEGF and the three VEGF-Rs immunohistochemically in 63 human fetal autopsy DA specimens of 11-38 wk gestation. Specimens were classified into different maturity stages according to their histologic appearance. VEGF and VEGF-Rs-staining was detected in all maturity stages. VEGF-staining was localized perinuclearly in all vascular layers and did not change during development. VEGF-R1 and VEGF-R3 expression was marked in the endothelium in early maturity stages and decreased during development. In contrast, -R2 predominated in the media in later developmental stages. Our results emphasize the importance of VEGF as a mediator during programmed proliferative degeneration of fetal DA and support the hypothesis that VEGF-R1 and VEGF-R3 are required for normal blood vessel development during embryogenesis. In contrast, VEGF-R2 is the predominant receptor in later angiogenic signaling.

Association between Platelet Counts before and during Pharmacological Therapy for Patent Ductus Arteriosus and Treatment Failure in Preterm Infants.

BACKGROUND: The role of platelets for mediating closure of the ductus arteriosus in human preterm infants is controversial. Especially, the effect of low platelet counts on pharmacological treatment failure is still unclear. METHODS: In this retrospective study of 471 preterm infants [<1,500 g birth weight (BW)], who were treated for a patent ductus arteriosus (PDA) with indomethacin or ibuprofen, we investigated whether platelet counts before or during pharmacological treatment had an impact on the successful closure of a hemodynamically significant PDA. The effects of other factors, such as sepsis, preeclampsia, gestational age, BW, and gender, were also evaluated. RESULTS: Platelet counts before initiation of pharmacological PDA treatment did not differ between infants with later treatment success or failure. However, we found significant associations between low platelet counts during pharmacological PDA therapy and treatment failure (p < 0.05). Receiver operating characteristic (ROC) curve analysis showed that platelet counts after the first, and before and after the second cyclooxygenase inhibitor (COXI) cycle were significantly associated with treatment failure (area under the curve of >0.6). However, ROC curve analysis did not reveal a specific platelet cutoff-value that could predict PDA treatment failure. Multivariate logistic regression analysis showed that lower platelet counts, a lower BW, and preeclampsia were independently associated with COXI treatment failure. CONCLUSION: We provide further evidence for an association between low platelet counts during pharmacological therapy for symptomatic PDA and treatment failure, while platelet counts before initiation of therapy did not affect treatment outcome.

Genetic deafness in a preterm infant with a critical postnatal course.

OBJECTIVE: We present a case of deafness in a preterm infant with several predisposing factors of an acquired hearing impairment that, however, turned out to have a genetic cause. We describe the severe postnatal course and review the relevant literature. DESIGN: Case report. SETTING: University-based tertiary neonatal intensive care unit. PATIENT: Preterm infant (gestational age, 26/37; wks). MEASUREMENTS AND MAIN RESULTS: A preterm infant exhibited hearing impairment after a complicated clinical course with pneumothoraces, a hemodynamically relevant patent ductus arteriosus, treatment with potentially ototoxic drugs, intraventricular hemorrhage, and periventricular leukomalacia. Despite the absence of a family history for deafness, genetic testing was performed. Surprisingly, genetic analysis revealed the presence of two compound heterozygous mutations in the patient's GJB2 gene as the cause for his early-onset nonsyndromic deafness. CONCLUSION: To elucidate the nature of a hearing disorder, it is worthwhile to consider a genetic cause, despite the fact that it may seem unlikely in a severely sick preterm infant with numerous risk factors for a postnatally acquired hearing impairment and without a positive family history.

Recent Advances in the Treatment of Preterm Newborn Infants with Patent Ductus Arteriosus.

A patent ductus arteriosus (PDA) is associated with several adverse clinical conditions. Several strategies for PDA treatment exist, although data regarding the benefits of PDA treatment on outcomes are sparse. Moreover, the optimal treatment strategy for preterm neonates with PDA remains subject to debate. It is still unknown whether and when PDA treatment should be initiated and which approach (conservative, pharmacologic, or surgical) is best for individual patients (tailored therapies). This article reviews the current strategies for PDA treatment with a special focus on recent developments such as oral ibuprofen, high-dose regimens, and the use of paracetamol (oral, intravenous).

Chlamydophila pneumoniae induces expression of toll-like receptor 4 and release of TNF-alpha and MIP-2 via an NF-kappaB pathway in rat type II pneumocytes.

BACKGROUND: The role of alveolar type II cells in the regulation of innate and adaptive immunity is unclear. Toll-like receptors (TLRs) have been implicated in host defense. The purpose of the present study was to investigate whether Chlamydophila pneumoniae (I) alters the expression of TLR2 and/orTLR4 in type II cells in a (II) Rho-GTPase- and (III) NF-kappaB-dependent pathway, subsequently (IV) leading to the production of (IV) pro-inflammatory TNF-alpha and MIP-2. METHODS: Isolated rat type II pneumocytes were incubated with C. pneumoniae after pre-treatment with calcium chelator BAPTA-AM, inhibitors of NF-kappaB (parthenolide, SN50) or with a specific inhibitor of the Rho-GTPase (mevastatin). TLR2 and TLR4 mRNA expressions were analyzed by PCR. Activation of TLR4, Rac1, RhoA protein and NF-kappaB was determined by Western blotting and confocal laser scan microscopy (CLSM) and TNF-alpha and MIP-2 release by ELISA. RESULTS: Type II cells constitutively expressed TLR4 and TLR2 mRNA. A prominent induction of TLR4 but not TLR2 mRNA was detected after 2 hours of incubation with C. pneumoniae. The TLR4 protein expression reached a peak at 30 min, began to decrease within 1-2 hours and peaked again at 3 hours. Incubation of cells with heat-inactivated bacteria (56 degrees C for 30 min) significantly reduced the TLR4 expression. Treated bacteria with polymyxin B (2 mug/ml) did not alter TLR4 expression. C. pneumoniae-induced NF-kappaB activity was blocked by TLR4 blocking antibodies. TLR4 mRNA and protein expression were inhibited in the presence of BAPTA-AM, SN50 or parthenolide. TNF-alpha and MIP-2 release was increased in type II cells in response to C. pneumoniae, whereas BAPTA-AM, SN50 or parthenolide decreased the C. pneumoniae-induced TNF-alpha and MIP-2 release. Mevastatin inhibited C. pneumoniae-mediated Rac1, RhoA and TLR4 expression. CONCLUSION: The TLR4 protein expression in rat type II cells is likely to be mediated by a heat-sensitive C. pneumoniae protein that induces a fast Ca2+-mediated NF-kappaB activity, necessary for maintenance of TLR4 expression and TNF-alpha and MIP-2 release through possibly Rac and Rho protein-dependent mechanism. These results indicate that type II pneumocytes play an important role in the innate pulmonary immune system and in inflammatory response mechanism of the alveolus.

Diastolic left ventricular function in preterm infants with a patent ductus arteriosus: a serial Doppler echocardiography study.

In very low birth weight neonates, a left-to-right shunt via persistent ductus arteriosus (PDA) may interact with diastolic left ventricular function, but specific changes of Doppler parameters have yet to be reported. In a serial transmitral Doppler study, we investigated the impact of a PDA on diastolic function parameters. Twenty-two patients with and without PDA were examined on day 3.8+/-1 and day 14+/-2 after birth. By the first examination, 13 out of 22 patients had a PDA; by the second examination, the number was still 8 out of 22. Peak early and atrial flow velocities (44.8+/-15 and 50.1+/-13 cm/s, respectively) were higher (p<0.05) for neonates with PDA compared to those with closed duct (30.9+/-6 and 34.2 cm/s, respectively). Isovolumic relaxation time (IVRT) was shorter in neonates with PDA (45+/-7 ms, N=21) compared to those with a closed duct (55.3+/-5 ms, N=23) (p<0.01). IVRT correlated inversely with cardiac index (R=-0.79, p<0.01). All observed changes reversed to the normal range after closure of the PDA. When premature infants with a PDA experience a preload challenge, early and atrial peak velocities increase and IVRT shortens significantly. This coincidence of elevated transvalvular pressure differences and decreased IVRT in neonates with immature diastolic function can best be explained as a result of left atrial pressure elevation. Consequently, pulmonary venous pressure must be elevated, with its inherent effect on pulmonary capillary physiology. Thus, the monitoring of left ventricular diastolic function adds significant information to the care of preterm infants with a PDA.

Soluble intercellular cell adhesion molecule-1 and L-selectin plasma concentrations and response to surfactant in preterm infants.

OBJECTIVE: To investigate whether plasma concentrations of soluble intercellular cell adhesion molecule (ICAM)-1 and L-selectin at 24 hrs of life are related to good or poor response to exogenous surfactant in preterm infants. DESIGN: Prospective study of markers of inflammation in circulating blood at 24 hrs of life. SETTING: Level III neonatal intensive care unit. PATIENTS: Twenty-nine preterm newborns suffering from severe respiratory distress syndrome (Fio(2) > 0.4) without signs of infection or fetal acidosis, and 17 healthy preterm newborns of similar gestational age serving as controls. INTERVENTIONS: Infants with respiratory distress were treated with natural surfactant at 0.3-5 hrs of life. A response to surfactant, defined as a decrease of Fio(2) >50% within 6 hrs after surfactant, was seen in 21 infants. MEASUREMENTS AND MAIN RESULTS: Soluble ICAM-1 and L-selectin concentrations were determined in plasma samples taken at 24 hrs of age. ICAM-1 was elevated (p <.001) in infants who responded poorly to surfactant (median, 392 ng/mL; range, 58.26-4884.24 ng/mL) compared with good responders (20.52 ng/mL, 2.32-138.58 ng/mL) or controls (21.91 ng/mL, 2.61-65.73 ng/mL), without differences between controls and good responders. L-selectin was lower (p =.004) in surfactant-treated infants (4.45 nmol/L, 2.0-10.4 ng/mL) than in controls (6.0/2.35-10.25 nmol/L) without differences between surfactant good and poor responders. However, infants requiring supplemental oxygen at 36 wks of gestational age had reduced L-selectin at 24 hrs of age (3.2/2.0-3.45 vs. 5.0/2.35-10.4 nmol/L, p =.004), whereas there was no difference in ICAM-1. CONCLUSIONS: In preterm infants with respiratory distress, a poor response to surfactant within 6 hrs of administration is associated with elevated circulating ICAM-1 concentrations at 24 hrs of age. Low plasma L-selectin at 24 hrs of age predicts prolonged requirement for supplemental oxygen.