Oral Clonidine-Based Strategy to Reduce Opiate Use During Cooling for Neonatal Encephalopathy: An Observational Study.

OBJECTIVE: To determine whether an enteral, clonidine-based sedation strategy (CLON) during therapeutic hypothermia (TH) for hypoxic-ischemic encephalopathy would decrease opiate use while maintaining similar short-term safety and efficacy profiles to a morphine-based strategy (MOR). STUDY DESIGN: This was a single-center, observational study conducted at a level IV neonatal intensive care unit from January 1, 2017, to October 1, 2021. From April 13, 2020, to August 13, 2020, we transitioned from MOR to CLON. Thus, patients receiving TH for hypoxic-ischemic encephalopathy were grouped to MOR (before April 13, 2020) and CLON (after August 13, 2020). We calculated the total and rescue morphine milligram equivalent/kg (primary outcome) and frequency of hemodynamic changes (secondary outcome) for both groups. RESULTS: The MOR and CLON groups (74 and 25 neonates, respectively) had similar baseline characteristics and need for rescue sedative intravenous infusion (21.6% MOR and 20% CLON). Both morphine milligram equivalent/kg and need for rescue opiates (combined bolus and infusions) were greater in MOR than CLON (P < .001). As days in TH advanced, a lower percentage of patients receiving CLON needed rescue opiates (92% on day 1 to 68% on day 3). Patients receiving MOR received a greater cumulative dose of dopamine and more frequently required a second inotrope and hydrocortisone for hypotension. MOR had a lower respiratory rate during TH (P = .01 vs CLON). CONCLUSIONS: Our CLON protocol is noninferior to MOR, maintaining perceived effectiveness and hemodynamic safety, with an apparently reduced need for opiates and inotropes.

Maturational effect of leptin on CO2 chemosensitivity in newborn rats.

BACKGROUND: Leptin augments central CO2 chemosensitivity and stabilizes breathing in adults. Premature infants have unstable breathing and low leptin levels. Leptin receptors are on CO2 sensitive neurons in the Nucleus Tractus Solitarius (NTS) and locus coeruleus (LC). We hypothesized that exogenous leptin improves hypercapnic respiratory response in newborn rats by improving central CO2 chemosensitivity. METHODS: In rats at postnatal day (p)4 and p21, hyperoxic and hypercapnic ventilatory responses, and pSTAT and SOCS3 protein expression in the hypothalamus, NTS and LC were measured before and after treatment with exogenous leptin (6 µg/g). RESULTS: Exogenous leptin increased the hypercapnic response in p21 but not in p4 rats (P ≤ 0.001). At p4, leptin increased pSTAT expression only in the LC, and SOCS3 expression in the NTS and LC; while at p21 pSTAT and SOCS3 levels were higher in the hypothalamus, NTS, and LC (P ≤ 0.05). CONCLUSIONS: We describe the developmental profile of the effect of exogenous leptin on CO2 chemosensitivity. Exogenous leptin does not augment central CO2 sensitivity during the first week of life in newborn rats. The translational implication of these findings is that low plasma leptin levels in premature infants may not be contributing to respiratory instability. IMPACT: Exogenous leptin does not augment CO2 sensitivity during the first week of life in newborn rats, similar to the developmental period when feeding behavior is resistant to leptin. Exogenous leptin increases CO2 chemosensitivity in newborn rats after the 3rd week of life and upregulates the expression of pSTAT and SOC3 in the hypothalamus, NTS and LC. Low plasma leptin levels in premature infants are unlikely contributors to respiratory instability via decreased CO2 sensitivity in premature infants. Thus, it is highly unlikely that exogenous leptin would alter this response.

L-citrulline attenuates lipopolysaccharide-induced inflammatory lung injury in neonatal rats.

BACKGROUND: Prenatal or postnatal lung inflammation and oxidative stress disrupt alveolo-vascular development leading to bronchopulmonary dysplasia (BPD) with and without pulmonary hypertension. L-citrulline (L-CIT), a nonessential amino acid, alleviates inflammatory and hyperoxic lung injury in preclinical models of BPD. L-CIT modulates signaling pathways mediating inflammation, oxidative stress, and mitochondrial biogenesis-processes operative in the development of BPD. We hypothesize that L-CIT will attenuate lipopolysaccharide (LPS)-induced inflammation and oxidative stress in our rat model of neonatal lung injury. METHODS: Newborn rats during the saccular stage of lung development were used to investigate the effect of L-CIT on LPS-induced lung histopathology and pathways involved in inflammatory, antioxidative processes, and mitochondrial biogenesis in lungs in vivo, and in primary culture of pulmonary artery smooth muscle cells, in vitro. RESULTS: L-CIT protected the newborn rat lung from LPS-induced: lung histopathology, ROS production, NFκB nuclear translocation, and upregulation of gene and protein expression of inflammatory cytokines (IL-1ß, IL-8, MCP-1α, and TNF-α). L-CIT maintained mitochondrial morphology, increased protein levels of PGC-1α, NRF1, and TFAM (transcription factors involved in mitochondrial biogenesis), and induced SIRT1, SIRT3, and superoxide dismutases protein expression. CONCLUSION: L-CIT may be efficacious in decreasing early lung inflammation and oxidative stress mitigating progression to BPD. IMPACT: The nonessential amino acid L-citrulline (L-CIT) mitigated lipopolysaccharide (LPS)-induced lung injury in the early stage of lung development in the newborn rat. This is the first study describing the effect of L-CIT on the signaling pathways operative in bronchopulmonary dysplasia (BPD) in a preclinical inflammatory model of newborn lung injury. If our findings translate to premature infants, L-CIT could decrease inflammation, oxidative stress and preserve mitochondrial health in the lung of premature infants at risk for BPD.

Remote ischemic conditioning in necrotizing enterocolitis: study protocol of a multi-center phase II feasibility randomized controlled trial.

PURPOSE: Remote ischemic conditioning (RIC) is a maneuver involving brief cycles of ischemia reperfusion in an individual's limb. In the early stage of experimental NEC, RIC decreased intestinal injury and prolonged survival by counteracting the derangements in intestinal microcirculation. A single-center phase I study demonstrated that the performance of RIC was safe in neonates with NEC. The aim of this phase II RCT was to evaluate the safety and feasibility of RIC, to identify challenges in recruitment, retainment, and to inform a phase III RCT to evaluate efficacy. METHODS: RIC will be performed by trained research personnel and will consist of four cycles of limb ischemia (4-min via cuff inflation) followed by reperfusion (4-min via cuff deflation), repeated on two consecutive days post randomization. The primary endpoint of this RCT is feasibility and acceptability of recruiting and randomizing neonates within 24 h from NEC diagnosis as well as masking and completing the RIC intervention. RESULTS: We created a novel international consortium for this trial and created a consensus on the diagnostic criteria for NEC and protocol for the trial. The phase II multicenter-masked feasibility RCT will be conducted at 12 centers in Canada, USA, Sweden, The Netherlands, UK, and Spain. The inclusion criteria are: gestational age < 33 weeks, weight ≥ 750 g, NEC receiving medical treatment, and diagnosis established within previous 24 h. Neonates will be randomized to RIC (intervention) or no-RIC (control) and will continue to receive standard management of NEC. We expect to recruit and randomize 40% of eligible patients in the collaborating centers (78 patients; 39/arm) in 30 months. Bayesian methods will be used to combine uninformative prior distributions with the corresponding observed proportions from this trial to determine posterior distributions for parameters of feasibility. CONCLUSIONS: The newly established NEC consortium has generated novel data on NEC diagnosis and defined the feasibility parameters for the introduction of a novel treatment in NEC. This phase II RCT will inform a future phase III RCT to evaluate the efficacy and safety of RIC in early-stage NEC.

Chronic Intermittent Hypoxia in Premature Infants: The Link Between Low Fat Stores, Adiponectin Receptor Signaling and Lung Injury.

Premature infants have chronic intermittent hypoxia (CIH) that increases morbidity, and the youngest and the smallest premature infants are at the greatest risk. The combination of lung injury from inflammation/oxidative stress causing low functional residual capacity combined with frequent short apneas leads to CIH. Adiponectin (APN) is an adipose-derived adipokine that protects the lung from inflammation and oxidative stress. Premature and small for gestational age (SGA) infants have minimal body fat and low levels of circulating APN. To begin to understand the potential role of APN in lung protection during lung development, we characterized the developmental profile of APN and APN receptors (AdipoR1 and AdipoR2) protein and mRNA expression in the newborn rat lung at fetal day (FD) 19, and postnatal days (PD) 1, 4, 7, 10, 14, 21, and 28. Protein levels in lung homogenates were measured by western blot analyses; relative mRNA expression was detected by quantitative PCR (qPCR); and serum high molecular weight (HMW) APN was measured using enzyme-linked immunosorbent assay (ELISA). Results: APN protein and mRNA levels were lowest at FD19 and PD1, increased 2.2-fold at PD4, decreased at PD10, and then increased again at PD21. AdipoR1 protein and mRNA levels peaked at PD1, followed by a threefold drop by PD4, and remained low until PD21. AdipoR2 protein and mRNA levels also peaked at PD1, but remained high at PD4, followed by a 1.7-fold drop by PD10 that remained low by PD21. Serum APN levels detected by ELISA did not differ from PD4 to PD28. To date, this is the first report characterizing APN and APN receptor protein and mRNA expression in the rat lung during development. The developmental stage of the newborn rat lung models that of the premature human infant; both are in the saccular stage of lung development. In the newborn rat lung, alveolarization begins at PD4, peaks at PD10, and ends at PD21. Importantly, we found that AdipoR1 receptor protein and mRNA expression is lowest during lung alveolarization (PD4 to PD21). Thus, we speculate that low levels of AdipoR1 during lung alveolarization contributes to the increased susceptibility to developing acute lung edema and chronic lung injury such as bronchopulmonary dysplasia (BPD) in premature human infants.

Mechanisms of modulation of cytokine release by human cord blood monocytes exposed to high concentrations of caffeine.

BACKGROUND: Serum caffeine concentrations >20 µg/ml (100 µmol/l) in infants treated for apnea of prematurity increases TNF-α and decreases IL-10, changes that perhaps are linked to comorbidities. We hypothesize that this proinflammatory cytokine profile may be linked to differential binding of caffeine to adenosine receptor subtypes (AR), inhibition of phosphodiesterases (PDEs), and modulation of toll-like receptors (TLR). METHODS: Lipopolysaccharide-activated cord blood monocytes (CBM) from 19 infants were exposed to caffeine (0-200 µmol/l) with or without previous exposure to A1R, A3R, or PDE IV antagonists to determine changes in dose-response curves. Cytokines levels (enzyme-linked immunosorbent assay (ELISA)), intracellular cyclic adenosine monophosphate (cAMP) accumulation (enzyme immunoassay (EIA)), and TLR gene expression (real time qRT PCR) were measured. RESULTS: Caffeine at ≤100 µmol/l decreased TNF-α levels (~25%, P = 0.01) and cAMP. All caffeine concentrations decreased IL-10 levels (17-35%, P < 0.01). A1R, A3R, and PDE blockades decreased TNF-α (31, 21, and 88%, P ≤ 0.01), but not IL-10. Caffeine further decreased TNF-α following A3R and PDE blockades. Caffeine concentrations directly correlated to TLR4 gene expression (r = 0.84; P < 0.001). CONCLUSION: Neither A3R, nor PDE blockades are involved in caffeine's modulation of cytokine release by CBM at any concentration. Besides A1R blockade, caffeine's upregulation of TLR4 may promote inflammation at high concentrations.

Effect of Lipopolysaccharide Exposure on Structure and Function of the Carotid Body in Newborn Rats.

Premature infants are vulnerable to infections and have unstable breathing (Di Fiore JM, Martin RJ, Gauda EB, Respir Physiol Neurobiol 189:213-222, 2013). Inflammation adversely modifies carotid body (CB) structure and chemosensitivity in adult animals. We determined the effect of inflammation on CB structure and function in newborn rat pups. Pups were given LPS (0.1 mg/kg; IP) or saline at postnatal day 2 (P2). At P9-10 (1 week after exposure) various studies were done including ventilation, carotid sinus nerve (CSN) activity and histology. Using whole body plethysmography, we found that LPS exposure attenuates the change in interbreath (IBI) interval in response to changes in oxygen tension 1 week after LPS exposure. The response of the CSN to hypoxia was attenuated and delayed in onset in LPS-treated animals as compared to controls. Histological sections of the CB were examined for inflammatory cells at P4 (n = 7) and P9-12 (n = 6). After LPS exposure, only mast cells were seen, often encircling the CB, and clustered within the CSN as it entered the CB. Mast cells per section (mean ± SEM) were higher at P9-12 in LPS (7.4 ± 1.5) vs saline (5.4 ± 1.4) exposed animals (p = 0.04). Surprisingly, more mast cells were seen at 7-10 days vs 48 h after LPS exposure. In a newborn model of inflammation, breathing is altered which is associated with changes in structure and function of the carotid body.

SARS-CoV-2 Infection of Young Infants during the Omicron Wave: A Case Series.

We describe the clinical course of 4 infants infected with severe acute respiratory syndrome coronavirus 2. All were admitted to our tertiary care neonatal intensive care unit during the Omicron variant wave in our region. All 4 infants, who were less than 3 months of age, including three born prematurely, presented with critical illness. However, their clinical presentation varied considerably. Of them, two infants presented with apnea, one with respiratory distress, and one with gastrointestinal manifestation. Our experience with these four infants provides evidence for a severe form of disease and varied clinical presentation in neonates and young infants speculated to be infected with Omicron variant.

PGC-1α activity and mitochondrial dysfunction in preterm infants.

Extremely low gestational age neonates (ELGANs) are born in a relatively hyperoxic environment with weak antioxidant defenses, placing them at high risk for mitochondrial dysfunction affecting multiple organ systems including the nervous, respiratory, ocular, and gastrointestinal systems. The brain and lungs are highly affected by mitochondrial dysfunction and dysregulation in the neonate, causing white matter injury (WMI) and bronchopulmonary dysplasia (BPD), respectively. Adequate mitochondrial function is important in providing sufficient energy for organ development as it relates to alveolarization and axonal myelination and decreasing oxidative stress via reactive oxygen species (ROS) and reactive nitrogen species (RNS) detoxification. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) is a master regulator of mitochondrial biogenesis and function. Since mitochondrial dysfunction is at the root of WMI and BPD pathobiology, exploring therapies that can regulate PGC-1α activity may be beneficial. This review article describes several promising therapeutic agents that can mitigate mitochondrial dysfunction through direct and indirect activation and upregulation of the PGC-1α pathway. Metformin, resveratrol, omega 3 fatty acids, montelukast, L-citrulline, and adiponectin are promising candidates that require further pre-clinical and clinical studies to understand their efficacy in decreasing the burden of disease from WMI and BPD in preterm infants.

Risk factors for prolonged mechanical ventilation in neonates following gastrointestinal surgery.

Background: Prolonged mechanical ventilation (MV) should be avoided in neonates. Noninvasive ventilation (NIV) can facilitate weaning from MV but has risks for patients immediately following foregut surgery due to the potential risk of anastomotic leak. We evaluated the risk factors for prolonged MV following intestinal surgery in neonates. Methods: We retrospectively reviewed 253 neonates undergoing intestinal surgery in 2017-2018 to identify risk factors for prolonged MV, and determine the correlation between NIV and anastomotic leak in a tertiary neonatal intensive care unit that performs the greatest number of neonatal surgeries in Ontario. Results: The most common diagnoses were necrotizing enterocolitis/spontaneous intestinal perforation (NEC/SIP) 21%, intestinal atresia 16%, esophageal atresia/tracheoesophageal fistula 14%, ano-rectal malformation 13%, malrotation/volvulus 11%, gastroschisis 9% and omphalocele 4%. The median (IQR) duration of MV post-surgery was 3 (1-8) days with 25.7 % (n=65) of neonates on MV for >7 days. Compared to infants on MV post-surgery for ≤7 days, those with MV>7 days were of lower gestational age, birth weight and weight at surgery, but a higher proportion underwent stoma creation, had a longer duration of opioid administration and higher rates of moderate to severe bronchopulmonary dysplasia (BPD) and mortality (P<0.05). Generalized linear regression analysis showed lower gestational age (GA) and longer opioid administration were associated with longer duration of MV (P<0.001), but indication for surgery, weight at surgery and stoma creation didn't correlate with longer duration of MV (P>0.05). Of the 122 patients handled by one-stage resection with primary anastomosis, 22.1% (n=27) received NIV with 74.1% (n=20) commenced on NIV after 7 days post-surgery, anastomotic leak was detected in 2.5 % (3/122) patients and didn't correlate with NIV. Conclusions: Lower GA and longer opioid administration were risk factors for prolonged MV in neonates following intestinal surgery. Further research is needed to investigate modifiable practices around pain assessment/ventilation in these patients, and the correlation between NIV and anastomotic leak.

SARS-CoV-2 Associated Respiratory Failure in a Preterm Infant and the Outcome after Remdesivir Treatment.

Severe coronavirus disease 2019 (COVID-19) occurs in approximately 10% of neonates infected with severe acute respiratory syndrome coronavirus 2. Guidelines for optimal management of severe COVID-19 in neonates do not exist. In this report, we describe a late-preterm neonate with severe COVID-19, requiring invasive mechanical ventilation who recovered following treatment with remdesivir and high dose dexamethasone.

Clonidine for sedation in infants during therapeutic hypothermia with neonatal encephalopathy: pilot study.

OBJECTIVE: To determine a safe dose of clonidine (CLON) to be used in infants with hypoxic ischemic encephalopathy (HIE) undergoing therapeutic hypothermia (TH). STUDY DESIGN: A pilot prospective study was performed to determine the effect of CLON on autonomic parameters, the pharmacokinetics (PK) of CLON, and the amount of morphine (MOR) given "as needed" for shivering and agitation in a cohort of infants (n = 12) with HIE undergoing TH compared to a historical control group (n = 28). RESULTS: The CLON group received less "as needed" MOR than the MOR-only group for agitation/shivering (p < 0.001), and the CLON vs. MOR-only group spent 92% vs. 79% of cooling time at the target core body temperature (CBT; p = 0.03, CLON vs. MOR). CONCLUSIONS: Intravenous CLON (1 mcg/kg Q8h) is well tolerated in infants treated with TH for HIE. CLON stabilizes CBT in the ideal range during cooling, which may be optimal for neuroprotection.

Correlation between serum caffeine levels and changes in cytokine profile in a cohort of preterm infants.

OBJECTIVE: To determine changes in cytokine levels associated with caffeine treatment in a cohort of preterm infants. STUDY DESIGN: For this observational prospective study, we collected clinical data from 26 preterm infants (≤ 30 weeks gestational age). In addition to caffeine levels, cytokine profiles in peripheral blood (PB) and tracheal aspirates (TA) were determined with enzyme-linked immunosorbent assay at birth, before and after (at 24 hours and 1 week) initiation of caffeine. Non-parametric statistics were applied. RESULTS: Included infants were 26.9 ± 1.7 weeks gestational age and weighed 985 ± 202 g. At birth, all cytokine concentrations were significantly greater in TA than PB. Serum caffeine levels were 11.1 µg/mL (interquartile range, 1.85) at approximately 24 hours post-load and 16.4 (8.7) µg/mL at 1 week on treatment. At approximately 24 hours post-load, interleukin (IL)-10 levels decreased by 47.5% (P = .01) in PB and 38.5% (P = .03) in TA, whereas other cytokine levels remained unchanged. At 1 week, caffeine levels were correlated (U-shaped) with changes in proinflammatory tumor necrosis factor-α (R(2) = 0.65; P = .0008), interleukin (IL)-1ß (R(2) = 0.73; P = .0007), and IL-6 (R(2) = 0.59; P = .003), whereas inversely correlated (linear) with the anti-inflammatory IL-10 (R(2) = 0.64; P = .0008). Altogether, caffeine, at serum levels ≥ 20 µg/mL, was associated with a proinflammatory profile after 1 week of treatment. CONCLUSIONS: Caffeine treatment for apnea of prematurity correlates with changes in cytokine profile. Caffeine levels ≥ 20 µg/mL are associated with a proinflammatory profile in our cohort of preterm infants.

Adiponectin and the regulation of gastric content volume in the newborn rat.

BACKGROUND: Oral intake is dependent on the gastric ability to accommodate the food bolus. Comparatively, neonates have a smaller gastric capacity than adults and this may limit the volume of their milk intake. Yet, we previously reported that the newborn rat gastric milk volume is greatest after birth and, when normalized to body weight, decreases with postnatal age. Such age-dependent changes are not the result of intake differences, but greater gastric accommodation and reduced emptying rate. AIM: Hypothesizing that breastmilk-derived adiponectin is the factor regulating gastric accommodation in neonates, we comparatively evaluated its effects on the rat fundic muscle tone at different postnatal ages. METHODS: In freshly dispersed smooth muscle cells (SMC), we measured the adiponectin effect on the carbachol-induced length changes. RESULTS: Adiponectin significantly reduced the carbachol-stimulated SMC shortening independently of age. In the presence of the inhibitor iberiotoxin, the adiponectin effect on SMC shortening was suppressed, suggesting that it is mediated via large-conductance Ca2+ sensitive K+ channel activation. Lastly, we comparatively measured the newborn rat gastric milk curd adiponectin content in one- and two-week-old rats and found a 50% lower value in the latter. CONCLUSION: Adiponectin, a major component of breastmilk, downregulates fundic smooth muscle contraction potential, thus facilitating gastric volume accommodation. This rodent's adaptive response maximizes breastmilk intake volume after birth.

Premature birth, homeostatic plasticity and respiratory consequences of inflammation.

Infants who are born premature can have persistent apnea beyond term gestation, reemergence of apnea associated with inflammation during infancy, increased risk of sudden unexplained death, and sleep disorder breathing during infancy and childhood. The autonomic nervous system, particularly the central neural networks that control breathing and peripheral and central chemoreceptors and mechanoreceptors that modulate the activity of the central respiratory network, are rapidly developing during the last trimester (22-37 weeks gestation) of fetal life. With advances in neonatology, in well-resourced, developed countries, infants born as young as 23 weeks gestation can survive. Thus, a substantial part of maturation of central and peripheral systems that control breathing occurs ex-utero in infants born at the limit of viability. The balance of excitatory and inhibitory influences dictates the ultimate output from the central respiratory network. We propose in this review that simply being born early in the last trimester can trigger homeostatic plasticity within the respiratory network tipping the balance toward inhibition that persists in infancy. We discuss the intersection of premature birth, homeostatic plasticity and biological mechanisms leading to respiratory depression during inflammation in former premature infants.

Leptin: Master Regulator of Biological Functions that Affects Breathing.

Obesity is a global epidemic in developed countries accounting for many of the metabolic and cardiorespiratory morbidities that occur in adults. These morbidities include type 2 diabetes, sleep-disordered breathing (SDB), obstructive sleep apnea, chronic intermittent hypoxia, and hypertension. Leptin, produced by adipocytes, is a master regulator of metabolism and of many other biological functions including central and peripheral circuits that control breathing. By binding to receptors on cells and neurons in the brainstem, hypothalamus, and carotid body, leptin links energy and metabolism to breathing. In this comprehensive article, we review the central and peripheral locations of leptin's actions that affect cardiorespiratory responses during health and disease, with a particular focus on obesity, SDB, and its effects during early development. Obesity-induced hyperleptinemia is associated with centrally mediated hypoventilation with decrease CO2 sensitivity. On the other hand, hyperleptinemia augments peripheral chemoreflexes to hypoxia and induces sympathoexcitation. Thus, "leptin resistance" in obesity is relative. We delineate the circuits responsible for these divergent effects, including signaling pathways. We review the unique effects of leptin during development on organogenesis, feeding behavior, and cardiorespiratory responses, and how undernutrition and overnutrition during critical periods of development can lead to cardiorespiratory comorbidities in adulthood. We conclude with suggestions for future directions to improve our understanding of leptin dysregulation and associated clinical diseases and possible therapeutic targets. Lastly, we briefly discuss the yin and the yang, specifically the contribution of relative adiponectin deficiency in adults with hyperleptinemia to the development of metabolic and cardiovascular disease. © 2020 American Physiological Society. Compr Physiol 10:1047-1083, 2020.

Recombinant adiponectin protects the newborn rat lung from lipopolysaccharide-induced inflammatory injury.

Preterm infants are at high risk for developing bronchopulmonary dysplasia and pulmonary hypertension from inflammatory lung injury. In adult models, adiponectin (APN)-an adipocyte-derived hormone-protects the lung from inflammatory injury and pulmonary vascular remodeling. Cord blood APN levels in premature infants born < 26 weeks gestation are 5% of the level in infants born at term. We previously reported the expression profile of APN and its receptors in neonatal rat lung homogenates during the first 3 weeks of postnatal development. Here, we characterize the expression profile of APN and its receptors in specific lung cells and the effects of exogenous recombinant APN (rAPN) on lipopolysaccharide-(LPS)-induced cytokine and chemokine production in total lung homogenates and specific lung cells. In vitro, rAPN added to primary cultures of pulmonary artery smooth muscle cells attenuated the expression of LPS-induced pro-inflammatory cytokines while increasing the expression of anti-inflammatory cytokines. In vivo, intraperitoneal rAPN (2 mg/kg), given 4 hr prior to intrapharyngeal administration of LPS (5 mg/kg) to newborn rats at postnatal day 4, significantly reduced gene and protein expression of the pro-inflammatory cytokine IL-1ß and reduced protein expression of the chemokines monocyte chemoattractant protein (MCP-1) and macrophage inflammatory protein-1 alpha (MIP-1α) in the lung. LPS-induced histopathological changes in the lung were also decreased. Moreover, rAPN given 20 hr after intrapharyngeal LPS had a similar effect on lung inflammation. These findings suggest a role for APN in protecting the lung from inflammation during early stages of lung development.

Caffeine modulates TNF-alpha production by cord blood monocytes: the role of adenosine receptors.

Caffeine, a nonspecific adenosine receptor (AR) antagonist is widely used to treat apnea of prematurity. Because adenosine modulates multiple biologic processes including inflammation, we hypothesized that AR blockade by caffeine would increase cytokine release from neonatal monocytes. Using cord blood monocytes (CBM), we investigated 1) the changes in AR mRNA profile by real time quantitative reverse-transcription polymerase-chain-reaction (qRT-PCR) and protein expression (western blot) after in vitro culture, caffeine or lipopolysaccharide (LPS) exposure, and 2) the modulation of cytokine release and cyclic adenosine monophosphate (cAMP) production by enzyme-linked immunosorbent assay (ELISA) induced by caffeine and specific AR antagonists: DPCPX(A1R), ZM241385(A2aR), MRS1754(A2bR), and MRS1220(A3R). After 48 h in culture, A2aR and A2bR gene expression increased 1.9 (p = 0.04) and 2.5-fold (p = 0.003), respectively. A1R protein expression directly correlated with increasing LPS concentrations (p = 0.01), with minimal expression preexposure. Only caffeine (50 microM) and DPCPX (10 nM) decreased tumor necrosis factor-alpha (TNF-alpha) release from LPS activated-CBM by 20 and 25% (p = 0.01) and TNF-alpha gene expression by 30 and 50%, respectively, in conjunction with a > or =2-fold increase in cAMP (p < 0.05). AR blockade did not modulate other measured cytokines. The induction of A1R after LPS exposure suggests an important role of this receptor in the control of inflammation in neonates. Our findings also suggest that caffeine, via A1R blockade, increases cAMP production and inhibits pretranscriptional TNF-alpha production by CBM.