Margarita M. Vasquez

Smad7 inhibits autocrine expression of TGF-β2 in intestinal epithelial cells in baboon necrotizing enterocolitis

Preterm infants may be at risk of necrotizing enterocolitis (NEC) due to deficiency of transforming growth factor-β 2 (TGF-β(2)) in the developing intestine. We hypothesized that low epithelial TGF-β(2) expression in preterm intestine and during NEC results from diminished autocrine induction of TGF-β(2) in these cells. Premature baboons delivered at 67% gestation were treated per current norms for human preterm infants. NEC was diagnosed by clinical and radiological findings. Inflammatory cytokines, TGF-β(2), Smad7, Ski, and strawberry notch N (SnoN)/Ski-like oncoprotein (SKIL) was measured using quantitative reverse transcriptase-polymerase chain reaction, immunoblots, and immunohistochemistry. Smad7 effects were examined in transfected IEC6 intestinal epithelial cells in vitro. Findings were validated in archived human tissue samples of NEC. NEC was recorded in seven premature baboons. Consistent with existing human data, premature baboon intestine expressed less TGF-β(2) than term intestine. TGF-β(2) expression was regulated in epithelial cells in an autocrine fashion, which was interrupted in the premature intestine and during NEC due to increased expression of Smad7. LPS increased Smad7 binding to the TGF-β(2) promoter and was associated with dimethylation of the lysine H3K9, a marker of transcriptional silencing, on the nucleosome of TGF-β(2). Increased Smad7 expression in preterm intestine was correlated with the deficiency of SnoN/SKIL, a repressor of the Smad7 promoter. Smad7 inhibits autocrine expression of TGF-β(2) in intestinal epithelial cells in the normal premature intestine and during NEC. Increased Smad7 expression in the developing intestine may be due to a developmental deficiency of the SnoN/SKIL oncoprotein.

Immunosuppressive properties of surfactant in alveolar macrophage NR8383

Abstract.Objective:To evaluate the anti-inflammatory effects of exogenous surfactants and surfactant phospholipid without surfactant proteins (SP-A and SP-D) on the lipopolysaccharide- (LPS) stimulated rat alveolar macrophage (AM) cell line NR8383.Methods:Exogenous surfactants (beractant, calfactant or colfosceril) and surfactant phospholipid (dipalmitoyl phosphatidylcholine, DPPC), standardized to phospholipid content of 25–1,000 μg/ml were incubated with LPS- (1 μg/ml) stimulated NR8383 AMs.Results:TNF-α and IL-1β secretion and nitric oxide (NO) formation following LPS stimulation were inhibited by treatment with surfactants or DPPC. Furthermore, LPS-dependent NO production and iNOS protein levels were significantly suppressed in cells pretreated for one hour with beractant compared to beractant added simultaneously with or following LPS. Additionally, LPS-stimulated oxidative burst, measured by flow cytometry, was significantly decreased by beractant. Finally, beractant inhibited the translocation of NF-κB from cytoplasmic into nuclear extract in LPS-stimulated NR8383 AMs.Conclusions:Exogenous surfactants and surfactant phospholipid inhibit secretion of proinflammatory cytokines and NO in NR8383 AMs. The inhibitory effects of beractant on oxygen radical and LPS-induced NO formation may result from unique mechanisms of decreasing cell signaling. The anti-inflammatory activity of surfactant products used in the treatment of neonatal respiratory distress syndrome (RDS) may depend upon the specific preparation or dose used.

Neonatal chlamydial pneumonia induces altered respiratory structure and function lasting into adult life

Respiratory dysfunction in adults has been correlated with neonatal Chlamydia trachomatis pneumonia in several studies, but a causal association has not been clearly demonstrated. In this study, we examined radial alveolar counts (RACs) by microscopy, and airway and parenchymal lung function using a small animal ventilator in juvenile (5 weeks age) and adult (8 weeks age) BALB/c mice challenged as neonates with Chlamydia muridarum (C. mur) on day 1 or day 7 after birth, representing saccular (human pre-term neonates) and alveolar (human term neonates) stages of lung development, respectively. Pups challenged with C. mur on either day 1 or 7 after birth demonstrated significantly enhanced airway hyperreactivity and lung compliance, both as juveniles (5 weeks age) and adults (8 weeks age), compared with mock-challenged mice. Moreover, mice challenged neonatally with Chlamydia displayed significantly reduced RACs, suggesting emphysematous changes. Antimicrobial treatment during the neonatal infection induced early bacterial clearance and partially ameliorated the Chlamydia-induced lung dysfunction as adults. These results suggest that neonatal chlamydial pneumonia, especially in pre-term neonates, is a cause of respiratory dysfunction continuing into adulthood, and that antimicrobial administration may be partially effective in preventing the adverse respiratory sequelae in adulthood. The results of our studies also emphasize the importance of prenatal screening and treatment of pregnant women for C. trachomatis in order to prevent the infection of neonates.

Induction of serum- and glucocorticoid-induced kinase-1 (SGK1) by cAMP regulates increases in α-ENaC

α‐ENaC expression and activity is regulated by a variety of hormones including β‐adrenergic agonists via the second messenger cAMP. We evaluated the early intermediate pathways involved in the up‐regulation of SGK1 by DbcAMP and whether SGK1 is a prerequisite for induction of α‐ENaC expression. Submandibular gland epithelial (SMG‐C6) cells treated with DbcAMP (1 mM) induced both SGK1 mRNA and protein expression. DbcAMP‐stimulated SGK1 mRNA expression was decreased by actinomycin D and mRNA and protein expressions were attenuated by PKA inhibitors (H‐89 and KT5720). Inhibition of PI3‐K with either LY294002 or dominant negative PI3‐K reduced DbcAMP‐stimulated SGK1 protein and mRNA levels, attenuated the phosphorylation of CREB (a cAMP‐activated transcription factor) and decreased α‐ENaC protein levels and Na+ transport. In addition, the combination of PKA inhibitors with dominant negative PI3‐K synergistically inhibited DbcAMP‐induced Na+ transport. Inhibition of SGK1 expression by siRNA decreased but did not obliterate DbcAMP‐induced α‐ENaC expression. Thus, in a cell line which endogenously exhibits minimal α‐ENaC expression, induction of SGK1 by DbcAMP occurs via the PI3‐K and PKA pathways. Increased α‐ENaC levels and function are partly dependent upon the early induction of SGK1 expression. J. Cell. Physiol. 217: 632–642, 2008.

TGF-β2, a Protective Intestinal Cytokine, Is Abundant in Maternal Human Milk and Human-Derived Fortifiers but Not in Donor Human Milk

OBJECTIVE This study compared cytokines (in particular transforming growth factor [TGF]-β2) and lactoferrin in maternal human milk (MHM), human-derived milk fortifier (HDMF), and donor human milk (DHM). MATERIALS AND METHODS MHM was randomly collected from breastfeeding mothers who had no infectious illness at the time of milk expression. HDMF and DHM were products derived from human milk processed by Holder pasteurization. MHM samples were collected at different times (early/late) and gestations (preterm/term). Lactoferrin was analyzed by western blotting, and cytokines were quantified using commercial enzyme-linked immunosorbent assays. Significance was determined using analysis of variance. RESULTS In the 164 samples analyzed, TGF-β2 concentrations in HDMF and preterm MHM (at all collection times) were fivefold higher than in DHM (p<0.05). Early preterm MHM had levels of interleukin (IL)-10 and IL-18, 11-fold higher than DHM (p<0.05). IL-6 in DHM was 0.3% of the content found in MHM. IL-18 was fourfold higher in early MHM versus late MHM regardless of gestational age (p<0.05). Lactoferrin concentration in DHM was 6% of that found in MHM. CONCLUSIONS Pasteurization decreases concentrations of most cytokines and lactoferrin in DHM. TGF-β2, a protective intestinal cytokine, has comparable concentrations in HDMF to MHM despite pasteurization.

Regulation of epithelial Na+ channel (ENaC) in the salivary cell line SMG-C6.

Glucocorticoids and mineralocorticoids modulate Na+ transport via epithelial Na+ channels (ENaC). The rat submandibular epithelial cell line, SMG-C6, expresses α-ENaC mRNA and protein and exhibits amiloride-sensitive Na+ transport when grown in low-serum (2.5%) defined medium, therefore, we examined the effects of altering the composition of the SMG-C6 cell growth medium on ENaC expression and function. No differences in basal or amiloride-sensitive short-circuit current (Isc) were measured across SMG-C6 monolayers grown in the absence of thyroid hormone, insulin, transferrin, or EGF. In the absence of hydrocortisone, basal and amiloride-sensitive Isc significantly decreased. Similarly, monolayers grown in 10% serum-supplemented medium had lower basal Isc and no response to amiloride. Adding hydrocortisone (1.1 μM) to either the low or 10% serum medium increased basal and amiloride-sensitive Isc, which was blocked by RU486, the glucocorticoid and progesterone receptor antagonist. Aldosterone also induced an increase in α-ENaC expression and Na+ transport, which was also blocked by RU486 but not by the mineralocorticoid receptor antagonist spironolactone. Thus, in the SMG-C6 cell line, hydrocortisone and aldosterone increased ENaC expression and basal epithelial Na+ transport. The absence of endogenous ENaC expression in culture conditions devoid of steroids makes the properties of this cell line an excellent model for investigating pathways regulating ENaC expression and Na+ transport.

NewB for newbies: A randomized control trial training housestaff to perform neonatal intubation with direct and videolaryngoscopy

Competency rates in neonatal intubation among pediatric residents are low and currently not meeting ACGME/AAP standards.

Conducting Quantitative Medical Education Research: From Design to Dissemination

Rigorous medical education research is critical to effectively develop and evaluate the training we provide our learners. Yet many clinical medical educators lack the training and skills needed to conduct high-quality medical education research. We offer guidance on conducting sound quantitative medical education research. Our aim is to equip readers with the key skills and strategies necessary to conduct successful research projects, highlighting new concepts and controversies in the field. We utilize Glassicks criteria for scholarship as a framework to discuss strategies to ensure that the research question of interest is worthy of further study and how to use existing literature and conceptual frameworks to strengthen a research study. Through discussions of the strengths and limitations of commonly used study designs, we expose the reader to particular nuances of these decisions in medical education research and discuss outcomes generally focused on, as well as strategies for determining the significance of consequent findings. We conclude with information on critiquing research findings and preparing results for dissemination to a broad audience. Practical planning worksheets and comprehensive tables illustrating key concepts are provided in order to guide researchers through each step of the process. Medical education research provides wonderful opportunities to improve how we teach our learners, to satisfy our own intellectual curiosity, and ultimately to enhance the care provided to patients.

Neonatal lead intoxication following maternal pica: A case report and review of the literature

Pica is a significant risk factor for lead exposure during pregnancy and may often go unrecognized. Lead exposure in the pregnant woman may be extremely toxic to the fetus due to unencumbered transport across the placenta. Long term neurological effects of in utero and neonatal lead exposure are largely unknown, and the efficacies of current therapies are controversial. We report a case of a pregnant woman with elevated blood lead levels secondary to pica that was not identified by a routine screening survey. Her full term baby girl had a blood lead level of 70.8 μg/dL. Neonatal therapy with double-volume exchange transfusion followed by a 3 day course of intravenous Edetate Calcium Disodium is described as well as the slow steady decline of her blood lead levels. Neurocognitive development to 18 months is also reported.

TRAFFICKING AND FUNCTION OF THE EPITHELIAL SODIUM CHANNEL ARE INHIBITED BY EPIDERMAL GROWTH FACTOR IN A RAT PAROTID GLAND CELL LINE.: 244

Background At the time of birth, the major pathway regulating lung fluid absorption is transepithelial Na+ movement via epithelial sodium channels (ENaCs). The density of ENaC proteins is high in the apical membranes of the type II alveolar cells and its expression and function are regulated by many agents, including steroids and growth factors. The rat parotid gland cell line (PAR-C5) expresses all ENaC subunit proteins (α, β, andγ), but monolayers grown in media containing EGF do not demonstrate significant ENaC function. Objective and Methods PAR-C5 cells were studied to examine the effect of EGF on ENaC function and trafficking. Cells were seeded on permeable filter supports in the presence or absence of EGF (20 ng/mL) and/or dexamethasone (0.1 μM). After the monolayers formed tight junctions as demonstrated by transepithelial resistances > 200 Ω.cm2 (mean resistance 513 Ω.cm2), permeable filters were mounted in Ussing chambers and Na+ transport was determined by the amiloride (10 μM)-sensitive component of the short-circuit current (Isc). α-ENaC protein content of similarly treated cells was examined by Western blot. α-ENaC localization in PAR-C5 cells was determined by confocal microscopy following staining with anti-α-ENaC antibody. Results The percent change in amiloride-sensitive Isc was 41.8 ± 5.0% in the absence versus 8.5 ± 5.5% in the presence of EGF (p = .0131). Dexamethasone increased the percent change in amiloride-sensitive Isc to 67.7 ± 12.1% in the absence and 60.3 ± 5.7% in the presence of EGF (p = .234). Despite differences in ENaC function, the amount of α-ENaC protein expression was similar in the presence or absence of EGF. However, PAR-C5 cells grown in the presence of EGF demonstrated less α-ENaC localization in the membrane portion compared with those cells grown in the absence of EGF. Conclusion These results suggest that EGF inhibits the intracellular trafficking of α-ENaC into the plasma membranes in the PAR-C5 cell line. Since animal in utero studies suggest that EGF supplementation may be used to reverse pulmonary hypoplasia, further investigation is needed to characterize the signaling mechanisms whereby EGF and other growth factors modulate intracellular ENaC trafficking in different cell types.