David M. Gourlay

Hypertonic Preconditioning Inhibits Macrophage Responsiveness to Endotoxin

Hypertonic saline has been shown to modulate cell shape and the response of components of the innate immune response. However, the effect of hypertonic saline on the macrophage remains unknown. We hypothesized that hypertonic preconditioning would impair subsequent inflammatory mediator signaling through a reduction in stress fiber polymerization and mitogen-activated protein kinase activity after LPS stimulation. Rabbit alveolar macrophages were stimulated with 100 ng/ml of LPS. Selected cells were preconditioned with 40–100 mM of NaCl, mannitol, or urea for 4 h and returned to isotonic medium before LPS stimulation. Cellular protein was harvested and subjected to Western blot analysis for the dually phosphorylated active forms of p38 and extracellular signal-related kinase (ERK) 1/2. TNF production was determined by an L929 bioassay, and stress fiber polymerization was evaluated by confocal microscopy. Preconditioning of macrophages with NaCl or mannitol resulted in dose-dependent reduction in ERK 1/2 phosphorylation with no effect on p38 phosphorylation. Urea preconditioning had no effect on either mitogen-activated protein kinase. A dose-dependent attenuation of TNF production was seen with NaCl and mannitol preconditioning (p < 0.05), but not with urea. NaCl and mannitol preconditioning resulted in failure of LPS-induced stress fiber polymerization, whereas urea did not. Extracellular hypertonic conditions (i.e., NaCl and mannitol) have an immunomodulatory effect on macrophages, demonstrated through failure of optimal stress fiber polymerization, ERK 1/2 activity, and TNF production. Intracellular hypertonic conditions (i.e., urea) had no significant effect. Hypertonic saline or mannitol resuscitation, therefore, may help protect against multiple-organ dysfunction syndrome as a result of this reduced proinflammatory responsiveness.

Modulation of macrophage responsiveness to lipopolysaccharide by IRAK-1 manipulation.

Local activation of the macrophage by endotoxin is essential for the eradication of invasive gram-negative infections. Circulating endotoxin at lower concentrations results in immune cell activation at distant sites leading to tissue injury. Although the cellular mechanisms involved in these potentially dissimilar events are incomplete, it appears that the proximal kinase IRAK-1 plays a role. Thus, sense and antisense IRAK-1 oligonucleotides were used to determine the role IRAK-1 plays in macrophage activation by systemic (1–100 ng/mL) and local (1000 ng/mL) concentration of lipopolysaccharide (LPS) within THP-1 cells. Within the sense group, 1–1000 ng/mL of LPS within the sense group resulted in cellular activation of ERK-1/2, p38, and JNK/SAPK and the nuclear activation of NF-&kgr;B and AP-1. This activation was associated with proinflammatory cytokine production and cellular spreading. Systemic concentrations of LPS within the antisense group were associated with significant attenuation of intracellular signaling, cytokine production, and cellular spreading compared with the sense group. Local concentrations of LPS within the antisense group, however, were associated only with a delay in intracellular signaling, with no effect on cytokine production or cell spreading compared with the sense group. Based on these results, it appears that IRAK-1 is essential to macrophage activation at systemic, but not local, concentrations of LPS. These data suggest that redundant pathways exist that are functional at higher concentrations of LPS. Therefore, IRAK-1 appears to be the central kinase involved in the activation of the macrophage at distant sites during septic shock but is not necessary for activation in areas of local infection.

Beyond feasibility: a comparison of newborns undergoing thoracoscopic and open repair of congenital diaphragmatic hernias

BACKGROUND Although both laparoscopic and thoracoscopic repair of congenital diaphragmatic hernia (CDH) have been described in the literature, neither appropriate selection criteria nor improved outcomes for minimally invasive repair over open repair have been clearly delineated. METHODS We reviewed our experience with neonatal CDH repair between 2004 and 2007 to determine clinical parameters that are associated with successful thoracoscopic CDH repair. We compared these patients to a similarly matched cohort of patients who had undergone an open neonatal CDH repair between 1999 and 2003. RESULTS From 2004 to 2007, 20 (61%) of 33 patients underwent successful neonatal thoracoscopic CDH repair. Characteristics common to all patients who underwent successful thoracoscopic repair included absence of congenital heart defects, no need for extracorporeal membrane oxygenation, ventilatory peak inspiratory pressure of less than 26 cmH(2)O, and oxygenation index less than 5 on the day of planned surgery. From 1999 to 2003, 40 patients underwent an open neonatal CDH repair, of which 18 (45%) patients would have matched our selection criteria for thoracoscopic repair. These 2 cohorts were similar in age, estimated gestational age, weight, APGAR scores, and oxygenation index at the time of surgery. The thoracoscopic cohort had statistically and clinically significant quicker return to full enteral feeds, had shorter duration on the ventilator postoperatively, and required less narcotic/sedation postoperatively. Less severe complications occurred in the thoracoscopic cohort. Adjusted total hospital charges were less for the thoracoscopic repair. CONCLUSIONS Successful thoracoscopic CDH repair can be expected in newborns, which has limited respiratory compromise. Thoracoscopic CDH repair is associated with lower morbidity and quicker recovery than traditional open repair and without increased risk of recurrence or complications.

Endothelial Microparticles Induce Inflammation in Acute Lung Injury

BACKGROUND Previously, we have shown that endothelial microparticles (EMPs) injected into mice induce acute lung injury (ALI) [1]. In this study, we hypothesize that EMPs induce ALI by initiating cytokine release in the lung, leading to recruitment and activation of neutrophils. MATERIALS AND METHODS C57BL/6J male mice (8-10 wk old) were intravenously injected with EMPs (200,000/mL), LPS (2 mg/kg), or both. Bronchoalveolar lavage (BAL) and serum levels of IL-1β and TNF-α were analyzed by enzyme-linked immunoassay (ELISA). Morphometric analysis was performed on H and E stained lung sections. Myeloperoxidase (MPO) levels were determined via an enzymatic assay and immunofluorescence of stained sections. RESULTS EMPs led to significantly increased pulmonary and systemic IL-1β and TNF-α levels, which correlated with increased neutrophil recruitment to the lung. MPO levels in the lungs were increased significantly following injection of EMPs or LPS, compared to PBS. In mice treated with EMPs and LPS either simultaneously or successively, the cytokine and MPO levels were significantly increased over that of either treatment alone. CONCLUSION EMPs contribute to lung injury through the initiation of a cytokine cascade that increases recruitment of neutrophils and subsequent release of MPO. Furthermore, treatment of mice with both EMPs and LPS induced greater lung injury than either treatment alone, suggesting that EMPs prime the lung for increased injury by other pathogens. Therapies aimed at reducing or blocking EMPs may be a useful strategy for attenuating lung injury.

The protective role of intestinal alkaline phosphatase in necrotizing enterocolitis.

BACKGROUND Enterocytes produce intestinal alkaline phosphatase (iAP), which detoxifies lipopolysaccharide (LPS), a mediator in necrotizing enterocolitis (NEC) pathogenesis. We hypothesize that aberrant expression or function of iAP contributes to the pathogenesis of NEC. MATERIALS AND METHODS Newborn Sprague Dawley rat pups were divided into three main groups. Control pups were breast fed, while two groups were exposed to intermittent hypoxia, LPS, and formula feeding for 4 d to induce NEC. Bovine iAP, with and without the presence of LPS, was administered orally to one of the NEC groups. The intestine was harvested and used to detect alkaline phosphatase (AP) activity and protein expression. Terminal ileum sections were used to grade intestinal injury and stained for AP. Comparisons were made with adult rat duodenum. RESULTS Compared with adult rats, control pups expressed significantly less AP protein but had 2-fold higher AP activity. NEC pup AP activity was significantly decreased compared to controls (P < or = 0.05), which paralleled both the AP protein expression and immunofluorescence assay results. Following iAP administration, immunofluorescence, protein expression, and activity of AP were significantly increased compared with NEC pups without iAP supplementation. All NEC pups had intestinal injury grades > or = 2 on a 4-point scale, while control and iAP-treated pups had grades < 0.25 (P < 0.001). CONCLUSIONS Enteral administration of iAP to rat pups with experimental NEC increased AP activity levels to that of controls, and appears to protect the intestine. This opens up a new area of study in NEC pathophysiology as well as a potential novel treatment strategy to prevent the development of NEC.

Intestinal alkaline phosphatase administration in newborns is protective of gut barrier function in a neonatal necrotizing enterocolitis rat model

BACKGROUND Previously, we have shown that supplementation of intestinal alkaline phosphatase (IAP) decreased severity of necrotizing enterocolitis (NEC)-associated intestinal injury. We hypothesized that IAP administration is protective of intestinal epithelial barrier function in a dose-dependent manner. METHODS Control rat pups were vaginally delivered and breast-fed. Premature rats were divided into 4 groups: formula fed with lipopolysaccharide and hypoxia (NEC) or additional daily bovine IAP 40, 4, or 0.4 U/kg (NEC + IAP 40 U, IAP 4 U, or IAP 0.4 U). RESULTS Necrotizing enterocolitis is associated with decreased IAP protein expression and activity. Supplemental IAP increases IAP activity in intestinal homogenates and decreased NEC injury score in a dose-dependent manner. Intestinal injury as measured by fluorescein isothiocyanate-dextran flux from ileal loops showed increased permeability vs control, but supplemental IAP reversed this. Tight junction proteins claudin-1, claudin-3, occludin, and zonula occludin 1 were elevated in the NEC and IAP-treated groups with differences in expression patterns. No differences in messenger RNA levels were observed on postinjury day 3. Intestinal alkaline phosphatase administration decreases intestinal NEC injury in a dose-dependent manner. CONCLUSION Early enteral supplemental IAP may reduce NEC-related injury and may be useful for preserving the intestinal epithelial barrier function.

Mesenteric Nitric Oxide and Superoxide Production in Experimental Necrotizing Enterocolitis

BACKGROUND A proposed mechanism of intestinal injury in necrotizing enterocolitis (NEC) involves vascular dysfunction through altered nitric oxide synthase (NOS) activity. We hypothesize that this dysfunction results in an imbalance in nitric oxide (*NO) and superoxide (O(2)(*-)) production by the intestinal vascular endothelium, which contributes to the intestinal injury seen in NEC. MATERIALS AND METHODS Neonatal rat pups were divided into two groups. Control pups were breast fed and housed with their mother. Experimental NEC pups were housed separately and either exposed to formula feeding and 5% to 10% hypoxia alone (FF/H) or with the addition of lipopolysaccharide (FF/H/LPS). Mesenteries from each group were analyzed for *NO and O(2)(*-) production with and without NOS inhibition by N(G)-monomethyl-L-arginine (L-NMMA). Western blot analysis for eNOS, phosphorylated eNOS (phospho-eNOS), and inducible NOS (iNOS) was performed, and each terminal ileum was graded for intestinal injury by histology. RESULTS Histology revealed mild intestinal injury (grade 1-2 on a 4-point scale) in the FF/H group and severe injury (grade 3-4) in the FF/H/LPS group. The FF/H cohort had significantly increased *NO and lower O(2)(*-) production, while the FF/H/LPS group shifted to significantly decreased *NO and increased O(2)(*-) production. L-NMMA inhibited >50% of O(2)(*-) production in all three groups but only inhibited *NO production in control and FF/H pups. Western blot analysis revealed increased levels of phospho-eNOS in FF/H pups and increased iNOS in FF/H/LPS pups. CONCLUSIONS This study demonstrates in the progression of NEC, intestinal ischemia is associated with a shift from *NO to O(2)(*-) production, which is NOS-dependent. Potentially greater injury results from impaired vasodilatation and over-production of reactive oxygen species.

Slow channel calcium inhibition blocks proinflammatory gene signaling and reduces macrophage responsiveness

BACKGROUND This study investigates the possible intracellular mechanisms responsible for calcium antagonist protection in tissue-fixed macrophages, a central modulator of the proinflammatory phenotype. METHODS Rabbit alveolar macrophages were exposed to lipopolysaccharide in the presence of different specific calcium antagonists. Cellular and nuclear protein were extracted and analyzed by Western blot for the phosphorylated forms of PYK2, ERK 1/2, and p38, and nuclear translocation of NF-kappaB and AP-1. Tumor necrosis factor-alpha (TNF-alpha) expression was measured by an L929 bioassay on cellular supernatants. Statistical analysis was performed by unpaired Students t tests. RESULTS Cells pretreated with 100 to 500 micromol/L of diltiazem or 50 to 100 micromol/L of verapamil, both slow channel calcium blockers, led to dose-dependent reductions in lipopolysaccharide-induced PYK2 and ERK 1/2 phosphorylation, and nuclear translocation of AP-1 when compared with controls (p < 0.05). Neither inhibitor had any significant effect on p38 or NF-kappaB translocation. EGTA an extracellular calcium chelator, had no significant effect on any intracellular process studied. A dose-dependent reduction in TNF-alpha production was demonstrated with diltiazem and verapamil (p < 0.05), with no effect induced by EGTA. CONCLUSION Slow channel calcium influx is essential for optimal intracellular signaling through PYK2 and ERK 1/2. This reduced intracellular signaling correlated with reduced AP-1 translocation and TNF-alpha production. Extracellular calcium chelation had no significant effect on intracellular signaling or TNF-alpha production. This study further elucidates the protective mechanism of action of calcium channel blockade by diltiazem and verapamil by reducing intracellular calcium release and down-regulating the excessive proinflammatory phenotype.

Operative vs nonoperative management for blunt pancreatic transection in children: multi-institutional outcomes.

BACKGROUND The management of traumatic pancreatic transection remains controversial. STUDY DESIGN A multi-institutional review from 1995 to 2012 was conducted comparing operative with nonoperative management for grades II and III blunt pancreatic injuries in patients younger than 18 years. RESULTS Fourteen pediatric trauma centers participated, yielding 167 patients; 57 underwent distal pancreatectomy and 95 were managed nonoperatively. Fifteen patients treated with operative drain placement only were studied separately. Patients undergoing resection had a shorter time to goal oral feeds (7.8 ± 0.7 days vs 15.1 ± 2.5 days; p = 0.007) and a lower rate of pseudocyst formation (0% vs 18%; p = 0.001). Pseudocyst formation resulted in a greater need for endoscopic and interventional radiologic procedures (26% vs 2%; p = 0.002) in the nonoperative group, as well as a longer time to complete resolution (38.6 ± 6.4 days vs 22.6 ± 5.0 days; p = 0.05) compared with resection. When looking at those patients with clear evidence of main duct injury at presentation, those undergoing resection also had fewer complications (33% vs 61%; p = 0.05) and fewer total days in-hospital (12.6 ± 8.4 days vs 17.5 ± 9.7 days; p = 0.04) compared with nonoperative management. CONCLUSIONS In children with blunt pancreatic injury, distal pancreatectomy is superior to nonoperative management with more rapid resumption of diet, fewer repeat interventions, and a shorter period to complete resolution. When the main duct is involved, the benefits to operative resection also include lower morbidity and fewer days of hospitalization. Therefore, assessing the status of the pancreatic duct is paramount in determining management.

Conservative management of giant omphalocele with topical povidone-iodine and its effect on thyroid function

PURPOSE The aim of the study was to evaluate topical povidone-iodine as a bridge to delayed fascial closure of giant omphaloceles with emphasis on its effect on thyroid function. METHODS Newborns from a single institution with giant omphaloceles treated with topical povidone-iodine for a 10-year period were reviewed. Recorded data included sex, associated anomalies, length of stay, frequency of povidone-iodine application, thyroid function tests, frequency of laboratory draws, and thyroid supplementation administration. RESULTS Six neonates with giant omphaloceles were treated with povidone-iodine. Thyroid function testing occurred weekly as inpatients and monthly as outpatients, with abnormal values normalized by the subsequent laboratory draw. One patient demonstrated persistent hypothyroidism and subsequently died secondary to cardiac complications, but this infants newborn thyroid screening suggested congenital hypothyroidism. Five patients remained euthyroid and ultimately achieved fascial closure without the need for a prosthetic implant. None of these patients had abnormal outpatient thyroid tests nor did they require thyroid hormone supplementation. CONCLUSION Topical povidone-iodine promotes escharification and epithelialization of the omphalocele sac. Because transient hypothyroidism may occur, thyroid function studies may guide inpatient therapy. After sac desiccation, systemic effects of iodine are minimal and thyroid supplementation is not necessary. Topical povidone-iodine is an effective initial strategy for giant omphaloceles and does not produce clinically significant hypothyroidism.