Background: Factors that regulate intestinal stem cell (ISC) proliferation and apoptosis are unknown. Results: Toll-like receptor 4 (TLR4) is expressed on ISCs and regulates their proliferation and apoptosis, which is critical in the pathogenesis of necrotizing enterocolitis (NEC). Conclusion: TLR4 regulates ISC proliferation and apoptosis. Significance: This is the first study showing that ISC regulation by microbial receptors contributes to NEC pathogenesis.

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by severe intestinal necrosis and for which breast milk represents the most effective protective strategy. Previous studies have revealed a critical role for the lipopolysaccharide receptor toll-like receptor 4 (TLR4) in NEC development through its induction of mucosal injury, yet the reasons for which intestinal ischemia in NEC occurs in the first place remain unknown. We hypothesize that TLR4 signaling within the endothelium plays an essential role in NEC development by regulating perfusion to the small intestine via the vasodilatory molecule endothelial nitric oxide synthase (eNOS). Using a unique mouse system in which we selectively deleted TLR4 from the endothelium, we now show that endothelial TLR4 activation is required for NEC development and that endothelial TLR4 activation impairs intestinal perfusion without effects on other organs and reduces eNOS expression via activation of myeloid differentiation primary response gene 88. NEC severity was significantly increased in eNOS −/− mice and decreased upon administration of the phosphodiesterase inhibitor sildenafil, which augments eNOS function. Strikingly, compared with formula, human and mouse breast milk were enriched in sodium nitrate-a precursor for enteral generation of nitrite and nitric oxide-and repletion of formula with sodium nitrate/nitrite restored intestinal perfusion, reversed the deleterious effects of endothelial TLR4 signaling, and reduced NEC severity. These data identify that endothelial TLR4 critically regulates intestinal perfusion leading to NEC and reveal that the protective properties of breast milk involve enhanced intestinal microcirculatory integrity via augmentation of nitrate-nitrite-NO signaling.neonatal inflammation | prematurity | infant formula | neonatal nutrition | sepsis N ecrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in the premature infant and is gradually increasing in frequency (1). The defining pathological feature of NEC is the presence of patchy areas of ischemia and necrosis of the small and large intestine (2). Although prematurity is the leading risk factor for NEC development, breast milk administration has been identified as the most important protective strategy (3). Importantly, the mechanisms that lead to the acute development of intestinal necrosis in the premature intestine and factors within breast milk that may prevent NEC remain largely unexplored.In seeking to understand the underlying biological mechanisms that lead to NEC, we and others have identified a critical role for the innate immune receptor toll-like receptor 4 (TLR4) in NEC pathogenesis, because mice deficient in TLR4 showed reduced mucosal inflammation and reduced intestinal necrosis in experimental NEC (4, 5). Microcirculatory perfusion of the premature intestine is primarily regulated by the vasodilator nitric oxide (NO), which is generated through the activity of endothelial NO synthase (eNOS) (6). ...

The fetal intestinal mucosa is characterized by elevated Toll-like receptor 4 (TLR4) expression, which can lead to the development of necrotizing enterocolitis (NEC)-a devastating inflammatory disease of the premature intestine-upon exposure to microbes. To define endogenous strategies that could reduce TLR4 signaling, we hypothesized that amniotic fluid can inhibit TLR4 signaling within the fetal intestine and attenuate experimental NEC, and we sought to determine the mechanisms involved. We show here that microinjection of amniotic fluid into the fetal (embryonic day 18.5) gastrointestinal tract reduced LPS-mediated signaling within the fetal intestinal mucosa. Amniotic fluid is abundant in EGF, which we show is required for its inhibitory effects on TLR4 signaling via peroxisome proliferator-activated receptor, because inhibition of EGF receptor (EGFR) with cetuximab or EGF-depleted amniotic fluid blocked the inhibitory effects of amniotic fluid on TLR4, whereas amniotic fluid did not prevent TLR4 signaling in EGFR-or peroxisome proliferator-activated receptor γ-deficient enterocytes or in mice deficient in intestinal epithelial EGFR, and purified EGF attenuated the exaggerated intestinal mucosal TLR4 signaling in wild-type mice. Moreover, amniotic fluid-mediated TLR4 inhibition reduced the severity of NEC in mice through EGFR activation. Strikingly, NEC development in both mice and humans was associated with reduced EGFR expression that was restored upon the administration of amniotic fluid in mice or recovery from NEC in humans, suggesting that a lack of amniotic fluid-mediated EGFR signaling could predispose to NEC. These findings may explain the unique susceptibility of premature infants to the development of NEC and offer therapeutic approaches to this devastating disease. N ecrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants (1). Although the underlying etiology of NEC remains incompletely understood, recent studies have identified a critical role for the LPS receptor, Toll-like receptor 4 (TLR4) in its pathogenesis. TLR4 activation within the intestinal epithelium leads to increased mucosal injury through accelerated enterocyte apoptosis as well as reduced healing through impaired intestinal restitution and proliferation (2), and mice lacking TLR4 (2, 3) show reduced NEC severity as the result of reduced injury and enhanced healing (4). These findings suggest that NEC develops in part in response to exaggerated TLR4 signaling in the intestinal mucosa and, by extension, that strategies may exist within the intestine that can limit TLR4 signaling and the propensity for NEC development. Given that the premature intestine is bathed in amniotic fluid throughout its development and that an abrupt lack of exposure to amniotic fluid is a natural consequence of early delivery, we hypothesized that amniotic fluid may exert a restraining influence on TLR4 signaling and that the absence of the anti-TLR4 signaling effects of the amniotic fluid places the p...

Necrotizing enterocolitis (NEC) develops in response to elevated Toll-like receptor-4 (TLR4) signaling in the newborn intestinal epithelium, and is characterized by TLR4-mediated inhibition of enterocyte migration and reduced mucosal healing. The downstream processes by which TLR4 impairs mucosal healing remain incompletely understood. In other systems, TLR4 induces autophagy, an adaptive response to cellular stress. We now hypothesize that TLR4 induces autophagy in enterocytes, and that TLR4-induced autophagy plays a critical role in NEC development. Using mice selectively lacking TLR4 in enterocytes(TLR4ΔIEC), and in TLR4-deficient cultured enterocytes, we now show that TLR4 activation induces autophagy in enterocytes. Immature mouse and human intestine showed increased expression of autophagy genes compared to full-term controls, and NEC development in both mouse and human was associated with increased enterocyte autophagy. Importantly, using mice in which we selectively deleted the autophagy gene ATG7 from the intestinal epithelium (ATG7ΔIEC), the induction of autophagy was determined to be required for and not merely a consequence of NEC, as ATG7ΔIEC mice were protected from NEC development. In defining the mechanisms involved, TLR4-induced autophagy led to impaired enterocyte migration both in vitro and in vivo, which in cultured enterocytes required the induction of RhoA-mediated stress fibers. These findings depart from current dogma in the field by identifying a unique effect of TLR4-induced autophagy within the intestinal epithelium in the pathogenesis of NEC, and identify that the negative consequences of autophagy on enterocyte migration play an essential role in its development.

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal-related mortality in premature infants, and it develops under conditions of exaggerated TLR4 signaling in the newborn intestinal epithelium. Because NEC does not develop spontaneously, despite the presence of seemingly tonic stimulation of intestinal TLR4, we hypothesized that mechanisms must exist to constrain TLR4 signaling that become diminished during NEC pathogenesis and focused on the intracellular stress response protein and chaperone heat shock protein-70 (Hsp70). We demonstrate that the induction of intracellular Hsp70 in enterocytes dramatically reduced TLR4 signaling, as assessed by LPS-induced NF-κB translocation, cytokine expression, and apoptosis. These findings were confirmed in vivo, using mice that either globally lacked Hsp70 or overexpressed Hsp70 within the intestinal epithelium. TLR4 activation itself significantly increased Hsp70 expression in enterocytes, which provided a mechanism of autoinhibition of TLR4 signaling in enterocytes. In seeking to define the mechanisms involved, intracellular Hsp70-mediated inhibition of TLR4 signaling required both its substrate-binding EEVD domain and association with the cochaperone CHIP, resulting in ubiquitination and proteasomal degradation of TLR4. The expression of Hsp70 in the intestinal epithelium was significantly decreased in murine and human NEC compared with healthy controls, suggesting that loss of Hsp70 protection from TLR4 could lead to NEC. In support of this, intestinal Hsp70 overexpression in mice and pharmacologic upregulation of Hsp70 reversed TLR4-induced cytokines and enterocyte apoptosis, as well as prevented and treated experimental NEC. Thus, a novel TLR4 regulatory pathway exists within the newborn gut involving Hsp70 that may be pharmacologically activated to limit NEC severity.

Propensity score matching yielded 116 pairs of non-hemiarch patients versus 116 hemiarch patients. Within the propensity score-matched cohort, there were no differences in postoperative stroke (1.7% versus 3.4%; p = 0.41), new postoperative dialysis (6.0% versus 5.2%; p = 0.78), postoperative renal insufficiency (27.6% versus 19.8%; p = 0.16), 30-day mortality (2.6% versus 3.4%; p = 0.701), or 1-year mortality (4.3% versus 4.3%; p = 1.00) CONCLUSIONS: Hemiarch replacement using DHCA with RCP does not increase the risk of operative complications compared with a normothermic, clamped-distal aortic anastomosis, and therefore its use should not be limited when planning complex multiprocedural reconstructions during elective ascending thoracic aortic replacement with concomitant cardiac surgery.

The 1:1 complex of 1,2-ethanediol with dimethyl sulfoxide was studied using density functional theory. A network of three hydrogen bonds holds the complex together, including two in which each methyl group donates to the same hydroxyl oxygen. Four lines of evidence support the existence of methyl-donated hydrogen bonds. The interaction energy is 36 +/- 5 kJ/mol using Becke's three parameter hybrid theory with the 1991 nonlocal correlation functional of Perdew and Wang, and a moderately large basis set (B3PW91/6-311++G**//B3PW91/6-31+G**). To determine the energy of each hydrogen bond, a relaxed potential energy scan was performed in a smaller basis set to break the weaker hydrogen bonds by forced systematic rotation of the methyl groups. Two cross-checking analyses show cooperative effects that cause individual hydrogen bond energies in the network to be nonadditive. When one methyl hydrogen bond is broken, the remaining interactions stabilize the complex by storing an additional 2-3 kJ/mol. With all hydrogen bonds intact, the O[bond]H...O[bond]S hydrogen bond contributes 26 +/- 2 kJ/mol stability, and each weak methyl bond stores 5 +/- 2 kJ/mol.

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