Transition from parenteral to enteral nutrition induces immediate diet-dependent gut histological and immunological responses in preterm neonates

Necrotizing enterocolitis (NEC) in preterm infants develops very rapidly from a mild intolerance to enteral feeding into intestinal mucosal hemorrhage, inflammation, and necrosis. We hypothesized that immediate feeding-induced gut responses precede later clinical NEC symptoms in preterm pigs. Fifty-six preterm pigs were fed total parenteral nutrition (TPN) for 48 h followed by enteral feeding for 0, 8, 17, or 34 h with either colostrum (Colos, n = 20) or formula (Form, n = 31). Macroscopic NEC lesions were detected in Form pigs throughout the enteral feeding period (20/31, 65%), whereas most Colos pigs remained protected (1/20, 5%). Just 8 h of formula feeding induced histopathological lesions, as evidenced by capillary stasis and necrosis, epithelial degeneration, edema, and mucosal hemorrhage. These immediate formula-induced changes were paralleled by decreased digestive enzyme activities (lactase and dipeptidylpeptidase IV), increased nutrient fermentation, and altered expression of innate immune defense genes such as interleukins (IL-1α, IL-6, IL-18), nitric oxide synthetase, tight junction proteins (claudins), Toll-like receptors (TLR-4), and TNF-α. In contrast, the first hours of colostrum feeding induced no histopathological lesions, increased maltase activity, and induced changes in gene expressions related to tissue development. Total bacterial density was high after 2 days of parenteral feeding and was not significantly affected by diet (colostrum, formula) or length of enteral feeding (8-34 h), except that a few bacterial groups (Clostridium, Enterococcus, Streptococcus species) increased with time. We conclude that a switch from parenteral to enteral nutrition rapidly induces diet-dependent histopathological, functional, and proinflammatory insults to the immature intestine. Great care is required when introducing enteral feeds to TPN-fed preterm infants, particularly when using formula, because early feeding-induced insults may predispose to NEC lesions that are difficult to revert by later dietary or medical interventions.     

Temporal proteomic analysis of intestine developing necrotizing enterocolitis following enteral formula feeding to preterm pigs

Necrotizing enterocolitis (NEC), a serious gastrointestinal inflammatory disease, frequently occurs in preterm neonates that fail to adapt to enteral nutrition. A temporal gel-based proteomics study was performed on porcine intestine with NEC lesions induced by enteral formula feeding. Functional assignment of the differentially expressed proteins revealed that important cellular functions, such as the heat shock response, protein processing; and purine, nitrogen, energy metabolism, were possible involved in the early progression of NEC.     

Enteral bile acid treatment improves parenteral nutrition-related liver disease and intestinal mucosal atrophy in neonatal pigs

Total parenteral nutrition (TPN) is essential for patients with impaired gut function but leads to parenteral nutrition-associated liver disease (PNALD). TPN disrupts the normal enterohepatic circulation of bile acids, and we hypothesized that it would decrease intestinal expression of the newly described metabolic hormone fibroblast growth factor-19 (FGF19) and also glucagon-like peptides-1 and -2 (GLP-1 and GLP-2). We tested the effects of restoring bile acids by treating a neonatal piglet PNALD model with chenodeoxycholic acid (CDCA). Neonatal pigs received enteral feeding (EN), TPN, or TPN + CDCA for 14 days, and responses were assessed by serum markers, histology, and levels of key regulatory peptides. Cholestasis and steatosis were demonstrated in the TPN group relative to EN controls by elevated levels of serum total and direct bilirubin and also bile acids and liver triglyceride (TG) content. CDCA treatment improved direct bilirubin levels by almost fourfold compared with the TPN group and also normalized serum bile acids and liver TG. FGF19, GLP-1, and GLP-2 were decreased in plasma of the TPN group compared with the EN group but were all induced by CDCA treatment. Intestinal mucosal growth marked by weight and villus/crypt ratio was significantly reduced in the TPN group compared with the EN group, and CDCA treatment increased both parameters. These results suggest that decreased circulating FGF19 during TPN may contribute to PNALD. Moreover, we show that enteral CDCA not only resolves PNALD but acts as a potent intestinal trophic agent and secretagogue for GLP-2.     

Elective cesarean delivery affects gut maturation and delays microbial colonization but does not increase necrotizing enterocolitis in preterm pigs

Although preterm birth and formula feeding increase the risk of necrotizing enterocolitis (NEC), the influences of cesarean section (CS) and vaginal delivery (VD) are unknown. Therefore, gut characteristics and NEC incidence and severity were evaluated in preterm pigs (92% gestation) delivered by CS or VD. An initial study showed that newborn CS pigs (n = 6) had decreased gastric acid secretion, absorption of intact proteins, activity of brush-border enzymes and pancreatic hydrolases, plasma cortisol, rectal temperature, and changes in blood chemistry, indicating impaired respiratory function, compared with VD littermates (n = 6). In a second experiment, preterm CS (n = 16) and VD (n = 16) pigs were given total parenteral nutrition (36 h) then fed porcine colostrum (VD-COL, n = 6; CS-COL, n = 6) or infant milk formula (VD-FORM, n = 10; CS-FORM, n = 10) for 2 days. Across delivery, FORM pigs showed significantly higher NEC incidence, tissue proinflammatory cytokines (IFN-gamma and IL-6), Clostridium colonization, and impaired intestinal function, compared with COL pigs. NEC incidence was equal for CS (6/16) and VD (6/16) pigs, CS pigs had decreased bacterial diversity and density, higher villus heights, and increased brush-border enzyme activities (lactase, aminopeptidases) compared with VD pigs. In particular, VD-FORM pigs showed reduced mucosal proportions, reduced lactase and aminopeptidases, and increased proinflammatory cytokine IL-6 compared with CS-FORM (P < 0.06). Despite the initial improvement of intestinal and metabolic functions following VD, gut function, and inflammation were similar, or more negatively affected in VD neonates than CS neonates. Both delivery modes exhibited positive and negative influences on the preterm gut, which may explain the similar NEC incidence.     

Plasma concentration of intestinal- and liver-FABP in neonates suffering from necrotizing enterocolitis and in healthy preterm neonates

Both early diagnostic and prognostic assessment of the acute abdomen in preterm infants are hampered by the lack of a sensitive and specific parameter for intestinal injury. In this prospective clinical study we wanted to estimate the value of intestinal (I-) and liver (L-) fatty acid binding protein (FABP) in diagnosing necrotizing enterocolitis (NEC). Using highly sensitive and specific sandwich ELISAs which employ recombinant human I- and L-FABP as standard proteins (limit of detection 0.1 ng/ml plasma), the L-FABP concentration (median 7.6 ng/ml) was determined to be about 3 fold that of I-FABP (median 2.52 ng/ml) in plasma of healthy preterm infants. I- and L-FABP concentrations significantly increased with birth weight (1.6 and 5.0 ng/ml per kg, respectively). At onset of symptoms, I-FABP concentration was significantly higher in infants who later developed severe NEC compared to healthy infants and those, whose illness remained confined to stage I or II. L-FABP was significantly elevated compared to the control group at onset of symptoms regardless of the further course of NEC. In conclusion, I-FABP appears to be a specific parameter for early detection of intestinal injury leading to severe NEC stage III. L-FABP, however, is a promising sensitive marker even for stage I of NEC.     

Nutritional modulation of the gut microbiota and immune system in preterm neonates susceptible to necrotizing enterocolitis

The gastrointestinal inflammatory disorder, necrotizing enterocolitis (NEC), is among the most serious diseases for preterm neonates. Nutritional, microbiological and immunological dysfunctions all play a role in disease progression but the relationship among these determinants is not understood. The preterm gut is very sensitive to enteral feeding which may either promote gut adaptation and health, or induce gut dysfunction, bacterial overgrowth and inflammation. Uncontrolled inflammatory reactions may be initiated by maldigestion and impaired mucosal protection, leading to bacterial overgrowth and excessive nutrient fermentation. Tumor necrosis factor alpha, toll-like receptors and heat-shock proteins are identified among the immunological components of the early mucosal dysfunction. It remains difficult, however, to distinguish the early initiators of NEC from the later consequences of the disease pathology. To elucidate the mechanisms and identify clinical interventions, animal models showing spontaneous NEC development after preterm birth coupled with different forms of feeding may help. In this review, we summarize the literature and some recent results from studies on preterm pigs on the nutritional, microbial and immunological interactions during the early feeding-induced mucosal dysfunction and later NEC development. We show that introduction of suboptimal enteral formula diets, coupled with parenteral nutrition, predispose to disease, while advancing amounts of mother's milk from birth (particularly colostrum) protects against disease. Hence, the transition from parenteral to enteral nutrition shortly after birth plays a pivotal role to secure gut growth, digestive maturation and an appropriate response to bacterial colonization in the sensitive gut of preterm neonates.     

Association of cholelithiasis with total parenteral nutrition and fasting in a preterm infant.

Cholelithiasis is uncommon in infants. A case of cholelithiasis in a preterm boy who required total parenteral nutrition (TPN) because of prolonged fasting is described. The diagnosis was confirmed by ultrasonography. Despite cholestasis, reintroduction of oral feeding and discontinuation of TPN resulted in the spontaneous disappearance of the gallstones.     

Enteral nutrients potentiate glucagon-like peptide-2 action and reduce dependence on parenteral nutrition in a rat model of human intestinal failure

Glucagon-like peptide-2 (GLP-2) is a nutrient-dependent, proglucagon-derived gut hormone that shows promise for the treatment of short bowel syndrome (SBS). Our objective was to investigate how combination GLP-2 + enteral nutrients (EN) affects intestinal adaption in a rat model that mimics severe human SBS and requires parenteral nutrition (PN). Male Sprague-Dawley rats were assigned to one of five groups and maintained with PN for 18 days: total parenteral nutrition (TPN) alone, TPN + GLP-2 (100 μg·kg(-1)·day(-1)), PN + EN + GLP-2(7 days), PN + EN + GLP-2(18 days), and a nonsurgical oral reference group. Animals underwent massive distal bowel resection followed by jejunocolic anastomosis and placement of jugular catheters. Starting on postoperative day 4, rats in the EN groups were allowed ad libitum access to EN. Groups provided PN + EN + GLP-2 had their rate of PN reduced by 0.25 ml/day starting on postoperative day 6. Groups provided PN + EN + GLP-2 demonstrated significantly greater body weight gain with similar energy intake and a safe 80% reduction in PN compared with TPN ± GLP-2. Groups provided PN + EN + GLP-2 for 7 or 18 days showed similar body weight gain, residual jejunal length, and digestive capacity. Groups provided PN + EN + GLP-2 showed increased jejunal GLP-2 receptor (GLP-2R), insulin-like growth factor-I (IGF-I), and IGF-binding protein-5 (IGFBP-5) expression. Treatment with TPN + GLP-2 demonstrated increased jejunal expression of epidermal growth factor. Cessation of GLP-2 after 7 days with continued EN sustained the majority of intestinal adaption and significantly increased expression of colonic proglucagon compared with PN + EN + GLP-2 for 18 days, and increased plasma GLP-2 concentrations compared with TPN alone. In summary, EN potentiate the intestinotrophic actions of GLP-2 by improving body weight gain allowing for a safe 80% reduction in PN with increased jejunal expression of GLP-2R, IGF-I, and IGFBP-5 following distal bowel resection in the rat.     

Potential prophylactic value of bovine colostrum in necrotizing enterocolitis in neonates: an in vitro study on bacterial attachment, antibody levels and cytokine production

Necrotizing enterocolitis (NEC) is an important disease of low birth-weight neonates. The immaturity of the gut mucosa may result in close contact between the host epithelium and microorganisms which are normally confined to the gut lumen. Damage of the mucosa due to endotoxin, cytokine production or other factors is believed to then occur. The aim of this study was to determine whether spray-dried bovine colostrum demonstrated potential in vitro as a prophylactic for NEC. Antiadherence was measured using a tissue culture assay and antibody levels against Enterobacteriaceae were determined by ELISA. The effect of bovine colostrum on the production of cytokines implicated in NEC was determined by a multiplex bead assay. Enterobacter cloacae, Klebsiella oxytoca, Escherichia coli, Serratia marcescens and Klebsiella pneumoniae ssp. pneumoniae were common in both NEC positive and NEC negative infants and IgA and IgG1 antibodies to these species were present in the bovine colostrum. Pretreatment with bovine colostrum produced a significant decrease (P<0.001) in attachment of bacteria to HT-29 cells. Bovine colostrum significantly increased the production of IL-8 in HT-29 cells and IL-8, IL-6 and TNF-alpha in THP-1 cells (P<0.001). The potential of bovine colostrum to increase the production of inflammatory mediators could limit its usefulness.     

Neutrophil extracellular trap inhibition increases inflammation,bacteraemia and mortality in murine necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease affecting primarily premature infants. The disease is characterized by intestinal inflammation and leucocyte infiltration, often progressing to necrosis, perforation, systemic inflammatory response and death. Neutrophil extracellular traps (NETs), denoting nuclear DNA, histone and antimicrobial protein release, have been suggested to play a role in NEC. This study aimed to determine the role of NETs in NEC and explore the effect of chloramidine, a NET inhibitor, on a murine NEC-like intestinal injury model. Blood and intestinal tissues were collected from infants diagnosed with ≥ Stage II NEC, and levels of nucleosomes and NETs, respectively, were compared with those of case-matched controls. In mice, NEC was induced with dithizone/Klebsiella, and mice in the treatment group received 40 mg/kg chloramidine. Bacterial load, intestinal histology, plasma myeloperoxidase and cytokine levels, and immunofluorescent staining were compared with controls. Nucleosomes were significantly elevated in both human and mouse NEC plasma, whereas NET staining was only present in NEC tissue in both species. Chloramidine treatment increased systemic inflammation, bacterial load, organ injury and mortality in murine NEC. Taken together, our findings suggest that NETs are critical in the innate immune defence during NEC in preventing systemic bacteraemia.     

Decline in intestinal mucosal IL-10 expression and decreased intestinal barrier function in a mouse model of total parenteral nutrition

Loss of intestinal epithelial barrier function (EBF) is a major problem associated with total parenteral nutrition (TPN) administration. We have previously identified intestinal intraepithelial lymphocyte (IEL)-derived interferon-gamma (IFN-gamma) as a contributing factor to this barrier loss. The objective was to determine whether other IEL-derived cytokines may also contribute to intestinal epithelial barrier breakdown. C57BL6J male mice received TPN or enteral nutrition (control) for 7 days. IEL-derived interleukin-10 (IL-10) was then measured. A significant decline in IEL-derived IL-10 expression was seen with TPN administration, a cytokine that has been shown in vitro to maintain tight junction integrity. We hypothesized that this change in IEL-derived IL-10 expression could contribute to TPN-associated barrier loss. An additional group of mice was given exogenous recombinant IL-10. Ussing chamber experiments showed that EBF markedly declined in the TPN group. TPN resulted in a significant decrease of IEL-derived IL-10 expression. The expression of several tight junction molecules also decreased with TPN administration. Exogenous IL-10 administration in TPN mice significantly attenuated the TPN-associated decline in zonula occludens (ZO)-1, E-cadherin, and occludin expression, as well as a loss of intestinal barrier function. TPN administration led to a marked decline in IEL-derived IL-10 expression. This decline was coincident with a loss of intestinal EBF. As the decline was partially attenuated with the administration of exogenous IL-10, our findings suggest that loss of IL-10 may be a contributing mechanism to TPN-associated epithelial barrier loss.     

南京地区早产儿坏死性小肠结肠炎病原体特点和益生菌干预效果及预后影响因素分析

目的探究南京地区早产儿坏死性小肠结肠炎(NEC)的病原体特点,并观察益生菌辅助治疗对患儿血清炎症因子水平的影响,分析影响NEC早产儿预后的因素。方法将2014年5月至2018年12月于东南大学附属中大医院就诊的87例NEC早产儿选为研究对象,分析其病原体特点,观察不同治疗方法对患儿血清炎症因子的影响,并通过Logistic回归分析影响NEC患儿预后的危险因素。结果革兰阴性菌是NEC早产儿的主要致病菌,主要包括肺炎克雷伯菌(25株,27.78%)和大肠埃希菌(21株,23.33%)。肺炎克雷伯菌和大肠埃希菌对美罗培南、亚胺培南/西司他丁、哌拉西林/他唑巴坦、多粘菌素和阿米卡星敏感。益生菌干预后的NEC早产儿血清IL-1β、IL-10、TNF-α水平及TLR4的表达均显著低于常规治疗患儿(均P0.05)。发病前3 d内输血、新生儿窒息、益生菌干预、非营养性吮吸、腹膜炎、败血症、加奶速度、PDA均是影响NEC早产儿预后的相关因素(均P0.05),其中发病前3 d内输血、新生儿窒息、加奶速度、PDA是影响NEC早产儿预后的独立危险因素(均P0.05)。结论革兰阴性菌是NEC早产儿的主要致病菌,益生菌干预可有效降低NEC早产儿血清炎症因子水平。对于NEC早产儿的治疗可选用碳青霉烯类、氨基糖苷类等抗生素,同时需关注影响NEC早产儿预后的独立危险因素。     

FRB domain of human TOR protein induces compromised proliferation and mitochondrial dysfunction in Leishmaniadonovani promastigotes

Visceral leishmaniasis (VL) or Kala-azar, the second-largest parasitic killer worldwide, is caused by Leishmania donovani. The drugs to treat VL are toxic and expensive. Moreover, their indiscriminate use gave rise to resistant strains. The high rate of parasite proliferation within the host macrophage cells causes pathogenesis. In the proliferative pathway, FRB domain of TOR protein is ubiquitously essential. Although orthologues of mTOR protein are reported in trypanosomatids and Leishmania but therein depth molecular characterization is yet to be done. Considerable protein sequence homology exists between the TOR of kinetoplastidas and mammals. Interestingly, exogenous human FRB domain was shown to block G1 to S transition in mammalian cancer cells. Thus, we hypothesized that expression of human FRB domain would inhibit the proliferation of Leishmaniadonovani. Indeed, promastigotes stably expressing wild type human FRB domain show 4.7 and 1.5 folds less intra- and extra-cellular proliferations than that of untransfected controls. They also manifested 2.65 times lower rate of glucose stimulated oxygen consumption. The activities of all respiratory complexes were compromised in the hFRB expressing promastigotes. In these cells, depolarized mitochondria were 2-fold more than control cells. However, promastigotes expressing its mutant version (Trp²⁰²⁷-Phe) has shown similar characteristics like untransfected cells. Thus, this study reveals greater insights on the conserved role of TOR in the regulation of the respiratory complexes in L. donovani. The slow growing variant of FRB expressing promastigotes will have great potential to be exploited as a prophylactic agent against leishmaniasis.     

Physiological effects of enteral and parenteral feeding on pancreaticobiliary secretion in humans

In the nutritional management of digestive disorders, it is important to know the relative secretory and metabolic responses to enteral and parenteral feeding. Twenty-seven healthy volunteers were studied while receiving either oral drinks or duodenal infusions of a complex formula diet, duodenal or intravenous infusions of elemental (protein as free amino acids, low fat) formulae, or saline. Pancreaticobiliary secretory responses were measured by nasoduodenal polyethylene glycol perfusion and aspiration, while monitoring blood hormone and nutrient levels. Diets were matched for protein (1.5 g x kg(-1) x d(-1)) and energy (40 kcal x kg(-1) x d(-1)). Compared with placebo, all oroenteral diets stimulated amylase, lipase, trypsin, and bile acid secretion and increased plasma concentrations of gastrin and cholecystokinin, whereas intravenous feeding did not. The complex formula produced a similar response whether given as drinks or duodenal infusions. Changing the duodenal formula to elemental reduced enzyme secretion by 50%, independently of CCK. Higher increases in plasma insulin, glucose, and amino acids were noted with intravenous feeding. Delivering food directly to the intestine by a feeding tube does not reduce pancreaticobiliary secretion. Enteral "elemental" formulae diminish, but only intravenous feeding avoids pancreatic stimulation. Intravenous administration impairs metabolic clearance.     

双歧杆菌对新生大鼠坏死性小肠结肠炎肠损伤及肠细胞凋亡影响

目的探讨添加双歧杆菌对新生鼠坏死性小肠结肠炎(necrotizing enterocolitis,NEC)模型肠损伤的保护作用。方法 32只新生SD大鼠按析因设计随机分成4组,每组动物8只。A1B1组为NEC模型组并添加双歧杆菌(109CFU/d),A1B2组为NEC模型组,但未添加双歧杆菌;A2B1组为对照组并添加双歧杆菌(109CFU/d),A2B2组为对照组,且未添加双歧杆菌。在出生48 h开始给予鼠配方奶人工喂养,100%氮气缺氧90 s,4℃冷刺激10 min,每天2次,连续3 d,建立新生大鼠NEC模型;在最后1次缺氧、冷刺激后24 h空腹断头处死小鼠,解剖留取十二指肠下端至直肠上端肠道组织,其中,回盲部近端肠管进行病理学检查及肠损伤评分,组织学评分≥2为NEC,其余肠管进行肠细胞凋亡率检测及电镜观察。采用流式细胞仪检测肠细胞凋亡率。SPSS 11.0统计学软件进行统计分析,α=0.05为显著性检验标准。结果造模后,A1B1组、A1B2组相继出现腹泻、腹胀、生长发育减慢和活动度减少,显微镜下可见肠黏膜坏死、黏膜下层出血以及肌肉层坏死等肠损伤表现,透射电镜显示肠黏膜出现大量凋亡细胞,形成凋亡小体,但A1B1组程度较轻。A1B1、A1B2、A2B1和A2B24组肠损伤组织病理评分(x±s)分别为2.04±0.52、3.38±0.55、0.33±0.36和0.38±0.33,肠细胞凋亡率分别为(23.97±10.48)%、(47.28±21.98)%、(11.42±4.75)%和(12.16±4.95)%;各组间肠损伤组织评分、肠细胞凋亡率差异有显著统计学意义(H分别为26.657、20.916,P均0.01);与A1B2组相比,A1B1组新生鼠肠损伤组织评分、肠细胞凋亡率均明显降低,但仍高于A2B1、A2B22个对照组(P均0.01);肠组织损伤评分值、肠细胞凋亡率均受到NEC造模和添加双歧杆菌两个因素的影响,NEC造模与补充双歧杆菌之间均存在交互作用。结论添加双歧杆菌可以降低新生鼠NEC肠损伤程度,可能通过抑制肠上皮细胞凋亡,从而降低新生大鼠发生NEC危险性。     

肠内营养联合益生菌对重症脑卒中伴胃肠功能障碍患者肠黏膜屏障的保护作用

目的 探讨肠内营养联合益生菌对重症脑卒中伴胃肠功能障碍患者肠黏膜屏障的保护作用,为该类患者的治疗提供参考.方法 选择2016年1月至2019年7月于我院住院的84例重症脑卒中伴胃肠功能障碍患者为研究对象,根据肠内营养支持时是否添加益生菌将患者分为联用组和单用组,各42例.两组患者均予控制颅内压、营养脑细胞、保护胃黏膜和...