Arginine-enriched total parenteral nutrition improves survival in peritonitis by normalizing NFkappaB activation in peritoneal resident and exudative leukocytes.

BACKGROUND: Enteral nutrition maintains peritoneal defense more effectively than parenteral nutrition, at least partly by preserving NFkappaB activation in peritoneal cells. We hypothesized that arginine (ARG)-enriched parenteral nutrition would normalize NFkappaB activation in peritoneal leukocytes, thereby improving the survival of peritonitis models. METHODS: A total of 105 ICR mice were randomized to chow (n=33), IV feeding of a standard (STD) total parenteral nutrition (STD-TPN) solution (ARG 0.3%) (n=35), or 1% ARG-TPN solution (n=37), and fed accordingly for 5 days.Experiment 1: Thirty mice were used for intranuclear NFkappaB measurement in peritoneal resident cells (PRCs). After incubation with lipopolysaccharide (LPS: 0, 1, 10 microg/mL) for 30 minutes, intranuclear NFkappaB activity was examined by laser scanning cytometry.Experiment 2: Fifty-one mice were injected with 2 mL of 1% glycogen intraperitoneally. Peritoneal exudative cells (PECs) were obtained at 2 or 4 hours after glycogen administration for NFkappaB measurement. Cytokine (TNFalpha, IL-10) levels in peritoneal lavage fluid were also determined by ELISA.Experiment 3: After 5 days of feeding, 24 mice underwent cecal ligation and puncture. Survival was observed up to 5 days. RESULTS: Experiment 1: Intranuclear NFkappaB levels in the ARG-TPN and chow groups increased dose-dependently after LPS stimulation, while the level in the STD-TPN group was unchanged.Experiment 2: Intranuclear NFkappaB level was significantly higher at 2 hours in the chow than in the STD-TPN group, whereas in the ARG-TPN mice the level was midway between those of the chow and STD-TPN groups. TNFalpha and IL-10 levels of the chow group were significantly higher than those of STD-TPN mice at 2 hours. TNFalpha was significantly higher in the ARG-TPN group than in the STD-TPN group, but the IL-10 level showed no recovery.Experiment 3: Survival times were significantly reduced in the STD-TPN as compared with the chow group, though ARG-TPN improved survival. CONCLUSION: ARG-enriched TPN is a surrogate for enteral feeding which maintains peritoneal defense by preserving NFkappaB activation in peritoneal resident and exudative leukocytes.

Parenteral feeding depletes pulmonary lymphocyte populations.

BACKGROUND: The effect of parenteral nutrition (PN) on lymphocyte mass in the lung is unknown, but reduced mucosal lymphocytes are hypothesized to play a role in the reduced immunoglobulin A-mediated immunity in both gut and lung. The ability to transfer and track cells between mice may allow study of diet-induced mucosal immune function. The objectives of this study are to characterize lung T-cell populations following parenteral feeding and to study distribution patterns of transferred donor lung T cells in recipient mice. METHODS: In experiment 1, cannulated male Balb/c mice are randomized to receive chow or PN for 5 days. Lung lymphocytes are obtained via collagenase digestion, and flow cytometric analysis is used to identify total T (CD3+) and B (CD45/B220+) cells. In experiment 2, isolated lung T cells from chow-fed male Balb/c mice are pooled and labeled in vitro with a fluorescent dye (carboxyfluorescein diacetate succinimidyl ester [CFSE]), and 1.1 x 10(8) CFSE+ cells (3.1 x 10(6) T cells) are transferred to chow-fed Balb/c recipients. Cells recovered from recipient lungs and intestinal lamina propria (LP) are analyzed by flow cytometry to determine CFSE/CD3+ T cells at 1, 2, and 7 days. In experiment 3, cells are transferred to PN-fed recipients. RESULTS: In experiment 1, PN significantly decreases lung T- and B-cell populations compared with chow feeding. In experiment 2, CFSE+ T-cell retention is highest on day 1 in lung and LP, and decreases on day 2. Cells are gone by day 7; 98.1% of retained donor lung T cells migrate to recipient lungs and 1.9% to the intestine on day 1. Similar results are seen in experiment 3 after transfer of cells to PN-fed recipients. CONCLUSIONS: PN reduces pulmonary lymphocyte populations consistent with impaired respiratory immunity. Transferred lung T cells preferentially localize to recipient lungs rather than intestine with maximal accumulation at 24 hours. Limited cross-talk of transferred lung T cells to the intestine indicates that mucosal lymphocyte traffic might be programmed to localize to specific effector sites.

Detrimental effects of early nutrition administration after severe gut ischemia-reperfusion.

BACKGROUND: Although early enteral nutrition after insult has many advantages, effects of early nutritional manipulation on outcome after gut ischemia-reperfusion (I/R) remain unclear. We hypothesize that early enteral nutrition would improve survival after severe gut I/R by reducing organ injury and leukocyte activation. MATERIALS AND METHODS: Mice were randomized to chow, intravenous (IV)-total parenteral nutrition (TPN), intragastric (IG)-TPN, or complex enteral diet (CED) for feeding after I/R. In experiment 1, 72 mice underwent both IV cannulation and gastrostomy before 45 or 10 min gut ischemia. At 12 (45 and 10 min ischemia) or 24 h (45 min ischemia) after I/R, mice were given one of the above diets. The chow group received IV saline and free access to chow was started at 12 or 24 h after I/R, i.e., no infusion of nutritional solutions. Survival was observed until 120 h. In experiment 2, 25 mice received one of the above diets at 12 h after 45 min gut I/R. Organ vascular permeability was assessed with Evans blue at 6 h after feeding. Reactive oxygen intermediate production with or without phorbol myristate acetate stimulation by circulating myeloid cells and expressions of CD11a and CD11b on these cells were also determined using flow cytometry. RESULTS: When feeding started at 12 h after 45 min ischemia, IV-TPN, IG-TPN, and CED significantly reduced survival time, as compared with chow. However, no significant difference was observed when feeding started at 24 h. There were no significant differences in survival times after 10 min ischemia among the four groups. Lung and small intestine vascular permeability was significantly higher in the IV-TPN group than in the other groups. There were no significant differences in reactive oxygen intermediate production or adhesion molecule expressions. CONCLUSION: Early nutrition administration after severe I/R reduces survival, possibly by increasing organ injury in IV-TPN and by other mechanisms in IG-TPN and CED.

Parenteral nutrition induces organ specific alterations in polymeric immunoglobulin receptor levels.

BACKGROUND: Secretory immunoglobulin A (IgA) prevents pathogen adherence at mucosal surfaces to prevent infection. Polymeric immunoglobulin receptor (pIgR), located on the basolateral surface of mucosal cells, binds dimeric IgA produced by B cells with the cooperation of T cells in the lamina propria. This IgA-pIgR complex is transported apically, where it is exocytosed as secretory IgA to the mucosal surface. Our prior work shows that parenteral nutrition (PN) impairs both airway and small intestine mucosal immunity by reducing T and B cells and IgA levels. This work examines intestinal and respiratory tissue-specific pIgR responses to PN. METHODS: Cannulated male Institute of Cancer Research mice were randomized to Chow (n = 10) or PN (n = 10). After 5 days, animals were sacrificed and lavages obtained from the small intestine, lung (BAL = bronchoalveolar lavage), and nasal airways (NAL). Small intestine, lung, and nasal passage tissues were also collected. Lavage and tissue homogenate IgA levels were quantified by enzyme-linked immunosorbent assay and pIgR by Western blot. RESULTS: PN group SIL and NAL IgA levels dropped significantly compared with Chow. PN significantly reduced pIgR levels in the SI while no pIgR change was noted in nasal passages and lung pIgR actually increased with PN. Tissue homogenate IgA levels did not change with PN in the SI while levels in the nasal passage and lung decreased. CONCLUSIONS: PN impairs airway mucosal immunity by reduction in IgA available for transport rather than via a reduction in pIgR levels. In the small intestine, diminished pIgR is implicated in the deterioration of antibody-mediated mucosal immunity.

Decreased enteral stimulation alters mucosal immune chemokines.

BACKGROUND: Migration of lymphocytes into and through the mucosal immune system depends upon adhesion molecules to attract circulating cells and chemokines to stimulate diapedesis into tissues. Decreased enteral stimulation significantly reduces mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) levels, an adhesion molecule critical for homing of T and B cells to Peyer's patches (PP), which reduces PP and intestinal T and B cells. We studied the effect of type and route of nutrition on tissue specific chemokines in PP (CXCL-12, -13 and CCL-19, -20 and -21), small intestine (SI; CCL-20, -25 and -28) and lung (CXCL-12, CCL-28). METHODS: Intravenously cannulated male Institute of Cancer Research (ICR) mice were randomized to chow or parenteral nutrition (PN) for 5 days. PP, SI, and lung chemokine mRNA levels were measured using real-time qRT-polymerase chain reaction, and analyzed semiquantitatively by the DeltaDeltaCt method. Protein levels were quantified using enzyme-linked immunosorbent assay (ELISA) techniques, and groups compared using Student's t-test. RESULTS: PP CXCL13 protein significantly decreased, whereas CCL21 protein increased significantly in the parenterally fed group. Parenteral feeding significantly decreased SI CCL20 and CCL 25 protein levels. CCL28 decreased significantly in the SI and lung of intravenously fed animals. mRNA levels changed in the opposite direction (compared with protein) for all chemokines except CCL28. CONCLUSIONS: Decreased enteral stimulation significantly alters key mucosal immune chemokine protein levels at multiple sites. In general, PN (and concomitant lack of enteral stimulation) results in decreased levels of chemokines that control lymphocyte migration within the mucosal immune system.

Parenteral nutrition inhibits tumor necrosis factor-alpha-mediated IgA response to injury.

BACKGROUND: Parenteral nutrition (PN) increases the incidence of pneumonia in severely injured patients compared with enteral feeding (ENT). Injury induces an innate airway IgA response in severely injured patients; similar responses occur in mice. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) stimulate the production of polymeric immunoglobulin receptor (pIgR), the protein required to transport immunoglobulin A (IgA) to mucosal surfaces. We have shown that PN alters levels of lung and nasal passage IgA and several IgA-stimulating cytokines. We hypothesized that TNF-alpha and IL-1beta blockade, as well as PN, would blunt the airway IgA response to injury. METHODS: Male Institute of Cancer Research (ICR) mice were randomized to uninjured controls (n = 10) or to intra-peritoneal phosphate-buffered saline (PBS) (n = 9), antagonistic TNF-alpha antibody (100 mcg, n = 7), or antagonistic IL-1beta antibody (50 mcg, n = 8) 30 min prior to surgical stress with laparotomy and neck incisions. Mice were sacrificed at 8 h for nasal and bronchoalveolar lavage (NAL, BAL) to measure IgA by enzyme-linked immunosorbent assay. In a separate experiment, 12 mice underwent intravenous cannulation followed by chow (n = 5) or PN (n = 7) feeding for 5 days prior to the same stress and IgA measurement. RESULTS: Injury significantly increased NAL and BAL IgA (225 +/- 104 ng) compared with baseline (145 +/- 38 ng; p = 0.01). Blockade of TNF-alpha eliminated the innate airway IgA response to injury (130 +/- 47 ng; p = 0.01), whereas IL-1beta blockade blunted and PN eliminated it completely. CONCLUSIONS: Tumor necrosis factor-alpha is involved in the respiratory IgA immune response to injury. Both TNF-alpha blockade and PN impair this innate response, and blockade of IL-1beta impairs it to a degree. We hypothesize that these cytokines blunt this response via their known effects on the polymeric immunoglobulin receptor (pIgR), whereas the PN-induced deficit likely is multifactorial.

Reversal of parenteral nutrition-induced gut mucosal immunity impairment with small amounts of a complex enteral diet.

BACKGROUND: Although parenteral nutrition (PN) prevents progressive malnutrition, lack of enteral nutrition (EN) during PN leads to gut associated lymphoid tissue (GALT) atrophy and dysfunction. Administering a small amount of EN with PN reportedly prevents increases in intestinal permeability. However, its effects on GALT remain unclear. We analyzed the minimum amount of EN required to preserve gut immunity during PN. METHODS: Male Institute of Cancer Research mice underwent jugular vein catheter insertion and tube gastrostomy. They were randomized into four groups to receive isocaloric and isonitrogenous nutritional support with variable EN to PN ratios (EN 0, EN 33, EN 66, and EN 100). EN was provided with a complex enteral diet. After 5 days of feeding, the mice were killed and whole small intestines were harvested. GALT lymphocytes were isolated and counted. Their phenotypes were analyzed by flow cytometry. IgA levels of small intestinal washings were analyzed with enzyme-linked immunosorbent assay. RESULTS: Body weight changes did not differ between any two of the groups. Peyer's patch lymphocyte numbers increased in proportion to EN amount, whereas lamina propria lymphocyte numbers were significantly higher in the EN 100 than in the EN 0 group, with no marked increases in the EN 33 and EN 66 groups. Small intestinal IgA levels increased EN amount-dependently and reached a plateau at EN 66. CONCLUSIONS: A small amount of EN partially reverses PN-induced GALT changes, suggesting beneficial but limited effects on gut mucosal immunity.

Lack of enteral nutrition blunts extracellular-regulated kinase phosphorylation in gut-associated lymphoid tissue.

The mitogen-activated protein kinase (MAPK) family (extracellular-regulated kinase [ERK], p38, etc.) of signal transduction proteins includes important intracellular mediators of inflammation, playing critical roles in host defense. Phosphorylations of ERK and p38 are responsible for cell proliferation, cell differentiation, and cell death. We hypothesized that impaired gut-associated lymphoid tissue (GALT) function in the absence of enteral nutrition is associated with reduced MAPK phosphorylation in GALT cells. Fifty-three male Institute of Cancer Research mice were randomized into 3 groups; ad libitum chow, intragastric (i.g.)-TPN, and intravenous (i.v.)-TPN. TPN groups were administered a standard TPN solution. After 5 days of feeding, lymphocytes from Peyer patches (PPs), the lamina propria (LP) cells, and intraepithelial (IE) spaces in the small intestine were isolated. GALT lymphocyte numbers were determined. The lymphocytes were incubated with or without 50 ng/mL of phorbol myristate acetate (PMA) for 15 min, and phosphorylated ERK (p-ERK) and p38 (p-p38) levels were determined using laser scanning cytometry. In PP (GALT inductive site) lymphocytes, p-ERK was increased after PMA in all three groups. However, ERK phosphorylation in GALT effector sites (IE and LP) was enhanced only in the enteral groups. p38 phosphorylation was not increased in any GALT sites, in any of the three groups, in response to PMA. In another set of mice (n = 33), in vitro LP lymphocyte proliferation was assessed with BrdU with or without PMA. Cell proliferation was increased or maintained at high level with PMA in the i.g.-TPN and chow group, but remained low in the i.v.-TPN group. In conclusion, lack of enteral feeding blunts ERK activation and cell proliferation in response to PMA stimulation in GALT effector sites, which may be an important mechanism underlying reduced GALT function. The influence of nutrition on GALT p38 phosphorylation must be assessed with other types and dosages of stimulants.

Effects of lymphotoxin beta receptor blockade on intestinal mucosal immunity.

BACKGROUND: Mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) directs lymphocyte migration into gut-associated lymphoid tissue (GALT) through Peyer's patches (PPs). Parenteral nutrition (PN) impairs mucosal immunity by reducing PPs MAdCAM-1 expression, T and B cells in GALT, and intestinal and respiratory immunoglobulin (Ig) A levels. We previously showed that PN reduces lymphotoxin beta receptor blockade (LTbetaR) in PPs and intestine, and that stimulation with LTbetaR agonist antibodies reverses these defects. To confirm that LTbetaR regulates transcription of MAdCAM-1 message and more fully understand the effects of LTbetaR on MAdCAM-1 function within the mucosal immune system, we studied the effect of LTbetaR blockade with a chimeric LTbetaR Ig-fusion protein on MAdCAM-1 mRNA levels, PP lymphocyte mass and IgA levels in the intestinal and respiratory tracts. METHODS: Mice were cannulated and killed 3 days after receiving chow + control Ig, chow + LTbetaR-Ig fusion protein (100 microg IV), or PN + control Ig. The PPs of half of the animals were processed for lymphocyte count, and the other half were processed for complementary DNA and subsequent polymerase chain reaction (PCR). mRNA levels of MAdCAM-1 were determined by real-time PCR; intestinal and respiratory IgA levels were measured by ELISA. RESULTS: PN significantly reduced PP lymphocyte mass, MAdCAM-1 mRNA, and intestinal IgA. As anticipated, LTbetaR blockade significantly decreased PP cells and MAdCAM-1 mRNA, but not intestinal IgA because chow feeding was maintained. Both LTbetaR blockade and PN decreased nasal IgA, but not significantly. CONCLUSIONS: LTbetaR blockade in chow animals significantly reduces transcription of MAdCAM-1 gene and PPs lymphocyte mass. These data implicate inadequate LTbetaR signaling as a major mechanism for decreased GALT cells with lack of enteral stimulation, and further establish the role of LTbetaR in the mucosal immune system.

Influence of adding fish oil to parenteral nutrition on gut-associated lymphoid tissue.

BACKGROUND: Lack of enteral nutrition reduces gut-associated lymphoid tissue (GALT) mass and function, a mechanism underlying the increased morbidity of infectious complications in severely injured or critically ill patients. Strategies to restore parenteral nutrition (PN)-induced changes of GALT mass and function have been pursued. However, the influences of adding fish oil to PN on gut immunity remain to be clarified. METHODS: Male Institute of Cancer Research (ICR) mice (n = 50) were randomized to 4 groups: ad libitum chow (chow), fat free PN (fat (-)-PN), PN + fish oil (FO-PN), and PN + safflower oil (SO-PN). The PN groups were given isocaloric and isonitrogenous PN solutions. The FO- and SO-PN groups received 20% of total calories from fat emulsions. After 5 days of feeding, lymphocytes from Peyer's patches (PPs), the intraepithelial space (IE), and the lamina propria (LP) of the entire small intestine were isolated. GALT lymphocyte numbers and phenotypes (CD4+, CD8+, alphabetaTCR+, gammadeltaTCR+, B220+ cells) were determined. Immunoglobulin A (IgA) levels of small intestinal washings were also measured by enzyme-linked immunosorbent assay. Another set of mice (n = 24) was used to determine plasma fatty acid compositions after feeding. RESULTS: Lymphocyte numbers from PPs and the LP and intestinal IgA levels were significantly lower in the PN groups than in the chow group, with no significant differences between any 2 PN groups. The FO- and SO-PN groups showed moderate recovery of IE cell numbers compared with the fat (-)-PN group. Omega-3 and omega-6 fatty acid levels were increased with fish and safflower oil additions, respectively, compared with the fat (-)-PN group. CONCLUSIONS: Adding fish oil to PN does not exacerbate PN-induced GALT changes but rather partially reverses these changes, with increased plasma omega-3 fatty acid levels.

Intestinal polymeric immunoglobulin receptor is affected by type and route of nutrition.

BACKGROUND: Secretory immunoglobulin A (SIgA) prevents adherence of pathogens at mucosal surfaces to prevent invasive infection. Polymeric immunoglobulin receptor (pIgR) is located on the basolateral surface of epithelial cells and binds dimeric immunoglobulin A (IgA) produced by plasma cells in the lamina propria. This IgA-pIgR complex is transported apically, where IgA is exocytosed as SIgA to the mucosal surface. Our prior work shows that mice fed intragastric (IG, an elemental diet model) and IV parenteral nutrition (PN) solution have reduced intestinal T and B cells, SIgA, and interleukin-4 (IL-4) compared with mice fed chow or a complex enteral diet (CED). Prior work also demonstrates a reduction in IgA transport to mucosal surfaces in IV PN-fed mice. Because IL-4 up-regulates pIgR production, this work studies the effects of these diets on intestinal pIgR. METHODS: Male Institute of Cancer Research (ICR) mice were randomized to chow (n = 11) with IV catheter, CED (n = 10) or IG PN (n = 11) via gastrostomy and IV PN (n = 12) for 5 days. CED and PN were isocaloric and isonitrogenous. Small intestine was harvested for pIgR and IL-4 assays after mucosal washing for IgA. IgA and IL-4 levels were analyzed by enzyme-linked immunosorbent assay and pIgR by Western blot. RESULTS: Small intestinal pIgR expression, IgA levels, and IL-4 levels decreased significantly in IV PN and IG PN groups. CONCLUSIONS: Lack of enteral stimulation affects multiple mechanisms responsible for decreased intestinal SIgA levels, including reduced T and B cells in the lamina propria, reduced Th-2 IgA-stimulating cytokines, and impaired expression of the IgA transport protein, pIgR.

Albumin infusion after reperfusion prevents gut ischemia-reperfusion-induced gut-associated lymphoid tissue atrophy.

BACKGROUND: Our recent study clarified that gut ischemia-reperfusion (I/R) causes gut-associated lymphoid tissue (GALT) mass atrophy, a possible mechanism for increased morbidity of infectious complications after severe surgical insults. Because albumin administration reportedly reduces hemorrhagic shock-induced lung injury, we hypothesized that albumin treatment prevents GALT atrophy due to gut I/R. METHODS: Male mice (n = 37) were randomized to albumin, normal saline, and sham groups. All groups underwent jugular vein catheter insertion. The albumin and normal saline groups underwent 75-minute occlusion of the superior mesenteric artery. During gut ischemia, all mice received normal saline infusions at 1.0 mL/h. The albumin group was given 5% bovine serum albumin in normal saline at 1.0 mL/h for 60 minutes after reperfusion, whereas the normal saline group received 0.9% sodium chloride at 1.0 mL/h. The sham group underwent laparotomy only. Mice were killed on day 1 or 7, and the entire small intestine was harvested. GALT lymphocytes were isolated and counted. Their phenotypes (alphabetaTCR, gammadeltaTCR, CD4, CD8, B220) were determined by flow cytometry. RESULTS: On day 1, the gut I/R groups showed significantly lower total lymphocyte and B cell numbers in Peyer's patches and the lamina propria than the sham group. However, the albumin infusion partially but significantly restored these cell numbers. On day 7, there were no significant differences in any of the parameters measured among the 3 groups. CONCLUSIONS: Albumin infusion after a gut ischemic insult may maintain gut immunity by preventing GALT atrophy.

Interleukin-7 dose-dependently restores parenteral nutrition-induced gut-associated lymphoid tissue cell loss but does not improve intestinal immunoglobulin a levels.

BACKGROUND: Without enteral nutrition, the mass and function of gut-associated lymphoid tissue (GALT), a center of systemic mucosal immunity, are reduced. Therefore, new therapeutic methods, designed to preserve mucosal immunity during parenteral nutrition (PN), are needed. Our recent study revealed that exogenous interleukin-7 (IL-7; 1 microg/kg twice a day) restores the GALT cell mass lost during intravenous (IV) PN but does not improve secretory immunoglobulin A (IgA) levels. Herein, we studied the IL-7 dose response to determine the optimal IL-7 dose for recovery of GALT mass and function during IV PN. We hypothesized that a high dose of IL-7 would increase intestinal IgA levels, as well as GALT cell numbers. METHODS: Male mice (n = 42) were randomized to chow, IL-7-0, IL-7-0.1, IL-7-0.33, IL-7-1 and IL-7-3.3 groups and underwent jugular vein catheter insertion. The IL-7 groups were fed a standard PN solution and received IV injections of normal saline (IL-7-0), 0.1, 0.33, 1, or 3.3 microg/kg of IL-7 twice a day. The chow group was fed chow ad libitum. After 5 days of treatment, the entire small intestine was harvested and lymphocytes were isolated from Peyer's patches (PPs), intraepithelial (IE) spaces, and the lamina propria (LP). The lymphocytes were counted and phenotypes determined by flow cytometry (alphabetaTCR, gammadeltaTCR, CD4, CD8, B cell). IgA levels of small intestinal washings were also examined using ELISA (enzyme-linked immunoabsorbent assay). RESULTS: IL-7 dose-dependently increased total lymphocyte numbers in PPs and the LP. The number of lymphocytes harvested from IE spaces reached a plateau at 1 microg/kg of IL-7. There were no significant differences in any phenotype percentages at any GALT sites among the groups. IgA levels of intestinal washings were significantly higher in the chow group than in any of the IL-7 groups, with similar levels in all IL-7 groups. CONCLUSIONS: Exogenous IL-7 dose-dependently reverses PN-induced GALT cell loss, with no major changes in small intestinal IgA levels. IL-7 treatment during PN appears to have beneficial effects on gut immunity, but other therapeutic methods are needed to restore secretory IgA levels.

Lack of enteral nutrition delays nuclear factor kappa B activation in peritoneal exudative cells in a murine glycogen-induced peritonitis model.

BACKGROUND: Early enteral nutrition is associated with a lower incidence of intraabdominal abscess in severely injured patients than parenteral nutrition (PN). We explored the underlying mechanisms by examining the influence of nutrition route on nuclear factor kappaB (NFkappaB) activation in peritoneal exudative cells (PECs) and peritoneal cytokine levels. METHODS: Thirty male Institute Cancer Research mice were randomized to chow (n = 10), IV PN (n = 10), or intragastric (IG) PN (n = 10) and fed for 5 days. PECs were harvested at 2 or 4 hours after intraperitoneal injection of 2 mL of 1% glycogen. Intranuclear NFkappaB activity in PECs was examined by laser scanning cytometry. Cytokine (tumor necrosis factor-alpha [TNF-alpha], macrophage inflammatory protein-2 [MIP-2], interleukin-10 [IL-10]) levels in peritoneal lavaged fluid were determined by enzyme-linked immunosorbent assay. RESULTS: Intranuclear NFkappaB at 2 hours was significantly higher in the chow and IG-PN groups than in the IV-PN group. TNF-alpha and IL-10 levels of the chow group were significantly higher than those of IV-PN mice at 2 hours, whereas those of IG-PN mice were midway between those of the chow and IV-PN groups. MIP-2 was significantly higher in the chow group than in the IG-PN and IV-PN mice at 2 hours. TNF-alpha levels correlated positively with intranuclear NFkappaB activity in PECs. CONCLUSIONS: Enteral nutrition may improve peritoneal defense by preserving early NFkappaB activation in PECs and cytokine responses.

Influences of long-term antibiotic administration on Peyer's patch lymphocytes and mucosal immunoglobulin A levels in a mouse model.

BACKGROUND: Long-term antibiotic administration is sometimes necessary to control bacterial infections during the perioperative period. However, antibiotic administration may alter gut bacterial flora, possibly impairing gut mucosal immunity. We hypothesized that 1 week of subcutaneous (SC) antibiotic injections would affect Peyer's patch (PP) lymphocyte numbers and phenotypes, as well as mucosal immunoglobulin A (IgA) levels. METHODS: Sixty-one male Institute of Cancer Research mice were randomized to CMZ (cefmetazole 100 mg/kg, administered SC twice a day), IPM (imipenem/cilastatin 50 mg/kg x 2), and control (saline 0.1 mL x 2) groups. After 7 days of treatment, the mice were killed and their small intestines removed. Bacterial numbers in the small intestine were determined using sheep blood agar plates under aerobic conditions (n = 21). PP lymphocytes were isolated to determine cell numbers and phenotypes (CD4, CD8, alphabetaTCR, gammadeltaTCR, B220; n = 40). IgA levels in the small intestinal and bronchoalveolar washings were also measured with ELISA. RESULTS: Antibiotic administration decreased both bacterial number and the PP cell yield compared with the control group. There were no significant differences in either phenotype percentages or IgA levels at any mucosal sites among the 3 groups. CONCLUSIONS: Long-term antibiotic treatment reduces PP cell numbers while decreasing bacterial numbers in the small intestine. It may be important to recognize changes in gut mucosal immunity during long-term antibiotic administration.

Exogenous interleukin 7 affects gut-associated lymphoid tissue in mice receiving total parenteral nutrition.

In the absence of enteral nutrient delivery, gut-associated lymphoid tissue (GALT) mass and function are reduced. The purpose of this study was to examine whether exogenous interleukin (IL)-7 treatment reverses intravenous (IV)-total parenteral nutrition (TPN)-induced changes in GALT, immunoglobulin (Ig) A levels, and gut barrier function. Eighty-nine mice were randomized to chow, TPN, or TPN + IL-7 (1 microg/kg, administered IV twice a day) and treated for 5 days. The entire small intestine was harvested and lymphocytes were isolated from Peyer's patches (PPs), intraepithelial (IE) spaces, and the lamina propria (LP). Small intestinal and bronchoalveolar IgA levels were measured. Proximal and distal small intestinal levels of IgA-stimulating (IL-10) and IgA-inhibiting (IFNgamma) cytokines were determined with enzyme-linked immunoabsorbant assay. Moreover, 1 x 10 live Pseudomonas aeruginosa were delivered by gavage and survival was observed. TPN decreased total cell yields from PPs, IE spaces, and the LP compared with the chow group. IL-7 treatment restored cell numbers. PP CD4+, PP CD8+, IE gammadeltaTCR+, and LP CD4+ cell numbers were higher in the TPN + IL-7 group than in the TPN group. Secretory IgA levels were lower in the TPN and TPN + IL-7 than in the chow group. In the distal small intestine, IFNgamma levels were similar in the three groups, whereas IL-10 levels were reduced in the TPN and TPN + IL-7 groups relative to the chow group. Survival times were reduced in the TPN compared with the chow group, but IL-7 treatment significantly improved survival. Thus, exogenous IL-7 does not improve secretory IgA levels, nor are there any remarkable effects on levels of gut IgA-mediating cytokines. However, IL-7 treatment during TPN reverses TPN-induced GALT atrophy and improves survival in a gut-derived sepsis model.

Route and type of nutrition influence nuclear factor kappaB activation in peritoneal resident cells.

Morbidity of intra-abdominal abscess is increased when severely injured patients are fed parenterally. Lack of enteral nutrition appears to impair peritoneal cavity host defense. Because the transcription factor nuclear factor kappaB (NFkappaB) regulates various genes involved in inflammatory responses and its activation is important for host defense, we hypothesized that enteral nutrition would preserve appropriate NFkappaB activation in peritoneal resident cells (PRCs), the first defense line against peritoneal contamination. Mice (n = 105) were randomized to chow (n = 38), intravenous (IV)-total parenteral nutrition (TPN) (n = 34), or intragastric (IG)-TPN (n = 33) for 5 days' feeding. In experiment 1, PRCs were harvested for measurement of intranuclear NFkappaB activity with or without in vitro lipopolysaccharide (LPS) stimulation using laser scanning cytometry and enzyme-linked immunoabsorbant assay. PRC numbers tended to be higher in enterally fed mice than in IV-TPN mice. The main PRC subpopulation was macrophages in all groups. NFkappaB activation was increased in response to LPS in chow mice, whereas there was no increase in the IV-TPN group. IG-TPN mice demonstrated moderate NFkappaB activation. In experiment 2, mice underwent cecal ligation and puncture (CLP). Survival was observed up to 5 days. In another set of mice, tumor necrosis factor (TNF) alpha levels of peritoneal lavaged fluid were measured 4 h after CLP. Survival times after CLP improved in the chow and IG-TPN groups compared with the IV-TPN group. TNFalpha levels were significantly higher in the chow than in the IV-TPN group. In conclusion, parenteral nutrition decreases PRC number and blunts NFkappaB activation in PRCs. These changes may impair host defense in the peritoneal cavity.

5-Fluorouracil infusion reduces gut-associated lymphoid tissue cell number and mucosal immunoglobulin A levels.

BACKGROUND: Anticancer drugs have been demonstrated to affect gut mucosal morphology and cause gastrointestinal symptoms. We hypothesized that even small doses of 5-fluorouracil (5-FU) would reduce gut-associated lymphoid tissue (GALT) mass and function. METHODS: Mice underwent IV cannulation and received continuous infusion of normal saline or 10 mg/kg of 5-FU for 5 days. GALT cell numbers, phenotypes, and mucosal immunoglobulin A (IgA) levels were measured. RESULTS: During the infusion, there were no significant differences in food intake or body weight change between the 2 groups. Cell yields from the intraepithelial space and lamina propria of the small intestine were lower in the 5-FU than the control group. The lamina propria CD4/CD8 ratio was reduced in the 5-FU compared with the control group. Intestinal and respiratory tract IgA levels were lower in the 5-FU than in the control group. CONCLUSIONS: A small dose of 5-FU reduces GALT cell number and mucosal IgA levels, regardless of food intake.

Dietary restriction compromises resistance to gut ischemia-reperfusion, despite reduction in circulating leukocyte activation.

BACKGROUND: Gut ischemia-reperfusion (gut I/R) accompanying severe surgical insults leads to neutrophil-mediated injury and is regarded as a triggering event in early multiple-organ failure. Our previous study demonstrated dietary restriction to down-regulate leukocyte activation. Therefore, we hypothesized dietary restriction might be beneficial in terms of surviving I/R. We also evaluated leukocyte activation and the level of organ glutathione, an antioxidative substance. METHODS: Institute of Cancer Research mice received chow, 170 (ad libitum), 119 (MR: mild restriction) or 68 (SR: severe restriction) g/kg per day for 7 days. Exp. 1: The mice (n = 59) underwent 15 or 45 minutes of gut ischemia and survival was observed. Exp. 2: The mice (n = 73) were killed before or 60 or 120 minutes after 15-minute ischemia. Reactive oxygen intermediate (ROI) production by circulating myeloid cells and CD11b expression was determined. Some mice were assessed for nuclear factor kappa B (NFkappaB) activation. Glutathione levels were measured in some of the small intestine and liver samples from each group. RESULTS: Dietary restriction decreased survival. Circulating myeloid cell priming and activation, in terms of ROI production and CD11b expression, were enhanced in the ad libitum group but not in the restricted groups. NFkappaB was activated only in the ad libitum group. Gut and hepatic glutathione levels were lower in the SR than in the ad libitum group. Dietary restriction caused histologic damages in gut, liver, and lung 120 minutes after reperfusion. CONCLUSIONS: Dietary restriction blunts leukocyte priming and activation after gut ischemic insult but worsens the outcome by, at least in part, decreasing antioxidative activities. Clinically, nutrition replenishment may be required to improve the outcome of gut hypoperfusion.

Effects of L-arginine infusion during ischemia on gut blood perfusion, oxygen tension, and circulating myeloid cell activation in a murine gut ischemia/reperfusion model.

BACKGROUND: Gut hypoperfusion is considered to be a mechanism for early multiple-organ failure after severe surgical insults. L-Arginine (ARG) may preserve gut microcirculation as a substrate of nitric oxide synthase, but simultaneously may enhance immune cell response. It remains unknown if ARG infusion during gut ischemia improves the outcome after gut ischemia-reperfusion (I/R). METHODS: Male Institute of Cancer Research mice were randomized to control and ARG groups. After i.v. cannulation, mice underwent 90 (Exp. 1) or 60 (Exp. 2 and 3) minutes of gut I/R. Control mice received normal saline infusion at 1 mL/h for 60 minutes during ischemia, whereas the ARG group was given 1% ARG hydrochloride solution. In Exp. 1, survival was observed for 72 hours (n = 35). In Exp. 2, blood perfusion and oxygen tension of the small intestine were measured (n = 9). In Exp. 3, peripheral blood was obtained at 2 or 4 hours after reperfusion (n = 22). Reactive oxygen intermediate (ROI) production by myeloid cells with or without phorbol myristate acetate (PMA) stimulation and expression of CD11a and CD11b on myeloid cells were examined using flow cytometry. RESULTS: Exp. 1: There was no significant difference in survival times (log rank test, p = .2). However, survival rates at 12 hours were 72% (13/18) for the control group and 35% (6/17) for the ARG group (p < .05 Fisher). Exp. 2: ARG infusion significantly improved gut blood perfusion ratio during ischemia but had no effect on oxygen tension. Exp. 3: In the ARG group, ROI production with PMA and CD11b expression at 4 hours were higher than those at 2 hours, whereas there were no significant changes in the control mice. CONCLUSIONS: ARG infusion improves intestinal blood perfusion during ischemia but primes and activates circulating myeloid cells excessively. Consequently, i.v. infusion of ARG during ischemia reduces survival rate.