Measuring platelet aggregation in dialysis patients with a whole blood aggregometer by the screen filtration pressure method.

A semi-automatic, whole-blood aggregometer based on the screen filtration pressure (SFP) method is now widely used clinically and in research, but has not been used with hemodialysis (HD) patients. We measured whole-blood platelet aggregation in HD patients by the SFP method. This retrospective cross-sectional study included 62 HD patients, of whom 47 were non-diabetic and 15 were diabetic; we also included a control group of healthy, non-uremic subjects. With the t-test, we examined differences in the platelet aggregation threshold index (PATI) in meaningful sub-groupings of the HD patients, depending on whether or not they had diabetes, and whether or not they had been given antiplatelet agents. Considering the non-diabetic HD patients first, their PATI values were significantly higher than those values in the control subjects (3.1 [1.0-5.2] vs. 1.8 [1.3-2.3] µM, P<0.001). The non-diabetic HD patients taking antiplatelet agents showed significantly (1.9 times) higher PATI values than the non-diabetic HD patients without antiplatelet agents (4.4 [1.8-7.0] vs. 2.3 [1.3-3.3] µM, P=0.003). We observed similar trends among diabetic HD patients. Whole-blood analysis by the SPF method seems to be a promising way of monitoring platelet function for HD patients.

Using the supercharge technique to additionally revascularize the gastric tube after a subtotal esophagectomy for esophageal cancer.

BACKGROUND: Maintaining sufficient blood flow to the gastric tube after a subtotal esophagectomy for esophageal cancer is crucial for decreasing esophagogastric anastomotic leakage. METHODS: After subtotal esophagectomy for esophageal cancer, the supercharge technique was performed in 21 esophageal reconstruction patients to additionally revascularize the gastric tube using the splenic artery and vein, external carotid artery, and internal jugular vein. Operative results of the supercharge group were retrospectively compared with those of the control group (patients not receiving the technique). RESULTS: Both operation time and operative blood loss in the supercharge group were significantly longer and larger than those of the control group. However, the incidence of anastomotic leakage was significantly lower in the supercharge group than in the control group. CONCLUSION: This practical supercharge technique reduces leakage during esophageal anastomosis.

Enteral feeding preserves gut Th-2 cytokines despite mucosal cellular adhesion molecule-1 blockade.

BACKGROUND: Parenteral nutrition (PN) decreases gut-associated lymphoid tissue (GALT), the intestinal IgA stimulating cytokines IL-4 and IL-10 in gut homogenates, intestinal IgA levels and the expression of Peyer patch (PP) mucosal cellular adhesion molecule-1 (MAdCAM-1), an adhesion molecule found on the high endothelial venules of PP and other tissues. IL-4 in PP stimulates MAdCAM expression in vitro. MAdCAM-1 blockade with MECA-367 reduces GALT cell populations to PN levels but maintains intestinal IgA levels if the animals are chow fed. This study compares IL-4 levels in PP of chow and PN fed mice and measures the effects of MAdCAM blockade on IL-4 and IL-10 levels in gut homogenates of chow fed mice. We hypothesized that in vivo IL-4 levels drop in PP of PN fed mice and IL-4 and IL-10 levels are maintained after MAdCAM-1 blockade in chow fed mice. METHODS: Exp 1: 18 mice received chow or PN for 5 days to determine PP IL-4 levels. Exp 2: 44 mice were randomized to chow + control monoclonal antibody (mAb), chow + MECA-367 (anti-MAdCAM-1 mAb) or PN for 4 days before measurement of IL-4 and IL-10 levels in gut homogenates. RESULTS: Exp 1: IL-4 levels in vivo were lower in PP of PN-fed mice than chow fed mice (92.0 +/- 15.1 pg/mL vs 251.1 +/- 14.8, p = .0003). Exp 2: IL-4 levels were significantly higher in chow + control mAb (187.1 +/- 44.1 pg/mL) and chow + MECA-367 (110.9 +/- 19.1 pg/mL) groups than PN mice (21.8 +/- 30.6 pg/mL, p < .02 vs chow + control or chow + MECA-367). IL-10 levels were significantly lower with PN (23.1 +/- 40.9 pg/mL) with chow+control (174.0 +/- 22.2 pg/mL p < .01), or chow + MECA-367 (181.7 +/- 23.1 pg/mL, p < .02 vs PN). CONCLUSIONS: PN-feeding reduces in vivo IL-4 levels in PP (consistent with lowered MAdCAM-1 expression) and IL-4 and IL-10 levels in gut homogenates compared with chow. Despite MAdCAM-1 blockade, enteral feeding preserved gut IL-4 levels and increased IL-10 levels consistent with preserved IgA levels.

l-selectin and alpha4beta7 integrin, but not ICAM-1, regulate lymphocyte distribution in gut-associated lymphoid tissue of mice.

BACKGROUND: Adhesion molecules on lymphocytes ( l -selectin and alpha4beta7) and endothelium (MAdCAM-1 and ICAM-1) direct lymphocytes into the gut-associated lymphoid tissue (GALT) of mice. Parenteral nutrition and MAdCAM-1 blockade reduce GALT cell mass. This study examined the effects on GALT cell mass of blockade of l -selectin, alpha4beta7, and ICAM-1 with saturating doses of monoclonal antibodies. METHODS: In experiment 1, l -selectin and alpha4beta7 expression were measured by flow cytometry in chow-fed mice. In experiment 2, 49 mice randomly received chow, parenteral nutrition, chow + intravenous (IV) anti-CD62L, chow + IV anti-LPAM-1, or chow + IV isotype control antibody. After 4 days, lymphocyte yields in GALT and respiratory and intestinal IgA levels were measured. In experiment 3, 27 mice randomly received chow, parenteral nutrition, chow + IV anti-ICAM-1 monoclonal antibody, or chow + IV isotype control antibody for 5 days. Lymphocyte counts and IgA levels were determined as in experiment 2. RESULTS: Some 80% of all circulating lymphocytes were positive for l -selectin and alpha4beta7. Lymphocyte counts in the Peyer's patches, lamina propria, and intraepithelial space were lower in the l -selectin and alpha4beta7 blockade groups (3.1, 1.8, and 0.9 x 10(6) and 2.1, 1.9, and 0.7 x 10(6) , respectively) than in the chow group (5.9, 3.0, and 1.7 x 10(6) ; P < .02 vs the l -selectin group and P <.001 vs the alpha4beta7 group) and similar to the levels in the parenteral group. Respiratory and intestinal IgA levels are maintained in all groups except the parenteral group ( P <.04 vs the chow group). ICAM-1 blockade did not influence cell counts or IgA levels. CONCLUSION: Most circulating lymphocytes have GALT homing potential. Their distribution into GALT is hindered by blockade of l -selectin or alpha4beta7, but not by ICAM-1.

The C-terminal heptapeptide of bombesin reduces the deleterious effect of total parenteral nutrition (TPN) on gut-associated lymphoid tissue (GALT) mass but not intestinal immunoglobulin A in vivo.

BACKGROUND: Bombesin, the amphibian analog of mammalian gastrin-releasing peptide, reverses total parenteral nutrition (TPN)-induced atrophy of gut-associated lymphoid tissue and increases intestinal and respiratory immunoglobulin A (IgA) levels. Structure-activity studies suggested that the biologically active portion of bombesin is a C-terminal heptapeptide (7AA). This study investigates the effect of 7AA on lymphocytes counts of the Peyer's patches (PP), the lamina propria (LP) and the intraepithelial layer (IE). METHODS: Forty-eight male mice were randomized to receive chow (n = 13), TPN only (n = 9), TPN + 15 microg 7AA 3 times per day (n = 13) or TPN + 150 microg 7AA 3 times per day (n = 13). After 5 days of feeding, PP, LP, and IE lymphocytes were determined. Intestinal IgA levels were measured with ELISA. Groups were compared with ANOVA. RESULTS: All TPN-fed mice lost more weight than mice fed chow (p < .04). Lymphocyte counts in PP, LP, and IE were significantly lower in the TPN group than in the 3 other groups but did not differ between the groups fed chow, TPN + 15 microg 7AA 3 times per day, or TPN + 150 microg 7AA 3 times per day. Intestinal IgA levels were higher in chow-fed mice (148.4 +/- 16.9) than in mice fed TPN (98.4 +/- 14.0, p = .008), TPN + 15 microg 7AA 3 times per day (96.9 +/- 7.7, p = .003) or TPN + 150 microg 7AA 3 times per day (87.3 +/- 6.7, p = .001). CONCLUSIONS: The C-terminal heptapeptide of bombesin prevented the TPN-induced decrease in intestinal lymphocyte populations but not the reduction in intestinal IgA levels.

Patients with postoperative infections have sticky neutrophils before operation.

Appropriate polymorphonuclear neutrophil (PMN) recruitment is essential for host defense against infection. We investigated the significance of the preoperative PMN adhesion-migration process, as assessed by the flow chamber method, on postoperative infectious complications. Thirty-one consecutive patients with gastrointestinal malignancies, 21 colorectal and 10 gastric, who were undergoing elective surgery were enrolled. PMNs, isolated preoperatively from each patient's venous blood, were perfused onto a tumor necrosis factor alpha-stimulated human umbilical vein endothelial cell (HUVEC) monolayer through the flow chamber. We evaluated the adherent PMN number, the migrated PMN number, and the stuck PMN number by directly inspecting PMN interactions with a HUVEC monolayer under continuous shear flow simulating postcapillary venules. The expression of adhesion molecules on circulating PMNs was also measured. Patients were grouped into an infectious and a noninfectious group according to the occurrence of postoperative infectious complications defined by the Centers for Disease Control criteria. Eleven patients developed postoperative infectious complications. Although the number of preoperative in vitro adherent PMNs in patients with postoperative infection was significantly higher than in those without postoperative infection (P = 0.01), migrated PMN number was similar in both groups. Stuck PMN number tended to be higher in the infectious group than in the noninfectious group. The migrated PMN number showed a significant positive correlation with the adherent PMN number in the noninfectious group but not in the infectious group. Preoperative CD31 expression on circulating PMNs was significantly lower in the infectious group than in the noninfectious group. Preoperative in vitro derangement of the PMN adhesion-migration process is closely associated with postoperative infectious complications.

Enteral feeding preserves mucosal immunity despite in vivo MAdCAM-1 blockade of lymphocyte homing.

OBJECTIVE: To determine the influence of route of nutrition on gut mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) expression and the effect of MAdCAM-1 blockade on gut-associated lymphoid tissue (GALT) lymphocyte populations and established respiratory antibacterial immunity. SUMMARY BACKGROUND DATA: Lymphocytes, sensitized to antigens in Peyer's patches, migrate via mesenteric lymph nodes and home to intestinal lamina propria. MAdCAM-1 located on endothelial cells regulates this trafficking. Experimentally, parenteral nutrition (PN) decreases GALT cell mass and mucosal immunity when compared with enteral feeding. METHODS: In experiment 1, MAdCAM-1 expression was quantified in 32 mice after 4 days of feeding chow, a complex diet, intragastric (IG)-PN, or PN. In experiment 2, MAdCAM-1 was measured in 102 mice 0, 4, 8, 12, 24, 48, or 72 hours after starting PN and at 0, 4, 8, 12, 24, or 48 hours after reinstituting chow following 5 days of PN. In experiment 3, 56 mice received chow, PN, chow + MECA-367 (anti-MAdCAM-1 mAb), or chow + Isotype control Ab (IsoAb) for 5 days, followed by Peyer's patches, lamina propria, and intraepithelial lymphocyte yield with respiratory and intestinal IgA levels. In experiment 4, 10 days after Pseudomonas immunization, mice received chow + MECA-367 or chow + IsoAb for 4 days followed by 1.2 x 108 Pseudomonas intratracheally. RESULTS: Diet and route affect MAdCAM-1 expression (chow > complex diet > IG-PN > PN). Decreased MAdCAM-1 expression occurred within hours of starting PN in Peyer's patches, but not mesenteric lymph nodes or the intestine, and recovered quickly with enteral refeeding. MAdCAM-1 blockade reduced all GALT populations. Blockade had little effect on IgA levels and partially impaired the late response of established respiratory immunity. CONCLUSIONS: Enteral feeding affects MAdCAM-1 expression. Complete MAdCAM-1 blockade reduces GALT lymphocytes to PN levels, but the chow feeding stimulus preserves IgA and early antibacterial resistance, implying the existence of non-MAdCAM-1 mechanisms to preserve mucosal immunity.

Brief refeeding rapidly reverses dietary restriction-induced nuclear factor-kappaB downregulation in peritoneal resident cells.

BACKGROUND: Malnutrition impairs host immunity, resulting in high mortality and morbidity, secondary to infections. Nuclear factor kappaB (NFkappaB) plays a critical role in host defense, but how quickly refeeding normalizes the impaired NFkappaB activity in peritoneal resident cells (PRCs) is unknown. Our aim was to investigate the effects of 1-day ad libitum refeeding on severe diet restriction-induced NFkappaB activity in PRCs. METHODS: Mice received chow, 146 g/kg per day (ad libitum) or 36.5 g/kg per day (severe diet restriction), for 7 days. One-half the mice in the diet-restricted group were then fed ad libitum for 1 day (refeeding). PRCs were harvested by peritoneal lavage. After incubation with tumor necrosis factor-alpha (TNF-alpha), nuclear translocation of NFkappaB in PRCs was investigated using laser scanning cytometry. RESULTS: The main subpopulation of PRCs was macrophages in all groups. Mean fluorescence intensity over the nuclear area at 0 or 100 ng/mL of TNF-alpha was 16 +/- 2 or 31 +/- 8* in the ad libitum, 20 +/- 4 or 19 +/- 3 in the severe diet-restricted, and 20 +/- 4 or 30 +/- 5* in the refeeding group, respectively (*p < .05 versus 0 ng/mL of TNF-alpha in each group versus 100 ng/mL of TNF-alpha in diet-restricted group). Cytoplasmic accumulation of NFkappaB was significantly increased after TNF-alpha stimulation in the refed group but not in the ad libitum group. CONCLUSIONS: The blunted NFkappaB activity in PRCs, after exposure to inflammatory stimuli, was restored after 1 day of refeeding, with increased accumulation of NFkappaB in the cytoplasm. Even brief nutritional replenishment in malnutrition may improve host defense by restoring NFkappaB activity and thereby improving macrophage functions.

Oral administration of Bifidobacterium longum culture condensate in a diet-restricted murine peritonitis model enhances polymorphonuclear neutrophil recruitment into the local inflammatory site.

Dietary restriction impairs polymorphonuclear neutrophil (PMN) recruitment into the local inflammatory site, resulting in susceptibility to infection. Probiotics enhance host immunity via conditioning host intestinal microflora. Oral administration of Bifidobacterium longum culture condensate (BCC) in a diet-restricted murine peritonitis model may enhance PMN recruitment into the inflammatory site. Male ICR mice (n = 40) were assigned in equal numbers to control or BCC groups and subjected to 75% restricted food intake for 7 d. During dietary restriction, controls received only standard mouse chow, whereas the BCC group received standard mouse chow containing 1% BCC. Mice were killed before (0 h) or after (2 or 4 h) intraperitoneal glycogen injection. Peritoneal lavage fluid and exudative cells were recovered by peritoneal lavage. Peritoneal exudative cell number was counted. Tumor necrosis factor-alpha, interleukin-6, macrophage inflammatory protein-2, and interleukin-10 concentrations in peritoneal lavage fluid were determined by enzyme-linked immunosorbent assay. CD11b, CD18, CD31, and CD62L expressions on circulating PMNs were measured by flow cytometry. Oral BCC administration upregulated PMN recruitment into the peritoneal cavity and increased peritoneal fluid cytokine concentrations as well as CD18 and CD62L expressions on circulating PMNs during glycogen-induced peritonitis. Oral BCC administration in a diet-restricted murine peritonitis model augmented PMN recruitment into the inflammatory site by upregulating cytokine concentrations in the local inflammatory site and adhesion molecule expression on circulating PMNs. Oral BCC administration may be a favorable modality for improving dietary restriction-induced host immunosuppression.

Glutamine improves impaired cellular exudation and polymorphonuclear neutrophil phagocytosis induced by total parenteral nutrition after glycogen-induced murine peritonitis.

Clinical and laboratory evidence shows that enteral feeding significantly reduces pneumonia and intra-abdominal abscess formation after celiotomy for severe trauma. Supplementation of total parenteral nutrition (TPN) with glutamine (GLN) supports impaired immunity induced by TPN in several animal and human studies. This work investigates the peritoneal cellular response and polymorphonuclear neutrophil (PMN) bactericidal function after mouse chemical peritonitis after TPN with and without GLN. Thirty-three mice received chow, TPN, or 2% GLN-supplemented TPN (GLN-TPN) for 5 days. All mice then received 2 mL of a 1% glycogen solution intraperitoneally to induce cell exudation, and peritoneal exudative cells (PECs) were recovered 4 h later. Total and differential PEC numbers, as well as PMN phagocytosis, reactive oxygen intermediate production (ROI), CD11b (integrin aM chain) expression, and CD16/32 (Fcgamma II/III receptor) expression were measured. PMN, macrophage, and lymphocyte cell numbers were significantly lower with TPN than with chow or GLN-TPN groups, with no differences between chow and GLN-TPN. TPN significantly lowered peritoneal PMN phagocytosis compared with chow (P < 0.05) and approached significance with GLN-TPN (P = 0.06). There were no significant differences in ROI production or CD11b and CD16/32 expression on peritoneal PMN. GLN supplementation improved the reduction in cell exudation and PMN phagocytosis induced by TPN after chemical peritonitis.

An experimental study on the effects of postburn dietary supplementation of enhanced nutrients / 中华烧伤杂志

<p><b>OBJECTIVE</b>To evaluate the effects of postburn dietary supplementation of arginine (Arg), omega-3 polyunsaturated fatty acid (omega-3PUFA) and glutamine (Glu) on the metabolism, immunology and wound healing in scalded rats.</p><p><b>METHODS</b>Thirty Sprague-Dawley (SD) rats inflicted with 30% total body surface area deep partial thickness scald on the back after the gastrostomy catheter was placed were employed as the model. The rats were randomly divided into A and B groups, and all of them received continuous isonitrogenous (25% protein, 12% fat, 63% carbohydrate), isocaloric (175 kcal/kg/day), and isovolemic intragastric tube feedings. The contents of Arg, omega-3PUFA, Glu in the dietary of B group were enriched. The parameters were measured on the 10th day after injury, including the response of spleen cells to ConA, the plasma levels of PGE(2), IL-2, albumin, transferrin, glucagons, cortisol in blood, the urinary content of vanillylmandelic acid (VAM) in 24-hour urine, the content of hydroxyproline, the ratio of type I to type III collagen in burn wounds, and the nitrogen content in the liver and in the jejunal mucosa, as well as the weight changes, skin delayed hypersensitivity test, and wound healing time.</p><p><b>RESULTS</b>It was revealed that the serum level of albumin, the nitrogen content in the liver and in the jejunal mucosa were obviously higher in B than those in A group. At the same time, there was no statistical difference in the plasma levels of cortisol and glucagons and urinary content of VAM between the two groups, nor in body weight changes. Meanwhile, the response of spleen cells to ConA and the skin delayed hypersensitivity induced by DNFB 14 days after injury in group B were also enhanced compared with those in group A. Although the expression of PGE2 from peritoneal macrophages was lower, the content of hydroxyproline from burn wounds in group B was significantly higher than that in group A, and the ratio of type l to type III collagen in group B was significantly lower than that in group A. Compared with group A, the wound healing time in group B was significantly shortened (P < 0.05).</p><p><b>CONCLUSION</b>The low-fat and high-protein feeding diet with enriched arginine, omega-3 PUFA, glutamine could benefit the nutritional status after burn injury, thus improve the immunological function and promote wound healing.</p>

Mucosal immunity preservation with bombesin or glutamine is not dependent on mucosal addressin cell adhesion molecule-1 expression.

BACKGROUND: Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is an adhesion molecule that directs naive T and B cells into Peyer's patches for sensitization and distribution to intestinal and extraintestinal sites. With no enteral stimulation, its expression drops rapidly in association with reduced Peyer's patch cell populations and increases rapidly with reinstitution of enteral feeding. Because both glutamine (GLN) and bombesin (BBS) preserve mucosal immunity, this study examined whether they preserve MAdCAM-1 expression. METHODS: In 2 separate experiments, animals were randomized to IV cannulation with chow, total parenteral nutrition (TPN), and (experiment 1) 15 microg/kg BBS 3 times per day or (experiment 2) an isocaloric, isonitrogenous 2% GLN-supplemented solution. After 5 days of feeding, MAdCAM-1 expression in Peyer's patches, spleen, and intestine was measured using a dual radiolabeled monoclonal antibody technique. RESULTS: MAdCAM-1 expression was not significantly improved from TPN levels either with BBS or GLN supplementation. Levels of MAdCAM-1 expression remained unchanged in non-Peyer's patch sites. CONCLUSIONS: Although MAdCAM-1 is considered the gateway molecule for cell entry into mucosal immunity, this does not seem to be the mechanism for mucosal immunity preservation in nonenterally fed mice receiving bombesin or glutamine.

Differences in neutrophil death among beta-lactam antibiotics after in vitro killing of bacteria.

Antibiotic therapy is an essential treatment for gram-negative bacterial infections. Antibiotic-induced endotoxin release and subsequent production of inflammatory cytokines reportedly depend on the type of antibiotic action. This study examined the effects of various beta-lactam antibiotics on cell death of human polymorphonuclear neutrophils (PMNs) cocultured with Escherichia coli (E. coli) in vitro. E. coli morphology after antibiotic treatment was determined. PMNs and E. coli were cocultured with antibiotics for 0, 4, or 12 h. Levels of endotoxin and cytokines (TNF-alpha, IL-1beta, and IL-6) in the supernatants were measured. The filtrates of antibiotic-treated E. coli supernatants were cocultured with PMNs for 0, 4, or 12 h. In all experiments, ampicillin (ABPC), cefazolin sodium (CEZ), cefoperazone sodium (CPZ), latamoxef sodium (LMOX), imipenem (IPM), and polymyxin B sulfate (PLB) were used at 30 microg/mL. PMNs were isolated from healthy volunteers. PMN cell death was assessed by flow cytometry and light microscopy. ABPC, CEZ, CPZ, and LMOX, which induce bacterial filament formation with lysis, caused PMN necrosis when cocultured with E. coli. In contrast, IPM, which induces bacterial spheroplast formation with lysis, caused PMN apoptosis. Levels of endotoxin, TNF-alpha and IL-6 in the supernatants with IPM and PLB were significantly lower than in those with other beta-lactam antibiotics. The filtrates of IPM- and PLB-treated E. coli supernatants induced PMN apoptosis, whereas those treated with other beta-lactam antibiotics increased PMN necrosis. Beta-lactam antibiotics have different impacts on the types of PMN cell death after E. coli killing. Underlying mechanisms and the clinical relevance of IPM-induced PMN apoptosis in severe gram-negative infection warrant further investigation.

Total parenteral nutrition supplementation with glutamine improves survival after gut ischemia/reperfusion.

BACKGROUND: Total parenteral nutrition (TPN) alters gut cytokines and mucosal immunity and increases intercellular adhesion molecule-1 (ICAM-1) expression, gut neutrophil levels, and mortality after gut ischemia. Supplementation of TPN with glutamine partially supports mucosal immunity by preserving respiratory and intestinal IgA levels, maintaining the proper IgA-stimulating cytokine milieu within the intestine, and reducing intestinal ICAM-1 expression and neutrophil accumulation. This work investigates whether glutamine supplementation of TPN affects mortality in mice after gut ischemic insult. METHODS: Thirty-eight mice were randomized to receive chow, TPN, or 2% glutamine-supplemented TPN (GLN-TPN) for 5 days. After feeding their respective diets, gut ischemia/reperfusion (I/R) was induced with superior mesenteric artery occlusion for 30 minutes followed by resuscitation with 1 mL saline. Survival was recorded until 72 hours after reperfusion. RESULTS: Survival time was significantly reduced in the TPN-fed mice compared with both chow-fed and GLN-TPN-fed mice (p < .05). Survival at 72 hours after reperfusion was also significantly lower in the TPN-fed mice than in the chow-fed and GLN-TPN-fed mice (p < .05) CONCLUSIONS: Glutamine supplementation of TPN significantly improves survival after gut I/R, suggesting modulation of the inflammatory response or improved gut tolerance to low-flow states.