Effects of whole blood leukoreduction on platelet function and hemostatic parameters.

AIMS/OBJECTIVES: The aim of this study was to evaluate the hemostatic consequences of whole blood leukoreduction (LR). BACKGROUND: Whole blood is being used for trauma resuscitation in the military, and an increasing number of civilian trauma centres across the nation. The benefits of LR, such as decreased infectious and transfusion-related complications, are well established, but the effects on hemostatic parameters remain a concern. METHODS: Twenty-four units of whole blood were assigned to one of the four groups: non-leukoreduced (NLR), leukoreduced at 1 h and a height of 33 in. (LR-1), leukoreduced at 4 h and a height of 33 in. (LR-4(33)), or leukoreduced at 4 h and a height of 28 in. (LR-4(28)). Viscoelastic parameters, platelet aggregation, cell counts, physiological parameters and thrombin potential were evaluated immediately before and after LR, and on days 1, 7, 14 and 21 following LR. RESULTS: The viscoelastic parameters and thrombin generation potential were unchanged between the groups. Platelet aggregation was reduced in the LR-1 group compared with NLR after 7 days. The LR-4(28) group also showed a trend of reduced platelet aggregation compared with NLR. Aggregation in LR-4(33) was similar to NLR throughout the storage time. Physiological and electrolyte changes over the whole blood storage period were not affected by LR. CONCLUSION: Our study shows that whole blood can be LR at 4 h after collection and a height of 33 in. while maintaining platelet count and without altering platelet function and hemostatic performance.

Intraoperative manometry to assess the esophagogastric junction during laparoscopic fundoplication and myotomy.

SUMMARY: Surgery for gastroesophageal reflux disease and achalasia is performed to alleviate symptoms by improving esophagogastric junction (EGJ) function. Intraoperative manometry was used to evaluate the pressure-length characteristics of the reconstructed EGJ during laparoscopic Nissen fundoplication and laparoscopic Heller myotomy. Intraoperative manometry was performed in 37 consecutive patients undergoing laparoscopic Nissen fundoplication (n = 22) or laparoscopic Heller myotomy (n = 15). Measurements were taken before surgery, after creation of the pneumoperitoneum, after completion of the myotomy in achalasia, and after creation of the fundoplication. Tracings were analyzed for pressure, length, and the integrated pressure-length relation (area under the curve [AUC]). Statistical comparison was made using paired t tests; intraoperative EGJ measurements did not correlate well with preoperative values for either pressure or length. Laparoscopic Nissen fundoplication significantly increased pressure, length, and AUC of the EGJ compared with prefundoplication values. Laparoscopic Heller myotomy significantly decreased EGJ pressure, length, and AUC. Creation of a Toupet fundoplication after myotomy did not significantly increase pressure, length, and AUC of the EGJ compared with postmyotomy values. Intraoperative manometry identified 2 of 15 achalasia patients (13%) with persistent areas of high pressure after initial myotomy that were corrected by extending the myotomy. Intraoperative manometry identifies mechanical changes created during EGJ surgery and may be a useful adjunct to improve outcomes of laparoscopic Nissen fundoplication and laparoscopic Heller myotomy.

Evaluation of endoscopic and laparoscopic training practices in surgical residency programs.

BACKGROUND: The ability to adequately train surgical residents in flexible and rigid endoscopy has become a difficult challenge for program directors. The American Board of Surgery requires residents to be familiar in these procedures but the methods for training have not been well defined nor formally outlined. The goals of this study were to evaluate resident experience in flexible endoscopy and laparoscopy and to investigate the specific methods used by surgical programs for the training of residents. METHODS: A survey was created by the authors and the Resident Education Committee of the Society of American Gastrointestinal Endoscopic Surgeons and was mailed to all program directors in general surgery in the United States based on the data base of the Association of Program Directors in Surgery (APDS). RESULTS: Ninety-six of 283 surveys were returned (33.9%). The surgeon played a greater role in flexible endoscopic training in 1998 as compared to 1988 (p=0.002). When analyzed by type of institution, community programs showed a similar trend but this was not seen in academic programs. Formal endoscopy rotations existed in 60% of programs but flexible endoscopy (5.2%) and laparoscopy (10.4%) fellowships were uncommon. No significant differences in the number of advanced laparoscopic procedures performed were found between academic and community programs. The presence of a laparoscopic fellow did not significantly decrease the number of cases per resident. CONCLUSION: According to our survey, surgery departments have a greater impact on flexible endoscopic training in 1998 than in 1988. This is likely due to the creation of formal endoscopy rotations and the hiring of fellowship trained endoscopic instructors. In addition, community programs have been able to provide adequate experience in both basic and advanced laparoscopic techniques as compared to academic programs. As with flexible endoscopy, however, formal laparoscopic rotations may be necessary to allow more intensive experience for each resident.

Dantrolene reduces serum TNFalpha and corticosterone levels and muscle calcium, calpain gene expression, and protein breakdown in septic rats.

The effects of dantrolene on serum TNFalpha and corticosterone levels and on muscle calcium, calpain gene expression, and protein breakdown were studied in rats with abdominal sepsis induced by cecal ligation and puncture. Treatment of rats with 10 mg/kg of dantrolene 2 h before and 8 h after induction of sepsis reduced serum TNFalpha and corticosterone, muscle calcium levels, mRNA levels for m- and mu-calpain, and the muscle specific calpain p94, as well as total and myofibrillar protein breakdown rates, determined as release of tyrosine and 3-methylhistidine, respectively, from incubated extensor digitorum longus muscles. The results support the concept that increased calcium concentrations may be an important mechanism of sepsis-induced muscle protein breakdown. The data also indicate that other mechanisms, in addition to reduced muscle calcium concentrations such as decreased levels of TNFalpha and glucocorticoids, may contribute to the anti-catabolic effects of dantrolene during sepsis. The observations are important from a clinical standpoint because they suggest that the catabolic response in skeletal muscle during sepsis may be prevented by treatment with a calcium antagonist.

Induction of the stress response increases interleukin-6 production in the intestinal mucosa of endotoxaemic mice.

Previous studies suggest that production of interleukin-6 (IL-6) is increased in the intestinal mucosa during sepsis and endotoxaemia. We tested the hypothesis that mucosal IL-6 production during endotoxaemia is increased further by the heat-shock (stress) response. The stress response was induced in mice by hyperthermia (rectal temperature of 42 degrees C for 3 min) or by intraperitoneal injection of sodium arsenite (10 mg/kg). At 2 h after induction of the stress response, groups of mice were injected subcutaneously with endotoxin (10 mg/kg) or sterile saline. IL-6 mRNA and protein levels in the jejunal mucosa were determined by an RNase protection assay and an ELISA respectively, and levels of hsp72 (heat-shock protein of 72 kDa) were determined by Western blot analysis. Hyperthermia and sodium arsenite increased hsp72 levels in the intestinal mucosa. IL-6 concentrations were increased in the jejunal mucosa of endotoxaemic mice, and this effect of endotoxaemia was potentiated by the stress response. Mucosal IL-6 mRNA levels were increased in endotoxaemic mice, and were increased further by the stress response. Thus it is concluded that mucosal IL-6 production during endotoxaemia may be further stimulated by the stress response. Increased IL-6 levels in the intestinal mucosa may be a potential mechanism by which the stress response exerts a protective effect during sepsis and endotoxaemia.

The transcription factor activator protein-1 is activated and interleukin-6 production is increased in interleukin-1beta-stimulated human enterocytes.

The intestinal mucosa is an active participant in the inflammatory and metabolic response to sepsis, endotoxemia, and other critical illness. The genes for various cytokines, e.g., interleukin 6 (IL-6), are regulated by multiple transcription factors, including nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1). In recent studies, treatment with IL-1beta of cultured Caco-2 cells, a human intestinal epithelial cell line, resulted in increased NF-kappaB DNA binding. The effect of IL-1beta on AP-1 activity in the enterocyte and the potential role of AP-1 in enterocyte IL-6 production are not known. We treated Caco-2 cells with IL-1beta and determined AP-1 activity by electrophoretic mobility shift assay (EMSA) and IL-6 production by enzyme-linked immunosorbent assay (ELISA). Treatment of Caco-2 cells with IL-1beta resulted in a dose- and time-dependent increase in AP-1 DNA binding. Supershift analysis suggests that activated AP-1 contained c-Jun, JunD, c-Fos, FosB, and Fra1 subunits. When Caco-2 cells were transiently transfected with an AP-1 luciferase reporter plasmid, stimulation with IL-1beta resulted in increased luciferase activity, suggesting that AP-1 DNA binding increased gene activation. Additional luciferase assays were performed with a plasmid containing a wild-type or AP-1-mutated IL-6 promoter. Stimulation of these cells with IL-1beta gave rise to results supporting the role of AP-1 in the regulation of IL-6 production. Geldanamycin, which has been shown in studies to inhibit AP-1 activation, blocked IL-1beta-induced AP-1 luciferase gene activation and IL-6 production. These results suggest that the AP-1 family of transcription factors is activated by IL-1beta in human enterocytes and that AP-1 may at least in part regulate IL-6 production in these cells.

Activation of NF-kappaB varies in different regions of the gastrointestinal tract during endotoxemia.

The transcription nuclear factor-kappaB (NF-kappaB) regulates a large number of genes involved in the inflammatory response to sepsis and endotoxemia. We recently found that NF-kappaB is activated in the jejunal mucosa during endotoxemia, but the response of NF-kappaB in other parts of the gastrointestinal tract is not known. We hypothesized that NF-kappaB is differentially activated in different regions of the gastrointestinal tract during endotoxemia. NF-kappaB DNA binding activity was determined by electrophoretic mobility shift assay in mucosa of the stomach, jejunum, ileum, and colon from endotoxemic and saline-injected mice. Cytoplasmic levels of the NF-kappaB inhibitory proteins IkappaB-alpha and IkappaB-beta were determined by Western blot analysis. Endotoxemia increased NF-kappaB activity in mucosa of stomach, jejunum, and ileum, with jejunum responding to smaller doses of endotoxin than the other parts of the gastrointestinal tract. NF-kappaB DNA binding activity was not induced in colonic mucosa, even following administration of high doses of endotoxin. IkappaB-alpha and IkappaB-beta levels decreased in jejunal mucosa of endotoxin injected mice, concomitant with activation of NF-kappaB. The results suggest that during endotoxemia, NF-kappaB is activated in mucosa of stomach and small intestine, but not in colon, and that the jejunum is particularly sensitive to endotoxin.

Induction of the stress response in vivo decreases nuclear factor-kappa B activity in jejunal mucosa of endotoxemic mice.

BACKGROUND: Results of previous studies suggest that the stress response protects cells and tissues by regulating proinflammatory mediators. The transcription factor nuclear factor-kappa B (NF-kappa B), normally sequestered in the cytoplasm by its inhibitory protein, I kappa B, regulates many genes involved in inflammatory responses to critical illness. Endotoxemia is associated with increased NF-kappa B activity in intestinal mucosa, but the effect of the stress response on endotoxin-induced NF-kappa B activation in intestinal mucosa is not known. HYPOTHESIS: Induction of the stress response inhibits NF-kappa B DNA binding activity in jejunal mucosa during endotoxemia. METHODS: The stress response was induced in mice by hyperthermia (42 degrees C) or injection with sodium arsenite (10 mg/kg). After 2 to 5 hours, mice were injected with endotoxin (lipopolysaccharide, 12.5 mg/kg) or a corresponding volume of sterile saline. One hour later, jejunal mucosa was harvested for preparation of nuclear and cytoplasmic extracts. RESULTS: Mucosal levels of heat shock protein-72 increased after hyperthermia or treatment with sodium arsenite, consistent with induction of the stress response. The increase in NF-kappa B DNA binding activity and decrease in I kappa B-alpha levels seen after endotoxin injection were inhibited by previous induction of the stress response. CONCLUSION: The protective effects of the stress response in vivo might, at least in part, be due to inhibited NF-kappa B activation.

Interleukin-1beta induces complement component C3 and IL-6 production at the basolateral and apical membranes in a human intestinal epithelial cell line.

In previous studies, stimulation of cultured enterocytes with IL-1beta resulted in production of IL-6 and complement component C3. The cellular mechanisms of these responses in the enterocyte are not fully understood. We tested the hypothesis that IL-1beta-induced C3 and IL-6 production is differentially regulated at the apical and basolateral membranes of the enterocyte. Caco-2 cells (a transformed human colonic carcinoma cell line) were grown in a 2-chamber system to full differentiation. The cells were treated with IL-1beta either at the apical or basolateral membrane, and C3 and IL-6 mRNA levels and release of C3 and IL-6 into the apical and basal chambers were determined. The release of C3 was greatest into the basal chamber regardless of whether the cells were stimulated at the apical or basolateral membrane. In contrast, the production of IL-6 was greatest at the cell membrane that was stimulated with IL-1beta. Stimulation of the Caco-2 cells with IL-1beta resulted in increased mRNA levels for C3 and IL-6 with no major differences noted when the cells were treated at the apical or basolateral membrane. The results suggest that enterocyte production and release of at least some acute phase proteins and cytokines are differentially regulated at the apical and basolateral membrane of the enterocyte after stimulation with IL-1beta.

IL-1beta induction of NF-kappaB activation in human intestinal epithelial cells is independent of oxyradical signaling.

IL-1beta stimulation of cultured epithelial cells induces the degradation of IkappaBalpha and the consequent nuclear translocation of NF-lambdaB, a critical proinflammatory transcription factor in the mucosal host immune response. The role of reactive oxygen intermediates, serine protease activity, and tyrosine kinase activity in the activation of NF-kappaB is weakly conserved across various cell lineages and has not been defined in human enterocytes, a major target of oxidant stress in sepsis, thermal injury, and hemorrhagic shock. We report here that in Caco-2BBe cells, a transformed human colon cancer cell line with features of small intestinal epithelial cells in culture, exposure to oxidant stress (hydrogen peroxide 1-10 mM) did not induce NF-kappaB activation. Similarly, scavenging of free radicals and oxidants by pyrrolidine dithiocarbamate and dimethyl sulfoxide did not block IL-1beta-induced IkappaBalpha degradation and NF-kappaB activation. Genistein, a nonspecific tyrosine kinase inhibitor, also had no effect on IL-1beta-mediated effects on NF-kappaB. Serine protease inhibition by tosyl-lysine-chloromethylketone and tosyl-phenylalanine-chloromethylketone inhibited IkappaBalpha degradation and NF-kappaB activation stimulated by IL-1beta. Our data highlight the strong divergence between epithelial and mononuclear cells in the signal transduction pathways relating IL-1beta stimulation and NF-kappaB nuclear translocation.

Burn injuries in rats upregulate the gene expression of the ubiquitin-conjugating enzyme E2(14k) in skeletal muscle.

Burn injuries are associated with muscle cachexia, which mainly reflects protein breakdown in the ubiquitin-proteasome pathway. Ubiquitination of proteins degraded by this mechanism is regulated by multiple enzymes, including the 14-kd ubiquitin-conjugating enzyme, E2(14k). In this study, burn injuries in rats resulted in increased levels of the 1.2 kilobase E2(14k) transcript in the white, fast-twitch extensor digitorum longus muscle with no changes or only minor changes in the red, slow-twitch soleus muscle, liver, and kidney. The results provide the first evidence that burn injuries upregulate the gene expression of E2(14k) in skeletal muscle and suggest that ubiquitin-proteasome-dependent muscle protein breakdown after thermal injuries may, at least in part, be regulated by E2(14k).

Implementation of a clinical pathway decreases length of stay and cost for bowel resection.

OBJECTIVE: To examine the effect of a clinical pathway for small and large bowel resection on cost and length of hospital stay. SUMMARY BACKGROUND DATA: Clinical pathways are designed to streamline patient care delivery and maximize efficiency while minimizing cost. Theoretically, they should be most effective in commonly performed procedures, in which volume and familiarity are high. METHODS: A clinical pathway to assist in the management of patients undergoing bowel resection was developed by a multidisciplinary team and implemented. Data about length of stay and cost was collected for all patients undergoing bowel resection 1 year before and 1 year after pathway implementation. Three groups were compared: patients undergoing bowel resection in the year prior to pathway implementation (prepathway), patients in the year after pathway implementation but not included on the pathway (nonpathway), and patients included in the pathway (pathway). RESULTS: The mean cost per hospital stay was $19,997.35 +/- 1244.61 for patients in the prepathway group, $20,835.28 +/- 2286.26 for those in the nonpathway group, and $13,908.53 +/- 1113.01 for those in the pathway group (p < 0.05 vs. other groups). Mean postoperative length of stay was 9.98 +/- 0.62 days (prepathway), 9.68 +/- 0.88 days for (nonpathway), and 7.71 +/- 0.37 days (pathway) (p < 0.05 vs. other groups). CONCLUSIONS: Implementation of the pathway produced significant decreases in length of stay and cost in the pathway group as compared to the prepathway group. These results support the further development of clinical pathways for general surgical procedures.

Use of omeprazole in the management of giant duodenal ulcer: results of a prospective study.

BACKGROUND: Giant duodenal ulcer (GDU) is generally thought to require surgical intervention. Proton pump inhibitors have beneficial effects in peptic ulcer disease, but their role in GDU disease is unknown. We examined the use of omeprazole in GDU management. METHODS: Twenty-eight patients were diagnosed with GDU. One patient required immediate operative intervention. The remaining 27 were placed on omeprazole (40 mg daily). When ulcer healing was documented by endoscopy, the patients were placed on oral histamine-2 receptor antagonist therapy. RESULTS: Of the 28 study patients, 20 (71.4%) did not require operative intervention, and 8 (28.6%) required operation for ulcer complications. Of the 15 patients with adherent clot or a visible vessel at initial endoscopy, 7 (46.7%) required operative intervention, as compared with 1 (7.7%) of the 13 patients without a visible vessel or adherent clot. This difference was statistically significant (P < .05). Twenty-three patients underwent antral biopsy and/or enzyme-linked immunosorbent assay for Helicobacter pylori, and 9 (39.1%) had a positive result. CONCLUSIONS: Omeprazole is effective in the treatment of GDU disease. An adherent clot or a visible vessel at endoscopy indicates a higher likelihood of complications requiring operation. The relatively low H pylori infection rate, as compared with other peptic ulcer disease, may indicate a different pathophysiology in GDU.

Stress response decreases the interleukin-1beta-induced production of complement component C3 in human intestinal epithelial cells.

Interleukin-1beta (IL-1beta) increases the production of complement component C3 in enterocytes. Heat shock regulates the response to cytokines and other inflammatory mediators in various cell types. We tested the hypothesis that the heat-shock response regulates IL-1beta-induced C3 production in the enterocyte. Cultured Caco-2 cells, a human intestinal epithelial cell line, were treated with sodium arsenite (10-500 microM) for 1 h or subjected to hyperthermia (43 degrees C) for 1-4 h, and allowed to recover for 1 h. The cells were then treated with IL-1beta (0.5 ng/ml) for up to 24 h, whereafter C3 levels were measured by ELISA and C3 mRNA by Northern blot analysis. Heat-shock protein of 72 kDa (hsp72) was determined by Western blot analysis. Treatment of the cells with sodium arsenite or subjecting them to hyperthermia induced the expression of hsp72. The IL-1beta-induced expression of C3 mRNA and C3 production were down-regulated by hyperthermia and sodium arsenite in a dose-dependent fashion. The results suggest that the stress response induced by hyperthermia or sodium arsenite decreases IL-1beta-induced C3 production in human enterocytes.

Complement component C3 production in IL-1beta-stimulated human intestinal epithelial cells is blocked by NF-kappaB inhibitors and by transfection with ser 32/36 mutant IkappaBalpha.

BACKGROUND: Recent studies suggest that interleukin-1beta (IL-1beta) stimulates the production of the acute phase protein complement component C3 in human intestinal epithelial cells. The transcription factor NF-kappaB activates different genes involved in the response to cytokines. It is not known if IL-1beta-induced C3 production in the enterocyte is regulated by NF-kappaB. MATERIALS AND METHODS: Cultured Caco-2 cells, a human intestinal epithelial cell line, were treated with one of the NF-kappaB inhibitors, tosyl-lys-chloromethylketone (TLCK), genistein, or pyrrolidine dithiocarbamate (PDTC), or with N-acetyl-leu-leu-norleucinal (LLnL), a proteasome inhibitor known to block the degradation of Ikappabeta, the cytosolic inhibitor of NF-kappaB. Following this treatment, the Caco-2 cells were stimulated with IL-1beta, and C3 levels in the culture medium were measured after 24 h by ELISA. C3 mRNA levels were determined after 4 h by Northern blot analysis. In other experiments, Caco-2 cells were transfected with a mutant IkappaBalpha in which serines 32 and 36 were substituted by alanine. This mutation prevents IkBalpha phosphorylation and subsequent NF-kappaB nuclear translocation. After transfection, the cells were stimulated with IL-1beta, and C3 levels in the culture medium were measured after 24 h. Cytosolic IkappaBalpha was determined by Western blot analysis. RESULTS: TLCK, genistein, and LLnL each inhibited IL-1beta-induced C3 production in a dose-dependent fashion. These responses were associated with decreased C3 mRNA levels. In contrast, PDTC did not influence C3 production or C3 mRNA in the Caco-2 cells. Transfection of the Caco-2 cells with the Ser 32/36 mutant IkBalpha resulted in maintained IkappaBalpha levels and decreased IL-beta-induced C3 production. CONCLUSIONS: IL-1beta-stimulated C3 production in the enterocyte may be regulated by NF-kappaB.

Nuclear factor-kappaB is activated in intestinal mucosa during endotoxemia.

BACKGROUND: The transcription factor nuclear factor-kappaB (NF-kappaB) regulates a large number of genes involved in the inflammatory response to critical illness. The intestinal mucosa plays an active role in the inflammatory and metabolic response to sepsis and endotoxemia, but it is not known if NF-kappaB is activated in the mucosa during these conditions. OBJECTIVE: To test the hypothesis that endotoxemia in mice activates NF-kappaB in intestinal mucosa. METHODS: Mice were injected subcutaneously with lipopolysaccharide, 12.5 mg/kg, or a corresponding volume of saline. At various intervals following injection, jejunal mucosa was harvested and nuclear and cytoplasmic fractions were prepared. The nuclear fractions were analyzed by electrophoretic mobility shift assay for NF-kappaB activation and by Western blot analysis for the NF-kappaB subunits p50 and p65. Cytoplasmic fractions were analyzed by Western blotting for the NF-kappaB inhibitory proteins IkappaB-alpha and IkappaB-beta. RESULTS: Electrophoretic mobility shift assay showed that NF-kappaB was activated in jejunal mucosa 1 hour after injection of lipopolysaccharide and persisted for at least 4 hours. The NF-kappaB subunits p50 and p65 were present in nuclear fractions of mucosa from endotoxemic mice at the corresponding time points. Cytoplasmic levels of the inhibitory proteins IkappaB-alpha and IkappaB-beta decreased during endotoxemia, and the proteins were nearly absent 60 minutes after injection of lipopolysaccharide. CONCLUSIONS: The results suggest that IkappaB is degraded and NF-kappaB is activated in intestinal mucosa during endotoxemia. The findings support the concept that the intestinal mucosa is an important component of the inflammatory response to sepsis and endotoxemia.