Risk factors for acute chest syndrome in children with sickle cell disease undergoing abdominal surgery.

BACKGROUND/PURPOSE: The reported incidence of acute chest syndrome (ACS) in children with sickle cell disease (SCD) is 15% to 20%. Our current objective was to assess risk factors and morbidity associated with ACS. METHODS: The authors reviewed the outcome of children with SCD undergoing abdominal surgery over a 10-year period. RESULTS: From 1991 to 2003, 60 children underwent laparoscopic cholecystectomy (LC; n = 29), laparoscopic splenectomy (LS; n = 28), or both (LB; n = 3). Mean age was 8.6 (0.7 to 20) years, and 35 (58%) were boys. Fifty-four (90%) had a preoperative hemoglobin greater than 10 g/dL, but only 22 (37%) received routine oxygen after surgery. No surgery was converted to an open procedure. Four children (6.6%), all of whom underwent either LS or LB, had ACS associated with an increased length of stay (7.4 +/- 2.4 days) but no mortality. Factors associated with the development of ACS were age (3.0 +/- 1.7 v 9.4 +/- 5.7 years; P =.03), weight (12.1 +/- 3.0 v 32.6 +/- 18.2 kg; P =.04), operative blood loss (3.2 +/- 0.5 v 1.4 +/- 1.2 mL/kg; P =.03), and final temperature in the operating room (OR; 36.2 +/- 0.4 v 37.6 +/- 0.4 degrees C; P =.01). ACS was not significantly related to duration of surgery, OR fluids, or oxygen usage. CONCLUSIONS: Younger children with greater blood and heat loss during surgery appear more prone to ACS. Splenectomy also seems to increase the risk of ACS. The authors' current incidence (6.6%) of ACS in children with SCD undergoing abdominal surgery is much lower than previously reported. This may be explained by the aggressive use of preoperative blood transfusion or more routine use of laparoscopy.

Cecal volvulus: a report of two cases occurring after the antegrade colonic enema procedure.

BACKGROUND: Many children with chronic constipation and fecal incontinence have benefited from the antegrade colonic enema (ACE) procedure. Routine antegrade colonic lavage often allows such children to avoid daytime soiling. This report describes 2 children in whom the ACE procedure was complicated by a cecal volvulus. METHODS: A retrospective review of 164 children with an ACE procedure was conducted. Two instances of cecal volvulus were identified. RESULTS: The first child presented with abdominal pain and difficulty intubating the ACE site. Over the subsequent day, his pain worsened, and radiographs depicted a colonic obstruction. At laparotomy, a cecal volvulus resulting in bowel necrosis was observed, and resection of the affected bowel and appendix (in the right lower quadrant) and end ileostomy was required. He subsequently had the stoma closed and a new ACE constructed with a colon flap. The second child presented with shock and evidence of an acute abdomen. At laparotomy, a cecal volvulus was noted, and ileocolic resection including the ACE stoma (located at the umbilicus) and an ileostomy and Hartmann pouch was performed. He had a protracted hospital course requiring ventilator and inotropic support. He currently is well and still has an ileostomy stoma. CONCLUSIONS: A high index of suspicion for a potentially life-threatening cecal volvulus should be maintained in children undergoing an ACE procedure who present with abdominal pain, evidence of bowel obstruction, or difficulty in advancing the ACE irrigation catheter.

Effect of pediatric surgical practice on the treatment of children with appendicitis.

OBJECTIVE: Acute appendicitis in children is managed by both general surgeons (GSs) and pediatric surgeons (PSs). Our objective was to investigate the economics of surgical care provided by either GSs or PSs for appendicitis. METHODS: The outcome of children within our state who underwent operative treatment for appendicitis (January 1994 to June 1997) by board-certified GSs were compared with the results of PSs. Data were sorted according to patient age and diagnosis according to the International Classification of Diseases, Ninth Revision. Analysis of variance was performed on continuous data, and chi(2) analysis was performed on nominal data; data are depicted as mean +/- standard error of the mean. RESULTS: GSs (n = 2178) managed older children when compared with PSs (n = 1018; 11.0 +/- 0.1 vs 9.1 +/- 0.1 years) and less frequently treated perforated appendicitis (18.8% vs 31.9%). Independent of diagnosis (simple or perforated appendicitis), younger children (0-4 years, 5-8 years, and 9-12 years) who were treated by PSs had a significantly shorter hospital stay and/or decreased hospital charge when compared with those who were treated by GSs. However, older children (13-15 years) seemed to have comparable outcomes. CONCLUSIONS: Younger children with appendicitis have reduced hospital days and charges when they are treated by PSs.

Characteristics of pediatric cervical spine injuries.

PURPOSE: The objective of this study was to assess the mechanisms and patterns of injury and outcome in children with cervical (C) spine trauma. METHODS: We reviewed the National Pediatric Trauma Registry between April 1994 and March 1999 and identified (by ICD-9 criteria) all cases of blunt trauma victims with cervical fractures, dislocations, and spinal cord injuries without radiographic abnormality (SCIWORA). Data are shown as mean +/- SEM. RESULTS: During the 5-year period, the incidence of blunt C-spine injury was 1.6% (n = 408 of 24,740 total entries). Mean age was 10.5+/-0.3 (1 to 20) years, and 59% were boys. Leading mechanisms were motor vehicle accidents (n = 179; 44%), sports (n = 66; 16%), and pedestrian injuries (n = 57, 14%). Younger (< or =10 years) children more often sustained high (C1 to C4) vs low (C5 to C7) injuries (85% v 57%; P<.01) and also had a higher incidence of dislocations (31% v 20%; P<.01) and cord injuries (26% v 14%; P<.01), whereas older children had more C-spine fractures (66% v 43%; P<0.01). Mortality rates (overall, 17%) were higher in younger children (n = 180) when compared with older children (n = 228; 30% v 7%; P<.01). Overall, the majority of deaths (93%) were associated with brain injuries. No children with cervical dislocations had neurologic sequelae. The preponderance of children with fractures (83%) also were without neurologic injury, whereas those associated with SCIWORA usually were (80%) partial. Overall, complete cord lesions were infrequent (4%). CONCLUSIONS: These data, representing the largest series to date, confirm that blunt C-spine injuries in children are rare. Patterns of injury vary significantly according to child age. Major neurologic sequelae in survivors is uncommon, does not correlate well with cord level, and rarely is complete.

Outcome of the antegrade colonic enema procedure in children with chronic constipation.

BACKGROUND: Chronic constipation and fecal incontinence in children related to pelvic trauma, congenital anomalies, or malignancy will eventually lead to significant social and psychologic stress. Maximal medical treatment (daily enemas and laxatives) can also be difficult to maintain in many children. METHODS: At our children's hospital, 11 children with chronic constipation or fecal incontinence or both underwent the antegrade colonic enema (ACE) procedure. The operation involved constructing a conduit into the cecum using either the appendix (n = 8) or a "pseudo-appendix" created from a cecal flap (n = 3). We report our surgical results. RESULTS: Mean child age was 9.6 (5 to 18) years. With a mean follow-up of 14 (6 to 24) months, 10 of the children (91%) had significant improvement and 7 children (64%) are completely clean with no soiling and controlled bowel movements after irrigation. CONCLUSIONS: Regular colonic lavage after the ACE procedure allows children with chronic constipation and fecal incontinence to regain normal bowel habits and a markedly improved lifestyle. This procedure should be considered before colostomy in children and adults for the treatment of fecal incontinence from a variety of causes.

Role of calcium homeostasis in gastric mucosal injury and protection.

Using a human gastric mucosal cell line, known as AGS cells, we determined the role that perturbations in intracellular Ca2+ concentration [Ca2+]i might play in cellular injury induced by various damaging agents. For deoxycholate (CD) and ethanol (EtOH) induced damage, a concentration related increase in [Ca2+]i was noted that preceded and closely paralleled the magnitude of injury. Thus, the higher the concentration of DC or EtOH, the more profound were the changes in [Ca2+]i and the resultant degree of cellular injury. Pretreatment with a low concentration of DC (50 microM; called a mild irritant) that was not damaging by itself attenuated injury induced by a damaging concentration (i.e. 250 microM) of DC, and appeared to elicit this protective action through mechanisms that resisted intracellular Ca2+ accumulation. Additional studies indicated that the mechanism of aspirin damage may be similar and that other protective agents such as prostaglandins and growth factors appear to mediate their protective properties through prevention of intracellular Ca2+ alterations. We propose that agents that prevent mucosal injury mediate this activity through a cellular response (involving active Ca2+ efflux) that subsequently provides a protective action by limiting the magnitude of intracellular Ca2+ accumulation.

Cytoskeleton as a target for injury in damaged intestinal epithelium.

This report summarizes the findings of a series of studies undertaken to discern the role of the cytoskeleton in intestinal injury and defense. Two established cell lines were used for these studies. IEC-6 cells (a rat intestinal cell line) were incubated in Eagle's minimal essential medium with and without 16, 16 dimethyl prostaglandin E(2) (dmPGE(2); 2.6 microM) for 15 minutes and subsequently incubated in medium containing 10% ethanol (EtOH). The effects on cell viability and the actin cytoskeleton were then determined. Using a similar protocol, Caco-2 cells (a human colonic cell line) were employed to assess the microtubule cytoskeleton under these conditions. In both cell lines, EtOH extensively disrupted the cytoskeletal component being evaluated coincident with adversely affecting cell viability. Pretreatment with dmPGE(2) increased cell viability and abolished the disruptive effects on both the actin and microtubule cytoskeleton in cells exposed to EtOH. Prior incubation with cytochalasin D, an actin disruptive agent, prevented the protective capabilities of dmPGE(2) in IEC-6 cells challenged with EtOH. Phalloidin, an actin stabilizing agent, demonstrated similar effects to that of dmPGE(2) by stabilizing the actin cytoskeleton and preserving cellular viability in IEC-6 cells in response to EtOH. In Caco-2 cells, taxol, a microtubule stabilizing agent, mimicked the effects of dmPGE(2) by increasing cell viability in cells exposed to EtOH and enhancing microtubular integrity. In contrast, pretreatment with colchicine, an inhibitor of microtubule integrity, prevented the protective effects of dmPGE(2). These findings support the hypothesis that the cytoskeleton may be a major target for injury in damaged intestinal epithelium, and that the protective action of dmPGE(2) is orchestrated through preservation of this target.

Nonsteroidal anti-inflammatory drugs attenuate proliferation of colonic carcinoma cells by blocking epidermal growth factor-induced Ca++ mobilization.

Numerous studies suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit colorectal carcinogenesis. We have previously reported that NSAIDs, in human colonic carcinoma cells (Caco-2), attenuate epidermal growth factor (EGF)-induced cellular proliferation through a process independent of their inhibitory effects on prostaglandin synthesis. Furthermore, separate studies have also suggested that NSAIDs inhibit EGF-induced store-operated Ca++ influx. Thus we developed the hypothesis that NSAIDs may limit the activity of EGF by altering intracellular Ca++ ([Ca++]i) mobilization. Serum-deprived Caco-2 cells were employed for all experimentation. [Ca++]i was measured with Fluo-3 and extracellular Ca++ influx was monitored by quenching Fluo-3 fluorescence with Mn++. Proliferation was quantitated with two assays: cellular nucleic acid and total protein content. Caco-2 cells exposed to EGF demonstrated an initial increase in [Ca++]i which was blocked by neomycin, an inhibitor of IPsubscript 3 generation, and the phospholipase C inhibitor U73122 but not U73343 (inactive control). This was followed by sustained extracellular Ca++ influx, which was attenuated with calcium-free buffer (-Ca++), the store- operated Ca++ channel blocker lanthanum, indomethacin, ibuprofen, and aspirin. In subsequent studies, cells were treated with either serum-free media or EGF +/- the aforementioned inhibitors, and again serum starved. Cells exposed to EGF +/- the inactive phospholipase C inhibitor U73343 demonstrated a significant increase in nucleic acid and protein. However, proliferation induced by EGF was not observed when [Ca++]i elevation was prevented by blocking either internal Ca++ store release via phospholipase C/IPsubscript 3 or sustained Ca++ influx through store-operated Ca++ channels. Sustained [Ca++]i elevation, as induced by EGF, appears to be required for mitogenesis. These data support our premise that one mechanism whereby NSAIDs may attenuate colonic neoplasia is by blocking EGF-induced Ca++ mobilization.

Epidermal growth factor-induced cytoprotection in human intestinal cells involves intracellular calcium signaling.

BACKGROUND: The mechanism(s) whereby epidermal growth factor (EGF) protects against cellular injury remains poorly understood. Previous data in our laboratory have suggested that EGF-induced cellular proliferation in human colonic carcinoma cells (Caco-2) may involve changes in intracellular calcium content ([Ca(2+)](i)). Our current objective was to determine if a similar process was involved with EGF-induced cytoprotection. METHODS: Postconfluent Caco-2 cells were employed for all experimentation. [Ca(2+)](i) was measured with Fluo-3 fluorescence. Injury was measured employing Ethidium homodimer 1 uptake and lactate dehydrogenase (LDH) release. RESULTS: Caco-2 cells pretreated, but not concomitantly treated, with EGF (10-100 ng/ml, 30-60 min) significantly attenuated cellular injury induced subsequently by 500 microM deoxycholate (DC). Cells exposed to 100 ng/ml EGF demonstrated an initial increase in [Ca(2+)](i) (1-5 min) which was blocked with neomycin, an inhibitor of inositol 1,4,5-trisphosphate (IP(3)) generation, and the phospholipase C (PLC) inhibitor U73122, but not U73343 (inactive control). This was followed by sustained extracellular Ca(2+) influx (5-20 min), which was attenuated with calcium-free buffer and the store operated Ca(2+) channel blocker La(3+). [Ca(2+)](i) then returned to baseline (20-30 min), a process blocked with the Ca(2+)-ATPase inhibitors quercetin and vanadate. The above treatments, which in and of themselves did not induce cellular injury, were repeated and cells were subsequently exposed to DC. All groups exposed to 500 microM DC demonstrated significant increases in both Ethidium Homodimer 1 uptake and LDH release. Both indices of injury were significantly decreased when cells were pretreated with EGF +/- the inactive PLC inhibitor U73343. However, protection induced by EGF was lost when any of its effects on changes in [Ca(2+)](i) were prevented: internal Ca(2+) store release via PLC and IP(3), sustained Ca(2+) influx through store operated Ca(2+) channels, or subsequent Ca(2+) efflux. CONCLUSION: Taken together, these data strongly suggest that the cytoprotective effects of EGF may involve Ca(2+) signaling.

Role of actin cytoskeleton in prostaglandin-induced protection against ethanol in an intestinal epithelial cell line.

Prostaglandins (PGs) protect a variety of gastrointestinal cells against injury induced by ethanol and other noxious agents. This investigation attempted to discern the mechanism of cytoprotection as it relates to the relationship between actin and PGs in IEC-6 cells (a rat intestinal epithelial cell line). IEC-6 cells were incubated in Dulbecco's modified Eagle's medium +/- 16,16-dimethyl prostaglandin E(2) (dmPG, 2.6 microM) for 15 min and subsequently incubated in medium containing 1, 2.5, 5, 7.5, and 10% ethanol (EtOH). Cells were then processed for immunocytochemistry using FITC-phalloidin in order to stain the actin cytoskeleton, and cell viability was determined by trypan blue exclusion. Quantitative Western immunoblotting of fractioned G-actin (nonpolymerized; S1) and F-actin (polymerized; S2) was also carried out. EtOH concentrations equal to and greater than 5% led to the collapse of the actin cytoskeleton as depicted by extensive disorganization and fragmentation. In addition, these same EtOH concentrations significantly decreased the S2 fraction and increased the S1 pool of actin. Preincubation with dmPG prevented collapse of the actin cytoskeleton, significantly increased the S2 polymerized fraction as determined by quantitative immunoblotting, and increased cell viability in EtOH-treated cultures. Prior incubation with cytochalasin D, an actin disruptive agent, not only reduced cell viability but also prevented the cytoprotective effects of dmPG. Phalloidin, an actin stabilizing agent, had effects similar to that of dmPG as demonstrated by stability of the actin cytoskeleton and increased cellular viability. Such findings indicate that PGs are important in the organization and stability of actin under in vitro conditions. These effects on actin may play an essential role in the mechanism of PG-induced cytoprotection.

Acute ovarian torsion in children.

BACKGROUND: Acute ovarian torsion (OT) is an uncommon cause of abdominal pain in children and is frequently confused with other conditions. METHODS: We reviewed the records (1983 to 1999) of all children treated for acute OT at our children's hospital. RESULTS: Mean child age (n = 51) was 12.5 +/- 0.3 years. Children presented with either right-sided (n = 29) or left-sided (n = 22) pain. Diagnosis of OT was confirmed preoperatively by ultrasound (73%) or computed tomography (CT) scan (10%) while nine children (17%) with right-sided pain underwent surgery for presumed appendicitis. Despite a relatively short time from diagnosis to surgery, all 51 children required salpingooophorectomy. Contralateral biopsy was performed in 29% and 57% had an appendectomy. Younger children more commonly had either a mature cystic teratoma or torsion with no underlying abnormality as an etiology compared with OT in older children that was more likely to result from either a follicular or corpus luteal cyst. Pathologic examination of the contralateral ovary and appendix was normal in all children who underwent biopsy and appendectomy. CONCLUSION: Ultrasonography with color doppler is helpful for differentiating acute OT from appendicitis. Although the twisted ovary can rarely be salvaged, the etiology is usually benign. Preoperative serum markers and contralateral ovary biopsy may be unnecessary.

Prostatorectal fistula associated with tubular colorectal duplication.

Tubular colonic duplications are exceedingly rare. The authors present an unusual case of a boy with a persistent prostatorectal fistula resulting from a tubular colorectal duplication. The current case is unique for 2 reasons: (1) the presence of a fistula without any concomitant genitourinary anomalies and (2) the existence of a prostatorectal fistula.

Treatment of refractory chylothorax with externalized pleuroperitoneal shunts in children.

BACKGROUND: Traditional therapy for refractory chylothorax in the pediatric population has included pleurodesis and thoracic duct ligation. These procedures are associated with high morbidity and questionable success rates. METHODS: We retrospectively reviewed our experience with 15 patients who underwent treatment for chylous effusions using pleuroperitoneal shunts with exteriorized pump chambers. Mean patient age at time of shunt placement was 2.1 (0.1 to 11.5) years and the most common indication (7 of 15) was refractory chylothorax following surgical correction of congenital heart disease. Mean chylothorax duration before shunt placement was 76 (5 to 810) days and shunts were in place for an average of 104 (12 to 365) days. A total of 19 chylous effusions (pleural or pericardial) were treated with shunts. RESULTS: Nine of 11 right-sided chylothoraces, 5 of 6 left-sided chylothoraces, and 2 of 2 chylopericardia resolved with shunt therapy (84% total). Pleuroperitoneal shunting failed to clear the effusion in 3 children. There were six episodes of shunt malfunction that were repaired and two episodes of infection. Inguinal or umbilical hernia developed in 4 patients. CONCLUSIONS: Externalized pleuroperitoneal shunting is a safe, effective, and minimally invasive treatment for children with refractory chylous effusions.

Calcium accentuates injury induced by ethanol in human gastric cells.

The mechanism(s) whereby ethanol induces cellular injury remains poorly understood. Furthermore, the role of calcium in gastric mucosal injury under in vitro conditions is poorly defined. The major objectives of this study were to (1) define the temporal relationship between intracellular calcium accumulation induced by ethanol and cellular injury, (2) characterize the mechanism(s) whereby ethanol increases cellular calcium content, and (3) determine whether calcium removal would attenuate ethanol-induced cellular injury. Human gastric cells (AGS) were used for all experiments. Sustained intracellular calcium accumulation induced by ethanol, but not transient changes, preceded and directly correlated with cellular injury. Cells exposed to damaging concentrations of ethanol demonstrated an initial calcium surge that appeared to be a consequence of inositol 1,4,5-triphosphate (IP3) generation and subsequent internal store release followed by a sustained plateau resulting from extracellular calcium influx through store-operated calcium channels. Finally, both morphologic (cellular injury) and functional (clearance of bovine serum albumin) changes induced by ethanol were significantly attenuated when extracellular Ca(+&plus) influx was prevented, and further decreased when intracellular Ca(++) stores were depleted. These data indicate that calcium plays a significant role in cellular injury induced by ethanol.

Rollover injuries in residential driveways: age-related patterns of injury.

BACKGROUND: The major objective of the present study was to determine the severity of nonfatal injuries sustained by children (<16 years old) when a motor vehicle rolls over them. We also sought to determine whether younger children (<24 months old) demonstrated different patterns of injury and/or a worse outcome, compared with older children (>24 months old). METHODS: We reviewed the medical records of 3971 consecutive admissions to a single trauma service at an urban children's hospital between March 1990 and October 1994. During this time period, 26 (0.7%) children presented with rollover injuries incurred by motor vehicles in residential driveways. Outcome was measured by length of both intensive care unit admission and hospitalization. RESULTS: Two children died shortly after admission and were excluded from the remainder of the study. Younger children (<24 months old) had significantly higher injury severity scores and lower pediatric trauma scale scores. Both the duration in the intensive care unit and the length of hospitalization were significantly longer in younger children, compared with children >24 months old. One explanation for these observations was that younger children had a significantly higher incidence of both head and neck and extremity injury but a similar incidence and severity of chest and abdominal trauma, compared with older children. Injuries requiring operative intervention were rare. CONCLUSION: Younger patients sustaining rollover injuries in the residential driveway have a worse outcome, in part, because of the head and neck or extremity injures that they incur. The majority of rollover injuries can be managed conservatively. pediatric trauma, driveway, pedestrian events, rollover injuries, injury severity score, pediatric trauma scale.

The impact of intraoperative culture on treatment and outcome in children with perforated appendicitis.

BACKGROUND: Most protocols for the operative treatment of perforated appendicitis use a routine culture. Although isolated studies suggest that routine culture may not be necessary, these recommendations generally are not based on objective outcome data. METHODS: The authors reviewed the records of 308 children who underwent operative treatment for perforated appendicitis between 1988 and 1998 to determine if information gained from routine culture changes the management or improves outcome. Inclusion criteria included either gross or microscopic evidence of appendiceal perforation. RESULTS: Mean patient age was 7.5 years, 51% were boys, and there was no mortality. The majority of children (96%) underwent culture that was positive for either aerobes (21%), anaerobes (19%), or both (57%). Antibiotics were changed in only 16% of the patients in response to culture results. The use of empiric antibiotics, as compared with modified antibiotics, was associated with a lower incidence of infectious complication, shorter fever duration, and decreased length of hospitalization. We also investigated the relationship between culture isolates and antibiotic regimens with regard to outcome. The utilization of antibiotics suitable for the respective culture isolate or organism sensitivity was associated with an increased incidence of infectious complication and longer duration of both fever and length of hospitalization. Finally, the initial culture correlated poorly with subsequent intraabdominal culture (positive predictive value, 11%). CONCLUSION: These outcome data strongly suggest that the practice of obtaining routine cultures can be abandoned, and empiric broad spectrum antibiotic coverage directed at likely organisms is completely adequate for treatment of perforated appendicitis in children.

Nonsteroidal anti-inflammatory drugs attenuate epidermal growth factor-induced proliferation independent of prostaglandin synthesis inhibition.

BACKGROUND: The mechanism(s) whereby nonsteroidal anti-inflammatory drugs (NSAIDs) attenuate colorectal tumor growth remains poorly understood. This study determined if NSAIDs decreased epidermal growth factor (EGF)-induced proliferation in human colonic tumor (Caco-2) cells and whether this process involved the inhibition of prostaglandin (PG) synthesis. METHODS: Caco-2 cells were serum-starved (48 h) and subsequently treated (48 h) with either serum-free media or EGF (10 ng/ml) +/- physiologic and noninjurious (as determined by LDH release) concentrations of aspirin, indomethacin, and ibuprofen. PG synthesis was measured by EIA. Proliferation was quantitated with two assays: cellular protein and nucleic acid content. RESULTS: NSAID treatment did not inhibit growth in cells treated with only serum-free media. Cells exposed to EGF demonstrated a significant increase in PGE2, protein, and nucleic acid. Levels of other eicosanoids (PGI2, TXA2) were minimal both before and after EGF treatment. Despite varying degrees of PGE2 inhibition, each NSAID group equally attenuated EGF-induced protein and nucleic acid synthesis. The correlation between PGE2 levels and protein (R2 = 0.56) or nucleic acid (R2 = 0.54) was poor. Finally, the addition of a physiologically appropriate concentration of exogenous PGE2 failed to reverse NSAID-induced growth inhibition. CONCLUSION: These data suggest that NSAIDs, independent of PG synthesis inhibition, attenuate EGF-induced proliferation in Caco-2 cells. This may provide one explanation for how NSAIDs limit colonic neoplasia.

Prostaglandins protect human intestinal cells against ethanol injury by stabilizing microtubules: role of protein kinase C and enhanced calcium efflux.

Prostaglandins (PG) protect gastrointestinal cells against damage induced by ethanol (EtOH) and other noxious agents, a process termed cytoprotection. The present study investigated the relationships between microtubule (MT) stability, protein kinase C (PKC) activation, and calcium efflux as a possible mechanism of PG's protective action using a human colonic cell line (Caco-2) exposed to known damaging concentrations of EtOH (7.5% and 10%). Preincubation of Caco-2 cells with 16,16-dimethyl-PGE2 (PG, 2.6 microM) significantly increased PKC activity in these cells. Pretreatment of Caco-2 cells with 50 microM OAG (a synthetic diacylglycerol and PKC activator) or 30 nM TPA (a direct PKC activator) prior to exposure to 7.5% or 10% EtOH for 5 min significantly reduced cell injury, as determined by trypan blue exclusion, and increased MT stability, as confirmed by confocal microscopy. Pretreatment of Caco-2 cells with 4 alpha-PDD (an inactive phorbol ester, 20 nM) failed to prevent cell injury and disruption of the MT cytoskeleton. Preincubation with staurosporine (a PKC inhibitor, 3 nM) abolished the protective effects of PG in cells exposed to 7.5% and 10% EtOH. Incubation of Caco-2 cells with A23187 (a Ca2+ ionophore), similar to 10% EtOH, caused a significant reduction in cell viability and MT stability. Preincubation with A23187 in combination with PG or OAG prior to subsequent exposure to EtOH significantly abolished the protective effects of PG or OAG pretreatment. Finally, pretreatment with OAG, TPA, or PG resulted in significant increases in calcium-45 efflux, which correlated with increased stability of the MT cytoskeleton. These data suggest that PG possesses direct protective effects against EtOH injury in Caco-2 cells and may act by stabilizing MT through the PKC signal transduction pathway and/or stimulation of calcium efflux from the cells.

A simplified method for studying hypoxia and reoxygenation injury under in vitro conditions.

A hypoxia chamber was constructed which allowed for the sequential sampling and blood gas analysis of buffer bathing cells in culture which were subjected to graded periods of hypoxia. Following hypoxia, the human fetal small intestinal cells (CCL-241) were placed into a normoxic environment for the remainder of a 24 h study period. A cytotoxicity assay revealed significant mortality in cells subjected to hypoxia and reoxygenation, but not in those subjected to hypoxia alone. Analysis of lactate dehydrogenase release into buffer samples also indicated a greater cellular injury among cells exposed to hypoxia and reoxygenation. Additionally, levels of lipid peroxidation products were found to be significantly elevated in cells exposed to periods of hypoxia followed by reoxygenation, but not hypoxia alone, as measured by a thiobarbituric acid fluorometric assay. This suggests that lipid peroxidation mediated by oxygen-derived free radical species is the mechanism of injury in these cells. This study demonstrates that such a chamber provides a more precise way to monitor hypoxia and is a useful tool for studying hypoxia and reoxygenation under in vitro conditions.