Contractile profile of esophageal and gastric fundus strips in experimental doxorubicin-induced esophageal atresia

Esophageal atresia (EA) is characterized by esophageal and gastric motility changes secondary to developmental and postsurgical damage. This study evaluated the in vitro contractile profile of the distal esophagus and gastric fundus in an experimental model of EA induced by doxorubicin (DOXO). Wistar pregnant rats received DOXO 2.2 mg/kg on the 8th and 9th gestational days. On day 21.5, fetuses were collected, sacrificed, and divided into groups: control, DOXO without EA (DOXO-EA), and DOXO with EA (DOXO+EA). Strips from the distal esophagus and gastric fundus were mounted on a wire myograph and isolated organ-bath system, respectively, and subjected to increasing concentrations of carbamylcholine chloride (carbachol, CCh). The isolated esophagus was also stimulated with increasing concentrations of KCl. In esophagus, the concentration-effect curves were reduced in response to CCh in the DOXO+EA and DOXO-EA groups compared to the control group (P<0.05). The maximum effect values (Emax) for DOXO+EA and DOXO-EA were significantly lower than control (P<0.05), but the half-maximal effective concentration (EC50) values were not significantly different when the three groups were compared (P>0.05). In response to KCl, the distal esophagus samples in the three groups were not statistically different with regard to Emax or EC50 values (P>0.05). No significant difference was noted for EC50 or Emax values in fundic strips stimulated with CCh (P>0.05). In conclusion, exposure of dams to DOXO during gestation inhibited the contractile behavior of esophageal strips from offspring in response to CCh but not KCl, regardless of EA induction. The gastric fundus of DOXO-exposed offspring did not have altered contractile responsiveness to cholinergic stimulation.

Contractile profile of esophageal and gastric fundus strips in experimental doxorubicin-induced esophageal atresia.

Esophageal atresia (EA) is characterized by esophageal and gastric motility changes secondary to developmental and postsurgical damage. This study evaluated the in vitro contractile profile of the distal esophagus and gastric fundus in an experimental model of EA induced by doxorubicin (DOXO). Wistar pregnant rats received DOXO 2.2 mg/kg on the 8th and 9th gestational days. On day 21.5, fetuses were collected, sacrificed, and divided into groups: control, DOXO without EA (DOXO-EA), and DOXO with EA (DOXO+EA). Strips from the distal esophagus and gastric fundus were mounted on a wire myograph and isolated organ-bath system, respectively, and subjected to increasing concentrations of carbamylcholine chloride (carbachol, CCh). The isolated esophagus was also stimulated with increasing concentrations of KCl. In esophagus, the concentration-effect curves were reduced in response to CCh in the DOXO+EA and DOXO-EA groups compared to the control group (P<0.05). The maximum effect values (Emax) for DOXO+EA and DOXO-EA were significantly lower than control (P<0.05), but the half-maximal effective concentration (EC50) values were not significantly different when the three groups were compared (P>0.05). In response to KCl, the distal esophagus samples in the three groups were not statistically different with regard to Emax or EC50 values (P>0.05). No significant difference was noted for EC50 or Emax values in fundic strips stimulated with CCh (P>0.05). In conclusion, exposure of dams to DOXO during gestation inhibited the contractile behavior of esophageal strips from offspring in response to CCh but not KCl, regardless of EA induction. The gastric fundus of DOXO-exposed offspring did not have altered contractile responsiveness to cholinergic stimulation.

Extended hiatoplasty: early experience with a simple technique to increase the intraabdominal esophageal length in complicated gastroesophageal reflux.

PURPOSE: The aim of this paper is to describe the experience of the Division of Pediatric Surgery of State University of Campinas Medical School with a simple technique of extended hiatoplasty to achieve intraabdominal placement of the distal esophagus in difficult situations. METHODS: From April 1997 to November 1999, 7 patients who had either complicated or recurrent gastroesophageal reflux (GER) underwent open (2 patients) or laparoscopic (5 patients) correction of GER that included an extended hiatoplasty. All had undergone previous unsuccessful clinical or surgical treatment. To investigate the severity of the disease, diagnostic endoscopy or barium swallow were performed before surgery. Postoperatively, these children underwent clinical evaluation or any additional diagnostic procedure deemed necessary. RESULTS: Using the extended hiatoplasty, a good length of intraabdominal esophagus could be achieved in every patient. No complications resulted from the procedure itself. There was a late instance of paraesophageal hernia with recurrence of GER attributable to disruption of the hiatoplasty, which was rerepaired through a laparoscopic approach. Symptomatic improvement was observed in all but the patient with caustic stricture. CONCLUSIONS: Extended hiatoplasty is a simple maneuver that may represent a good option to increase the length of intraabdominal esophagus in patients with a short esophagus secondary to severe GER disease, being associated with a high success rate and low morbidity. J Pediatr Surg 36:555-558.

Beckwith-Wiedemann syndrome and virilizing cortical adrenal tumor in a child.

The authors report a case of a virilizing adrenal tumor that developed in a 2-year-old child with Beckwith-Wiedemann syndrome (BWS). He had a fetal diagnosis of omphalocele and a history of neonatal adrenal cysts. The importance of prenatal diagnosis of BWS and postnatal follow-up of tumors is discussed. The differential diagnosis of adrenal pathologies occurring in BWS also is reviewed.

The involvement of macrophage-derived tumour necrosis factor and lipoxygenase products on the neutrophil recruitment induced by Clostridium difficile toxin B.

Clostridium difficile (Cd) toxins appear to mediate the inflammatory response in pseudomembranous colitis and/or colitis associated with the use of antibiotics. In contrast to Cd Toxin A (TxA), Cd Toxin B (TxB) has been reported not to promote fluid secretion or morphological damage in rabbits and hamsters and also does not induce neutrophil chemotaxis in vitro. However, TxB is about 1000 times more potent than TxA in stimulating the release of tumour necrosis factor-alpha (TNF-alpha) by cultured monocytes. In the present study, we investigated the ability of TxB to promote neutrophil migration into peritoneal cavities and subcutaneous air-pouches of rats. We also examined the role of resident peritoneal cells in this process as well as the inflammatory mediators involved. TxB caused a significant and dose-dependent neutrophil influx with a maximal response at 0.1 microgram/cavity after 4 hr. Depleting the peritoneal resident cell population by washing the peritoneal cavity or increasing this population by pretreating the animals with thioglycollate blocked and amplified the TxB-induced neutrophil migration, respectively. Pretreating the animals with MK886 (a lipoxygenase inhibitor), NDGA (a dual cyclo- and lipoxygenase inhibitor) or the glucocorticoid, dexamethasone, but not with indomethacin (a cyclo-oxygenase inhibitor), or BN52021 (a platelet-activating factor antagonist), inhibited the neutrophil migration evoked by TxB. Pretreatment with dexamethasone or the administration of anti-TNF-alpha serum into the air-pouches also significantly reduced the TxB-induced neutrophil migration. Supernatants from TxB-stimulated macrophages induced neutrophil migration when injected into the rat peritoneal cavity. This effect was attenuated by the addition of either MK886 or dexamethasone to the macrophage monolayer and by preincubating the supernatants with anti-TNF-alpha serum. TxB also stimulated the release of TNF-alpha by macrophages. Overall, these results suggest that TxB induces an intense neutrophil migration which is mediated by macrophage-derived TNF-alpha and lipoxygenase products.

Role of tumor necrosis factor and nitric oxide in the cytotoxic effects of Clostridium difficile toxin A and toxin B on macrophages.

Clostridium difficile, the bacterium involved in antibiotic-associated colitis, produces two exotoxins, toxin A (TxA) and toxin B (TxB). Although these toxins are well recognized as being cytotoxic to several mammalian cell types, the mechanisms involved are not fully understood. The aim of the present investigation was to examine the cytotoxicity of TxA and TxB to peritoneal macrophages in culture and to investigate whether tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) are involved in the process. As a control, the effect of E. coli LPS was also investigated. TxA, TxB and LPS were dose-dependently cytotoxic to macrophage monolayers, with TxB being the most potent. All of the toxins stimulated the release of TNF-alpha from macrophages. TxB was again the most active in inducing this response. The TNF-alpha released appears to be involved in the action of LPS and TxA, but not of TxB, since a mAb against TNF-alpha inhibited the cytotoxicity of the former two but had no effect on the latter. NO is not involved in the effects of TxA and TxB since these toxins did not induce the production of this mediator in macrophages, even in the presence of IFN-gamma. In addition, L-imino-ethyl-L-ornithine (L-NIO), a NO synthase inhibitor, did not modify the macrophage death caused by TxA or TxB. Although LPS was able to induce the production of high amounts of NO, NO did not mediate the LPS cytotoxicity since L-NIO did not influence the degree of macrophage death caused by LPS. TxA and TxB therefore appear to exert cytotoxic effects on cultured macrophages by different mechanisms. TNF-alpha is involved in TxA and LPS-mediated cytotoxicity but not in the toxicity caused by TxB. NO is not involved in the killing action of any of these toxins.

Role of pertussis toxin A subunit in neutrophil migration and vascular permeability.

The anti-inflammatory activity of pertussis toxin (Ptx) was compared to that of a noncatalytic mutant of pertussis toxin (9K/129G; Ptxm), which contains two amino acid substitutions in the A protomer, by using a rat model of inflammation. The toxins were administered intravenously 1 h prior to the injection of inflammatory stimuli. Ptx, but not Ptxm, inhibited neutrophil migration into peritoneal cavities in response to formyl-methionyl-leucyl-phenylalanine and lipopolysaccharide. The inhibitory effect of Ptx on neutrophil migration could not be explained by the ability of the toxin to induce leukopenia or neutropenia. The increase in skin vascular permeability induced by leukotriene B4, a powerful neutrophil chemotactic agent, was also inhibited only by Ptx. On the other hand, the increase in skin vascular permeability induced by histamine was potentiated by both toxins. These data show that Ptx inhibits neutrophil-mediated inflammation in vivo and that this effect is dependent on the ADP-ribosyltransferase activity of the A protomer.

Pertussis toxin from Bordetella pertussis blocks neutrophil migration and neutrophil-dependent edema in response to inflammation.

Pertussis toxin (Ptx) is a hexameric protein with classical AB architecture produced by Bordetella pertussis. The aim of this study was to investigate the effect of Ptx on migration of polymorphonuclear leukocytes to site of inflammation and on cell-dependent edema. Ptx was purified from the supernatant of the culture medium of B. pertussis using hydroxylapatite chromatography and fetuin affinity chromatography. Ptx induced a maximal clustering of Chinese hamster ovary cells at concentrations as low as 0.1 ng/ml. Intravenous injection of Ptx (400 ng) significantly blocked the neutrophil migration induced by 200 ng of lipopolysaccharide (LPS from E. coli O111:B4; 2.27 +/- 0.13 vs 0.61 +/- 0.16 per 10(6) neutrophils/ml; P < 0.001; N = 5) and by 200 ng of formyl-methionyl-leucyl-phenylalanine (fMLP; 2.53 +/- 0.45 vs 0.75 +/- 0.14 per 10(6) neutrophils/ml; P < 0.01; N = 6) into the peritoneal cavities of male Wistar rats (weighing 150-180 g). In addition, Ptx (400 ng) pretreatment also blocked the edema induced by intraplantar injection of 100 micrograms carrageenin (delta increase in volume: 0.667 +/- 0.087 vs 0.313 +/- 0.058 ml; P < 0.01; N = 5) but not the edema induced by 100 micrograms dextran (delta increase in volume: 0.537 +/- 0.06 vs 0.385 +/- 0.076 ml; P > 0.05; N = 5). These data demonstrate that Ptx blocked neutrophil migration induced by a direct fMLP stimulus of a site of inflammation. In addition, this toxin blocks the indirect stimulus of LPS on neutrophil migration.(ABSTRACT TRUNCATED AT 250 WORDS)

Pertussis toxin from Bordetella pertussis blocks neutrophil migration and neutrophil-dependent edema in response to inflammation

Pertussis toxin (Ptx) is a hexameric protein with classical AB architecture produced by Bordetella pertussis. The aim of this study was to investigate the effect og Ptx on migration of polymorphonuclear leukocytes to site of inflamation and on cell- dependent edema. Ptx was purified from the supernatant of the culture medium of B. pertussis using hydroxylapatite chromatography and fetuin affinity chromatography. Ptx induced a maximal clusterin of Chinese hamster ovary cells at concentration as low as 0.1 ng/ ml. Intravenous inection of Ptx (400 ng) significantly blocked the neutrophil migration induced by 200 ng of lipopolysaccharide (LPS from E. coli O111:B4; 2.27 ñ 0.13 vs 0.61 ñ 0.16 per 10**6 neutrophils/ml; P < 0.001; N = 5) and by 200 ng of formylmethionyl-leucyl-phenylalanine(fMLP; 2.53 ñ 0.45 vs 0.75 ñ 0.14 per 10**6 neutrophils/ml; P < 0.01; N=6) into the peritoneal cavities of male Wistar rats (eighing 150-180). In addition, Ptx (400ng) pretreatment also blocked the edema induced by intraplantar injection of 100 µg carrageenin ( increase in volume: 0.667 ñ 0.087 vs 0.313 ñ 0.058 ml; P < 0.01; N = 5) but not the edema induced by 100 µg dextran ( increase in volume: 0.537 ñ 0.06 vs 0.385 ñ 0.076 ml; P > 0.05; N = 5). These data demonstrate that Ptx blocked neutrophil migration induced by a direct f MLP stimulus of a site of inflammation. In addition, this toxin blocks the indirect stimulus of LPS on neutrophil migration. Furthermore, Ptx also inhibits the neutrophil-dependent edema induced by carrageenin, but not the edema induced by dextran that is in part dependent on basophil cells. These results warrant further studies on the mechanisms of Ptx inhibition of neutrophil-dependent edema and cell migration