Role of substance P in several models of bladder inflammation

Substance P (SP) is a peptide found in the sensory nervous system which has multiple biologic effects including stimulation of muscle contraction, pain nociception, immune cell functions, plasma extravasation and a constellation of inflammatory effects. Here we investigate the role of SP in several animals models of bladder inflammation. Using the female Lewis rat, inflammation was induced using either xylene, lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (polyIC). Inflammation occurred rapidly (4 h) and was maintained in each model for at least 7 days. Each of these protocols decreased the bladder content of immunoreactive SP by approximately 50%, suggesting enhanced release. There was no change in the urinary frequency of these animals over 3 weeks, suggesting that urinary frequency changes are not mediated by acute inflammation. We also found that the SP receptor (NK₁) antagonist, (?)CP96345, was unable to block the inflammation produced by polyIC, suggesting that SP is not an obligatory mediator of immune cell stimulation in this model.     

Simple suction drainage: an adjunct to septal surgery

Postoperative hematoma is a complication of septal surgery that can be associated with significant morbidity and possibly even mortality in some cases. A simple suction drainage technique using a scalp vein cannula and vacuum tube can prevent a septal hematoma without the need for bulky nasal packing (with its attendant nasal obstruction and risk of bleeding on removal). The technique is also useful for postoperative drainage of an established septal hematoma or abscess secondary to trauma.     

Is diagnostic peritoneal lavage for blunt trauma obsolete?

Diagnostic peritoneal lavage was 97 percent accurate, with a 2 percent false positive rate and a 1 percent false negative rate in this series of 414 patients. The ease, safety, and accuracy of diagnostic peritoneal lavage justify its continued use in evaluating these patients. Recent studies show computerized tomography (CT) can be highly accurate in detecting intra-abdominal injuries after blunt trauma. We reviewed our experience with diagnostic peritoneal lavage (DPL) to evaluate whether the accuracy, safety, speed, and cost justified its continued use. Four hundred fifteen DPLs were performed on 414 patients from February 1, 1983, through December 31, 1987. All DPLs were done by the open technique. The lavage was considered grossly positive if 10 cc gross blood were aspirated. If there were greater than 100,000 red blood cells (RBC)/mm3, greater than 500 white blood cells (WBC)/mm3, elevated amylase or bilirubin, or bacteria or vegetable fibers the lavage was microscopically positive. There were no cases with elevated bilirubin, amylase, or presence of bacteria. All four cases with "rare vegetable fibers" were false positive. Six DPLs were for penetrating trauma to the lower chest or back. There were 291 negative lavages, including five false negatives (1%), and 124 positive DPLs, including seven false positives (2%), resulting in a crude accuracy of 97 percent. Three of the five false negative lavages had a ruptured diaphragm as the only intra-abdominal injury. There was one minor complication. DPL was usually performed in the trauma resuscitation room during the secondary survey. At our institution, the total fees for DPL are +185 less than the fees for CT. DPL is accurate, rapid, safe, and avoids the disruption of patient care that results in the radiology suite. DPL remains our procedure of choice for evaluating blunt abdominal trauma in the adult.     

Microelectrode measurements of the effects of basolateral adenosine in polarized human intestinal epithelial cells in culture

 Activation of the basolateral receptor for adenosine in HT-29cl.19A cells, by 100 μM adenosine, increased the equivalent short-circuit current (ΔI _sc= 24±2 μA/cm²), depolarized the intracellular potential (ΔV _a= 26±2 mV) and decreased the fractional apical membrane resistance (ΔfR _a=–0.48). The changes in all parameters reached their peak values simultaneously. This suggests that the primary action of the adenosine-activated pathway is on only one membrane. Bumetanide inhibited the transepithelial response and repolarized the cell potential. After preincubation with 100 μM forskolin, application of 300 μM adenosine caused a significant further change in V _a, I _sc, the transepithelial potential (V _t) and fR _a. Together with the results from ion-replacement studies, the observations indicate that adenosine activates channels other than the cystic fibrosis transmembrane conductance regulator (CFTR). The rank order of potencies of adenosine and adenosine analogues implies that the receptor is of the A₂ subtype. Preincubation with 4-bromophenacyl bromide (4-BPB) inhibited the effect of an adenosine analogue by 50%, indicating that activation of phospholipase A₂ may be involved in the adenosine-induced response. Received: 5 August 1998 / Received after revision: 12 October 1998 / Accepted: 5 November 1998     

Neurotensin stimulation of mast cell secretion is receptor-mediated, pertussis-toxin sensitive and requires activation of phospholipase C

Pretreatment of isolated rat serosal mast cells with U-73122, an aminosteroid inhibitor of phospholipase C, inhibited histamine secretion in response to neurotensin (NT). This inhibition reached a maximum after 1 h of pretreatment at 37 degrees C and was dependent upon the concentration of U-73122 (IC50 approximately 0.2 microM). The inactive analog, U-73343, had no effect on the secretory response to NT. Pretreatment of mast cells with U-73122 also blocked histamine secretion in response to substance P (SP), mastoparan (MP), compound 48/80, or amidated NT (NT-NH2). Stimulation of mast cells by NT was accompanied by a rise in the level of intracellular free calcium and a rapid (within seconds) increase in the level of inositol trisphosphate (IP3) which was inhibited by pretreatment of the cells with U-73122. Pretreatment of isolated mast cells with pertussis toxin (PTx) blocked histamine release in response to NT as well as to all peptides tested. PTx had no effect on histamine secretion elicited by anti-IgE stimulation of sensitized mast cells. Pretreatment of mast cells with SR 48692, a NT-receptor antagonist, had no effect on histamine release induced by MP. At a high concentration (100 nM) SR 48692 partially inhibited the response to NT-NH2. These results, together with our earlier findings with SR 48692, indicate that the signal transduction pathway in mast cells activated by NT requires a specific NT-receptor, the activation of phospholipase C, and the involvement of a PTx sensitive G protein. The peptides SP and MP, and compound 48/80, while also requiring the activation of PLC and a PTx sensitive G protein, are not inhibited by the NT-R antagonist, SR 48692, suggesting that they exert their actions either via a different mast cell receptor or via a receptor-independent mechanism.     

Formation of histamine-releasing activity from albumin by medium conditioned by endotoxin-stimulated rat peritoneal macrophages.

Incubation of bovine serum albumin (BSA), rat serum albumin or rat plasma with medium conditioned by endotoxin stimulated rat peritoneal macrophages produced an activity that released histamine from isolated rat serosal mast cells. The amount of histamine-releasing activity (HRA) produced increased with the length of the incubation period, with the concentration of albumin, with the number of macrophages stimulated, and with the duration of exposure of the macrophages to endotoxin. Moreover, the formation of the HRA showed a dependency on the pH of the incubation medium with an optimum at pH 4.5. Boiling the medium conditioned by stimulated macrophages before its incubation with albumin or including the acid protease inhibitor, pepstatin with the conditioned medium prevented the formation of HRA. The generation of HRA was not inhibited by pretreatment of the macrophages with the inhibitor of protein synthesis, cycloheximide. Media from macrophages not stimulated with endotoxin failed to generate HRA. Histamine release from mast cells in response to the HRA was inhibited by pretreatment of the cells with antimycin A and deoxyglucose or by preincubation in Ca-free Locke's solution containing a calcium chelating agent. When injected intradermally into anesthetized Evan's Blue treated rats, the generated HRA produced a change in vascular permeability that was prevented by the H1 antagonist, diphenhydramine. Treatment of the HRA with carboxypeptidase A reduced its ability to stimulate histamine release from mast cells. Histamine-Releasing Peptide (HRP), a neurotensin-related octapeptide, shown previously by us to be formed by the action of cathepsin D or pepsin on albumin, was identified by radioimmunoassay in acid:acetone extracts of the histamine-releasing activity. It is concluded that the formation of HRA is due to the actions of enzymes released from macrophages acting on albumin. It is suggested that such histamine-releasing activity could be formed during the later stages of the inflammatory response and that HRP is one of the peptides present.     

A neurotensin antagonist, SR 48692, inhibits colonic responses to immobilization stress in rats.

We previously reported that short-term immobilization stress of rats causes increased colonic mucin release, goblet cell depletion, prostaglandin E2 secretion, and colonic mast cell activation, as well as increased colonic motility. The purpose of this study was to investigate whether neurotensin (NT), a peptide expressed in both brain and digestive tract, participates in these responses. Rats were pretreated with SR 48692 (1 mg/kg, i.p.), an NT antagonist, 15 min before immobilization (30 min). The administration of the antagonist significantly inhibited stress-mediated secretion of colonic mucin, prostaglandin E2, and a product of rat mast cells, rat mast cell protease II (P < 0.05), but did not alter the increase in fecal pellet output caused by immobilization stress. Immobilization stress also resulted in a quantifiable decrease in the abundance of NT receptor mRNA in rat colon compared with that in colonic tissues from nonimmobilized rats as measured by densitometric analysis of in situ hybridization studies (P < 0.03). We conclude that the peptide NT is involved in colonic goblet cell release and mucosal mast cell activation after immobilization stress.     

Alcohol and fatty acid stimulation of neurotensin release from rat small intestine

We have previously reported that neurotensin (NT) is released from the small intestine and elevated in the hepatic-portal circulation in response to the perfusion of the small intestine with a micellar solution of oleic acid. In order to determine the minimum acyl chain length and whether the presence of a carboxylic acid is necessary for the stimulation of NT release, the small intestine of anesthetized rats was perfused with test solutions of fatty acids of 2-, 4-, 8-, or 18-carbons or fatty alcohols of 2-, 4-, or 8-carbons at a concentration of 1 mM prepared in 2.4 mM taurodeoxycholate in 0.9% NaCl. Blood samples, collected from the superior mesenteric vein immediately before the start of the test perfusion and at 15-min intervals thereafter, were extracted immediately and radioimmunoassayed for NT-like immunoreactivity (NTLI) with a C-terminal-directed antiserum. Perfusions of fatty acids with 4 or more carbons and alcohols of 2 or more carbons resulted in a significant elevation (P less than 0.05) in plasma levels of NTLI above the values obtained before the onset of perfusion. Perfusions with ethanol resulted in a value of 4.3 +/- 0.03 mg/dl (SEM) in blood from the superior mesenteric vein while there was no increase in ethanol levels in the peripheral circulation. Perfusion with taurodeoxycholate and 0.9% NaCl alone had no significant effect on plasma levels of the NTLI. In order to characterize the chemical nature of the elevated NTLI, plasma samples from animals perfused with test solution were collected, extracted, pooled, and subjected to HPLC. NT and its N-terminal metabolite, NT(1-8), were quantitated. NT was defined as material having the same retention time as synthetic NT standard and having comparable measurements using N- and C-terminal-directed antisera. Perfusions of fatty acids of four or more carbons and alcohols of two or more carbons resulted in a 2- to 4-fold increase of both NT and NT(1-8) levels in plasma. It is particularly interesting that perfusion with ethanol (2-carbons) causes an elevation in plasma NT, because perfusion with acetic acid (2-carbons) does not increase NTLI. The fact that perfusion of ethanol is effective in releasing intestinal NT suggests that NT may mediate some of the biological effects observed after the consumption of alcohol.     

Failure to control an outbreak of qnrA1-positive multidrug-resistant Enterobacter cloacae infection despite adequate implementation of recommended infection control measures

A large outbreak with an aminoglycoside-resistant Enterobacter cloacae (AREC) clone occurred at the University Medical Center Utrecht beginning in 2001 and continued up through the time that this study was completed. This clone (genotype I) contains a conjugative R plasmid carrying the qnrA1, bla(CTX-M-9), and aadB genes, encoding resistance to quinolones, extended-spectrum beta-lactamases, and aminoglycosides, respectively. The aim of this study was to determine whether this clone was more transmissible than other AREC strains. Therefore, the dissemination of this genotype and of other E. cloacae strains was studied. In addition, infection control measures taken were evaluated. Pulsed-field gel electrophoresis analysis divided the 191 AREC strains into 42 different genotypes, of which 5 (12%) involved at least three patients. Aside from this outbreak (133 patients), only two other small outbreaks occurred, showing that the infection control measures were successful for all strains but one. Among 324 aminoglycoside-susceptible E. cloacae strains, 34/166 (20%) genotypes were identified from at least three patients, but only 4 involved small outbreaks. The outbreak strain was also detected in 11 of 15 other Dutch hospitals and caused outbreaks in at least 4. Evaluation of infection control measures showed that the outbreak strain disseminated throughout the hospital despite adequate implementation of internationally accepted guidelines on the control of multidrug-resistant Enterobacteriaceae (MRE). In conclusion, some MRE strains are better able to spread than others, and these strains may not be controlled by the current infection control guidelines. Strategies to identify such strains in an early phase and adapted guidelines for such "superbugs" are needed to prevent these clones from becoming endemic.