Ricardo Saban

Inhibition of Allergic Reactions with Antibodies to IgE

Numerous clinical studies show that direct interference with the IgE response leads to a decrease or elimination of allergic symptoms. The aim of these studies was to design a therapy aimed at decreasing IgE levels in order to ameliorate atopic disease. To this end, a murine monoclonal antibody, MAE11, directed against IgE was identified, which had all the properties necessary to interfere with IgE responses, but lacked the harmful side effects of inducing receptor cross-linking. The antibody was selected on the basis of its ability to bind circulating IgE at the same site as the high-affinity receptor, thus blocking the binding of IgE to mast cells and basophils. To allow for possible chronic administration and to avoid the problems of antigenicity, MAE11 was humanized. The best of several humanized variants, version 25 (rhumAb-E25) was selected since it possessed binding affinity and biological activity comparable to MAE11. Clinical studies are underway to determine the safety and efficacy of this treatment for allergic rhinitis and asthma.

MAST CELLS MEDIATE THE SEVERITY OF EXPERIMENTAL CYSTITIS IN MICE

PURPOSEnWe hypothesized that experimental cystitis induced by substance P (SP) or E. coli lipopolysaccharide (LPS) would be less severe in mice rendered mast cell deficient by genetic manipulation.nnnMATERIALS AND METHODSnTwo strains of mast-cell deficient mice (WBB6F1- kitW/kitW-v or kitW/kitW-v and WCB6F1-Sl/Sld or Sl/Sld) and their congenic, normal (+/+) counterparts were used. Cystitis was induced in female mice by intravenous injection of SP (0.1 ml.; 10(-6) M) or E. coli LPS (0.1 ml.; 2 mg./ml.), and inflammation was assessed by Evans blue dye extravasation. In a separate group of kitW/kitW-v and congenic normal mice, cystitis was induced by intravesical infusion of SP (0.05 ml.; 10(-5) M) or E. coli LPS (0.05 ml.; 100 microg./ml.) and compared with intravesical pyrogen-free saline (0.05 ml.; 0.9%). Severity of cystitis was determined by histological evaluation of the bladder wall 24 hours after intravesical infusions.nnnRESULTSnIntravenous SP or LPS stimulated increased plasma extravasation in congenic normal mice but not in mast cell-deficient mice. Intravesical SP or LPS resulted in increased edema, leukocytic infiltration, and hemorrhage within the bladder wall in congenic normal mice, but the only histological evidence of inflammation in the bladders of kitW/kitW-v mice was increased hemorrhage in response to LPS.nnnCONCLUSIONSnThis study indicates that mast cells modulate the inflammatory response of the bladder to SP and LPS in mice. Although clinical trials of the use of antihistamines to treat or prevent cystitis have not been successful, these results suggest that therapies directed toward preventing mast cell activation may yet prove effective in treating cystitis.

Involvement of leukotrienes, TNF-alpha, and the LFA-1/ICAM-1 interaction in substance P-induced granulocyte infiltration.

Substance P (SP) has been shown to mediate granulocyte infiltration into the mouse skin by inducing mast cell degranulation. In this study, using a variety of specific inhibitors, we investigated the cascade of events involved in the response of neutrophils and eosinophils to SP. The prostaglandin inhibitor, indomethacin, had little effect on SP‐induced leukocyte migration. In contrast, pretreatment with the leukotriene (LT) synthesis inhibitor, A‐64077, completely blocked neutrophil but not eosinophil migration in response to SP. Participation of tumor necrosis factor α (TNF‐α) and LFA‐1/ICAM‐1 interaction was confirmed by inhibition of SP‐induced leukocyte migration by pretreatment of mice with monoclonal antibodies to TNF‐α, LFA‐1, and ICAM‐1. Moreover, alteration in leukocyte migration by indomethacin was found to depend on the concentration of TNF‐α used. Indomethacin did not alter the number of leukocytes induced by low concentrations of TNF‐α (0.1 ng), but reduced the number of cells stimulated with high TNF‐α concentrations (1.0 ng). These results support the concept that SP modulates in vivo neuroinflammatory responses, as measured by granulocyte migration, initiating a cascade of events that includes LT production, TNF‐α secretion, and engagement of LFA‐1 and ICAM‐1. J. Leukoc. Biol. 61: 445–451; 1997.

A guinea pig model for study of bladder mast cell function : histamine release and smooth muscle contraction

To study the function of mast cells in bladder tissue, guinea pigs were sensitized with ovalbumin by intraperitoneal injections, bladder tissue strips were superfused, and tissue contractile force and histamine release were studied. Upon challenge with ovalbumin, bladder tissue contracted 64 +/- 4% (mean +/- S.E.M.) of the maximum carbachol contraction and released 14.1 +/- 1.6% of the total tissue histamine content. Incubation of sensitized bladder tissue with indomethacin led to an increased force and duration of the contraction while incubation with nordihydroguaiaretic acid combined with pyrilamine reduced histamine release and abolished the contraction. Tissue histamine content was significantly higher in the bladder neck than in the dome, and significantly elevated following sensitization. Histochemical studies of bladder tissue demonstrated mast cell degranulation in antigen challenge experiments. In addition, a group of guinea pigs were sensitized to ovalbumin through bladder instillations. With this model, study of the functional characteristics of bladder mast cells and the acute actions of mast cell products on the bladder microenvironment, should now be feasible.

Cyclooxygenase-2 expression is up-regulated in obstructed human ureter

PURPOSEnProstanoids produce significant effects on ureteral function and are synthesized by cyclooxygenase (COX) enzymes. COX is found in the 2 isoforms COX-1 (a constitutive form) and COX-2 (an inducible form). Due to the side effects associated with COX-1 inhibition there is great interest in selective COX-2 inhibition. We determined if COX-2 messenger (m)RNA and protein expression are regulated during ureteral obstruction.nnnMATERIALS AND METHODSnmRNA analysis was performed using excess ureteral segments from 6 patients undergoing reconstructive procedures for chronic ureteral obstruction and 8 (normal ureter) undergoing donor nephrectomy after providing informed consent. All ureteral segments were snap frozen in liquid nitrogen and stored at -70C. RNA was isolated from the segments using phenol extraction and complementary DNA was synthesized by reverse transcription with murine leukemia virus reverse transcriptase (Promega Corp., Madison, Wisconsin). Semiquantitative reverse transcriptase-polymerase chain reaction was performed using specific COX-2 gene primers with ribosomal S26 primers serving as the housekeeping gene. The polymerase chain reaction product was quantified by agarose gel electrophoresis and phospho-imaging. The ratio of COX-2-to-S26 mRNA was compared. Additional segments were homogenized and total protein was extracted, separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. These membranes were Western blotted for COX-2 and glyceraldehyde-3-phosphate dehydrogenase (housekeeping protein) with specific primary and secondary antibodies.nnnRESULTSnThe mean ratio of COX-2-to-S26 mRNA plus or minus standard error at 20, 22 and 24 cycles of amplification was 0.22 +/- 0.04 in the 8 normal ureters compared with 1.01 +/- 0.21 in the 6 obstructed ureters (unpaired Students t test p = 0.004). Similarly the mean ratio of COX-2-to-glyceraldehyde-3-phosphate dehydrogenase protein on immunoblotting was 0.15 +/- 0.02 in the 8 normal ureters compared with 0.59 +/- 0.10 in the 6 obstructed ureters (p = 0.003).nnnCONCLUSIONSnThese data indicate that COX-2 mRNA and protein levels are up-regulated in chronically obstructed human ureters. Using selective COX-2 inhibitors may be useful for treating prostanoid induced effects associated with ureteral obstruction.

In vitro effects of bladder mucosa and an enkephalinase inhibitor on tachykinin induced contractility of the dog bladder

Tachykinin-induced contractility of smooth muscle strips from dog bladders was studied in vitro, and the presence of substance P-like immunoreactivity and neurokinin A and neurokinin B-like immunoreactivity was examined in bladder sections. Nerve fibers with substance P-like immunoreactivity were present in the mucosa, submucosa and smooth muscle. Fibers were also found in nerves, intramural ganglia, and around blood vessels. Neurokinin A-like immunoreactivity had similar distribution, and no neurokinin B-like immunoreactivity was observed. Removal of the mucosa significantly enhanced the sensitivity and the maximum responses to the tachykinins. After removing the mucosa, the sensitivity to these tachykinins increased 0.4 to 0.5 log units (p less than 0.02). The responses to carbachol were not altered by mucosa removal. The leftward shifts of the concentration-response curves for neurokinin A were of similar magnitude after removal of the mucosa, and after pretreatment with phosphoramidon (10 microM), an enkephalinase inhibitor, in the presence of mucosa. However, phosphoramidon did not alter the sensitivity of the bladder strips to neurokinin B, and slightly changed the sensitivity to substance P (0.2 log units). Additional shifts of the substance P and neurokinin A curves to the left were observed in the presence of phosphoramidon when the mucosa was removed (0.6 and 0.5 log units, p less than 0.005). The order of potency for the tachykinins (neurokinin A greater than substance P) was not altered by mucosa removal, addition of phosphoramidon, or both. Neurokinin A was degraded by enkephalinase located in the bladder mucosa and addition of phosphoramidon or mucosa removal resulted in an inhibition or loss of enkephalinase activity. It is concluded that the responses to neurokinin A, which acts on NK-2 type of receptors, prevail on the dog bladder.

Evaluation of ureteric contraction: a comparison among ring, spiral-cut and longitudinal segments.

To determine the optimal contractile response of isolated ureters to inflammatory mediators and neurotransmitters by evaluating four common methods of ureteric suspension.

Response of the isolated guinea pig bladder to exogenous and endogenous leukotrienes.

Noninfectious urinary bladder inflammation is a poorly understood phenomenon, and the participation of leukotrienes (LTs) in the pathogenesis of bladder inflammation is unclear. Leukotrienes are synthesized by the bladder, and exogenous LTs induce contraction of isolated bladder segments. LTD4 and LTC4 were more potent contractile agents than LTE4. Leukotriene-induced contractions were blocked by ICI 198,615 (10(-6) M. and 10(-7) M.) a specific LT receptor antagonist. In the presence of indomethacin (5 x 10(-6) M.), bladder contraction in response to LTD4 was increased. Endogenous LT release was studied using an experimental model of cystitis. Antigen (ovalbumin 10(-6) to 10(-2) mg./ml.) challenge of bladder segments isolated from actively sensitized animals induced release of LT, prostaglandin D2 and histamine. A-64077 (Zileuton), a 5-lipoxygenase (5-LO) inhibitor, significantly reduced contraction of sensitized bladder tissue in response to antigen challenge in a concentration-dependent manner and abolished LT release. These data indicate that the guinea pig urinary bladder produces sulfidopeptide-LTs that can be released upon specific stimulation. Furthermore, LTs activate specific receptors promoting bladder contraction. Our findings suggest that specific 5-LO inhibitors or LT-receptor antagonists might be useful in treating or preventing bladder inflammation.

Effect of Octreotide, A Somatostatin Analogue, on Release of Inflammatory Mediators from Isolated Guinea Pig Bladder

OBJECTIVEnSomatostatin has been demonstrated to inhibit inflammation under certain circumstances. We hypothesized that in vivo treatment with octreotide, a long-acting analogue of somatostatin analogue, would diminish the capacity of inflammatory peptides to stimulate in vitro release of inflammatory mediators by the bladder.nnnMETHODSnFemale guinea pigs were injected with octreotide (20 mg./kg. i.m.) prior to euthanasia. Control guinea pigs received no treatment prior to euthanasia. Urinary bladders were removed and incubated with substance P (SP, 10 microM), neurokinin A (NKA, 10 microM), or bradykinin (BK, 10 microM) in the presence or absence of indomethacin (50 microM), and release of histamine, prostaglandins (PGE2 and PGF2 alpha), and leukotriene (LTB4) was determined.nnnRESULTSnSensory peptides and BK induced time-dependent release of histamine and eicosanoids from isolated urinary bladder. Blockade of cyclooxygenase with indomethacin (50 microM) abolished peptide-induced prostaglandin release but enhanced LTB4 release. In vivo octreotide pretreatment decreased peptide-induced histamine release, had no effect on PGE2 or PGF2 alpha release, and LTB4 release. However, octreotide prevented the increase in LTB4 release in tissues incubated with indomethacin.nnnCONCLUSIONSnThese results indicate that somatostatin has the capacity to suppress the release of histamine and prevents potentiation of LTB4 release by indomethacin by the guinea pig bladder in response to pro-inflammatory peptides, indicating that somatostatin may be useful in preventing or treating some forms of cystitis.

IN VITRO CONTRACTILE EFFECTS OF NEUROKININ RECEPTOR BLOCKADE IN THE HUMAN URETER

PURPOSEnWe identified the predominance of neurokinin-2 receptors and evaluated the inhibition of spontaneous contraction via the blockade of neurokinin-2 receptors in human ureteral segments.nnnMATERIALS AND METHODSnExcess ureteral segments from human subjects undergoing donor nephrectomy or reconstructive procedures were suspended in tissue baths containing Krebs buffer. After spontaneous contractions were recorded, tissues were incubated with 1 microM. solutions of phosphoramidon and captopril (to inhibit peptide degradation) and either the neurokinin-1 receptor antagonist CP 99,994, the neurokinin-2 receptor antagonist SR 48,968, the neurokinin-3 receptor antagonist SR 142,801 or dimethyl sulfoxide (control) for 1 hour. Contraction magnitude and frequency were again recorded and compared with spontaneous levels. Concentration-response curves to the tachykinins substance P, and neurokinins A and B were determined in the presence and absence of antagonists.nnnRESULTSnNeurokinin A increased contractility at lower concentrations than substance P or neurokinin B (p <0.013). Neurokinin-2 receptor blockade produced a 100-fold rightward shift of the concentration-response curves (p <0.013), while neurokinins 1 and 3 receptor blockade had no effect. SR 48,968 significantly reduced contractility during the 1-hour incubation period, causing a 97% reduction in spontaneous rates compared with a 29% reduction in control tissues. CP 99,994 and SR 142,801 had no significant effect.nnnCONCLUSIONSnNeurokinin-2 is the predominant receptor subtype responsible for tachykinin induced contraction of human ureteral smooth muscle. In vitro treatment with the neurokinin-2 antagonist SR 48,968 reduces the spontaneous contraction rate by 97% in vitro. Neurokinin-2 receptor antagonists may have clinical applications for ureteral disease.