Proteinase-activated receptor-2 and hyperalgesia: A novel pain pathway.

Using a combined pharmacological and gene-deletion approach, we have delineated a novel mechanism of neurokinin-1 (NK-1) receptor-dependent hyperalgesia induced by proteinase-activated receptor-2 (PAR2), a G-protein-coupled receptor expressed on nociceptive primary afferent neurons. Injections into the paw of sub-inflammatory doses of PAR2 agonists in rats and mice induced a prolonged thermal and mechanical hyperalgesia and elevated spinal Fos protein expression. This hyperalgesia was markedly diminished or absent in mice lacking the NK-1 receptor, preprotachykinin-A or PAR2 genes, or in rats treated with a centrally acting cyclooxygenase inhibitor or treated by spinal cord injection of NK-1 antagonists. Here we identify a previously unrecognized nociceptive pathway with important therapeutic implications, and our results point to a direct role for proteinases and their receptors in pain transmission.

Spectrum of ST-T-wave patterns and repolarization parameters in congenital long-QT syndrome: ECG findings identify genotypes.

BACKGROUND: Congenital long-QT syndrome (LQTS) is caused by mutations of genes encoding the slow component of the delayed rectifier current (LQT1, LQT5), the rapid component of the delayed rectifier current (LQT2, LQT6), or the Na(+) current (LQT3), resulting in ST-T-wave abnormalities on the ECG. This study evaluated the spectrum of ST-T-wave patterns and repolarization parameters by genotype and determined whether genotype could be identified by ECG. METHODS AND RESULTS: ECGs of 284 gene carriers were studied to determine ST-T-wave patterns, and repolarization parameters were quantified. Genotypes were identified by individual ECG versus family-grouped ECG analysis in separate studies using ECGs of 146 gene carriers from 29 families and 233 members of 127 families undergoing molecular genotyping, respectively. Ten typical ST-T patterns (4 LQT1, 4 LQT2, and 2 LQT3) were present in 88% of LQT1 and LQT2 carriers and in 65% of LQT3 carriers. Repolarization parameters also differed by genotype. A combination of quantified repolarization parameters identified genotype with sensitivity/specificity of 85%/70% for LQT1, 83%/94% for LQT2, and 47%/63% for LQT3. Typical patterns in family-grouped ECGs best identified the genotype, being correct in 56 of 56 (21 LQT1, 33 LQT2, and 2 LQT3) families with mutation results. CONCLUSIONS: Typical ST-T-wave patterns are present in the majority of genotyped LQTS patients and can be used to identify LQT1, LQT2, and possibly LQT3 genotypes. Family-grouped ECG analysis improves genotype identification accuracy. This approach can simplify genetic screening by targeting the gene for initial study. The multiple ST-T patterns in each genotype raise questions regarding the pathophysiology and regulation of repolarization in LQTS.

Accurate and rapid assessment of MHC antigen upregulation following cytokine stimulation.

Three human tumour cell lines, A431 (cervical), Scaber (bladder) and Fen (bladder), were studied using immunohistochemical staining (IC), radiobinding (RB), immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA) and dot-blot (DB) techniques in order to assess major histocompatibility antigen (MHC) induction in response to interferon-gamma (IFN-gamma). Induction of class II antigens by IFN-gamma was observed on all three cell lines using all techniques. Monoclonal antibody (Mab) staining showed that both Scaber and A431 lines were positive for intact class I (Mab W6/32), class I free heavy chain (Mab HC10) and beta 2 microglobulin (beta 2-m) (Mab BBM.1), while Fen cells were positive only with HC10. The IP technique demonstrated the presence of a 45 kDa band on precipitation of the Fen lysate with HC10 Mab, whereas no such band was observed when W6/32 was used. The DB technique confirmed the negative reaction with W6/32 and BBM.1 Mabs, while HC10 showed positive staining which was upregulated by IFN-gamma. Transfection of the Fen cells with the beta 2-m gene resulted in the surface expression of fully assembled class I molecules. The DB technique showed upregulation of class I antigens following IFN-gamma stimulation, while RB detected no significant increase in cell surface expression (t test; p = 0.104). The binding values for transfected Fen cells before and after IFN-gamma stimulation were 2000 +/- 48 and 2161 +/- 156 cpm respectively. These results demonstrate that the DB technique facilitates an accurate assessment of cytokine induced antigens, corrected against a background of total cellular protein synthesis. The ease of execution, simplicity, non-radioactive nature and economy make it the method of choice for routine screening prior to the selection of suitable patients for cytokine therapy.

Glycosylation and the activation of proteinase-activated receptor 2 (PAR(2)) by human mast cell tryptase.

1. Human mast cell tryptase appears to display considerable variation in activating proteinase-activated receptor 2 (PAR(2)). We found tryptase to be an inefficient activator of wild-type rat-PAR(2) (wt-rPAR(2)) and therefore decided to explore the factors that may influence tryptase activation of PAR(2). 2. Using a 20 mer peptide (P20) corresponding to the cleavage/activation sequence of wt-rPAR(2), tryptase was as efficient as trypsin in releasing the receptor-activating sequence (SLIGRL.). However, in the presence of either human-PAR(2) or wt-r PAR(2) expressing cells, tryptase could only activate PAR(2) by releasing SLIGRL from the P20 peptide, suggesting that PAR(2) expressed on the cells was protected from tryptase activation. 3. Three approaches were employed to test the hypothesis that PAR(2) receptor glycosylation restricts tryptase activation. (a) pretreatment of wt-rPAR(2) expressing cells or human embryonic kidney cells (HEK293) with vibrio cholerae neuraminidase to remove oligosaccharide sialic acid, unmasked tryptase-mediated PAR(2) activation. (b) Inhibiting receptor glycosylation in HEK293 cells with tunicamycin enabled tryptase-mediated PAR(2) activation. (c) Wt-rPAR(2) devoid of the N-terminal glycosylation sequon (PAR(2)T25(-)), but not rPAR(2) devoid of the glycosylation sequon located on extracellular loop-2 (PAR(2)T224A), was selectively and substantially (>30 fold) more sensitive to tryptase compared with the wt-rPAR(2). 4. Immunocytochemistry using antisera that specifically recognized the N-terminal precleavage sequence of PAR(2) demonstrated that tryptase released the precleavage domain from PAR(2)T25(-) but not from wt-rPAR(2). 5. Heparin : tryptase molar ratios of greater than 2 : 1 abrogated tryptase activation of PAR(2)T25(-). 6. Our results indicate that glycosylation of PAR(2) and heparin-inhibition of PAR(2) activation by tryptase could provide novel mechanisms for regulating receptor activation by tryptase and possibly other proteases.

Tryptase and agonists of PAR-2 induce the proliferation of human airway smooth muscle cells.

Airway remodeling with smooth muscle cell (SMC) hyperplasia is a feature of chronic asthma. We investigated the potential for tryptase, the major secretory product of human mast cells, to act as a growth factor for human airway SMCs. Because this serine protease can activate proteinase-activated receptor-2 (PAR-2), we also examined the actions of SLIGKV, a peptide agonist of PAR-2. Incubation with lung tryptase provoked a twofold increase in [(3)H]thymidine incorporation; a similar increase in cell numbers was found when we used the MTS assay. The effect was catalytic site dependent, being abolished by the protease inhibitors leupeptin and benzamidine and by heat inactivation of the enzyme. Tryptase-induced DNA synthesis was inhibited by preincubation of the cells with pertussis toxin, calphostin C, or genistein. Transduction mechanisms are thus likely to involve a pertussis toxin-sensitive G protein, protein kinase C, and tyrosine kinase. SLIGKV elicited a response on SMCs similar to that of tryptase. Tryptase could provide an important stimulus for SMC proliferation in asthmatic airways, by acting on PAR-2.

Accurate quantitative analysis of cellular proteins using a computerised scanning-aided dot blot technique.

The aim of this study was to use a dot blot technique (DB) aided by a computerised scanning programme (CSDBT) to assess expression of cellular proteins in various tumour cell lines. Two groups of proteins, i.e. major histocompatibility complex (MHC) Class I and cell adhesion molecules (e.g. ICAM-1) were investigated. The constitutive expression of Class I antigens was measured and found to vary for different tumour lines. Exposure of SKV14 (SV40 transformed epithelial cells) and 5637 lines (bladder) to interferons (IFN) alpha or gamma resulted in upregulation of Class I antigens. IFN gamma, but not IFN alpha, induced ICAM-1 on all the lines studied. Thus, the stimulatory indices (SI) for Class I antigens in the case of SKV14, J82 (bladder) and 5637 lines after IFN gamma and IFN alpha stimulation were 2.02, 1.77, 4.68 and 1.65, 1.36, 2.08 respectively. The corresponding values for ICAM-1 were 1.65, 1.87, 3.43 and 1.75, 1.0, 0.96. In all the cases, the P values (using a paired t-test), except ICAM-1 for IFN alpha-stimulated J82 cells, were below 0.05. Following exposure of cells to combinations of drugs and IFN alpha, the percentage inhibition for Class I and ICAM-1 in the case of SKV14 and 5367 lines in the presence of cycloheximide (10 micrograms/mL) were 82%, 85% and 57%, 45% respectively. The corresponding values for indomethacin (10 micrograms/mL) were -23%, -52% and -20%, -24%. In all cases, the P values (using a paired t-test) were below 0.05. To our knowledge this is the first report describing a computerised scanning programme for assessing the presence of cellular proteins. The results were consistent with those obtained by radiobinding and immunocytochemistry. This simple and sensitive approach highlighted two important issues: (a) that it can be used to detect minor changes, under various conditions, of cellular proteins whether cytoplasmic or otherwise; and (b) that the accuracy of the measurements was enhanced by the use of computer scanning.

Arrhythmia discrimination using hemoglobin spectroscopy in humans.

BACKGROUND: Inappropriate therapies are frequently delivered by implantable cardioverter-defibrillators (ICDs). We have investigated muscle perfusion as a means of augmenting arrhythmia discrimination by using implanted near-infrared spectroscopy. OBJECTIVE: To evaluate hemodynamic stability by monitoring muscle perfusion from within the ICD pocket, in fresh tissue and inside the scar capsule on preexisting ICD generators, during induced cardiac arrhythmias, in humans. METHODS: The sensor was implanted on or under the pectoral muscle, during ICD defibrillation threshold testing. A microvascular oxygenation trend indicator (O2 Index) was computed during 74 induced ventricular fibrillation and 34 normal sinus rhythm episodes in 34 patients and also during 28 atrial and 90 ventricular overdrive pacing episodes as simulations of supraventricular and ventricular tachycardias, respectively. RESULTS: On average, the change in oxygenation, based on the O2 Index, was statistically significant (P <.003) from baseline within 3 seconds following cardiac arrest. An optimized O2 Index, used for detecting the hemodynamic trend, exhibited a decreasing trend during ventricular fibrillation (P <.0001) and was different from that during normal sinus rhythm (P <.0001). The sensitivity for the detection of ventricular fibrillation was 100%, and the specificity for the rejection of normal sinus rhythm was 82% in the presence of scar tissue on the optical sensor. For a 35-mm Hg drop in the mean arterial pressure as the threshold for hemodynamic instability, the specificity for the rejection of hemodynamically stable atrial and ventricular pacing episodes was 93% and 71%, respectively. CONCLUSION: An implantable near-infrared spectroscopic sensor may be useful for hemodynamic monitoring during cardiac arrhythmias to prevent inappropriate therapy.

Mechanism of dye response and interference in the Bradford protein assay.

Bradford Coomassie brilliant blue G-250 protein-binding dye exists in three forms: cationic, neutral, and anionic. Although the anion is not freely present at the dye reagent pH, it is this form that complexes with protein. Dye binding requires a macromolecular form with certain reactive functional groups. Interactions are chiefly with arginine rather than primary amino groups; the other basic (His, Lys) and aromatic residues (Try, Tyr, and Phe) give slight responses. The binding behavior is attributed to Van der Waals forces and hydrophobic interactions. Assay interference by bases, detergents, and other compounds are explained in terms of their effects upon the equilibria between the three dye forms.

Measuring plant protein with the Bradford assay : 1. Evaluation and standard method.

The suitability of the Bradford protein assay for measuring plant protein was evaluated and a standard method developed. The assay involves extraction of dried, fresh, or frozen plant material in 0.1 NaOH for 30 min. Replicate 100-µl aliquots of centrifuged supernatant are assayed with 5 ml Bio-Rad Bradford dye reagent (Coomassie brilliant blue G-250) diluted 1:4 and containing 3 mg/ml soluble polyvinylpyrollidone. Absorbance at 595 nm is recorded after 15 min against an NaOH blank. Samples are calibrated against a ribulose 1,5-diphosphate carboxylase-oxygenase standard in NaOH. Procedures for plant preparation, extraction stability, the effects of phenol removal and quinone formation, and assay recovery are evaluated. Assay absorbance stability and techniques for increasing absorbance stability are reported. Changes in protein quality are briefly discussed.

Amiodarone is safe and highly effective therapy for supraventricular tachycardia in infants.

BACKGROUND: The clinical effectiveness of amiodarone must be weighed against the likelihood of adverse effects. Adverse effects are less common in children than in adults, yet there have been no large studies assessing the efficacy and safety of amiodarone in the first 9 months of life. We sought to assess the safety and efficacy of amiodarone as primary therapy for supraventricular tachycardia in infancy. METHODS: We evaluated the clinical course of 50 consecutive infants and neonates (1.0+/-1.5 months, 35 male) treated with amiodarone for supraventricular tachyarrhythmias between July 1994 and July 1999. At presentation, congenital heart disease, congestive heart failure, or ventricular dysfunction were present in 24%, 36%, and 44% of the infants, respectively. Infants received a 7- to 10-day load of amiodarone at either 10 or 20 mg/kg/d. If this failed to control the arrhythmia, oral propranolol (2 mg/kg/d) was added. Patients were followed up for 16.0+/-13.0 months, and antiarrhythmic drugs were discontinued as tolerated. RESULTS: Rhythm control was achieved in all patients. Of the 34 patients who have reached 1 year of age, 23 (68%) have remained free of arrhythmia, despite discontinuation of propranolol and amiodarone. Growth and development remained normal for age. Higher loading doses of amiodarone were associated with an increase in the corrected QT interval, but no proarrhythmia was seen. There were no side effects necessitating drug withdrawal. CONCLUSIONS: Amiodarone is an effective and safe therapy for tachycardia control in infancy.

Human mast cell tryptase stimulates the release of an IL-8-dependent neutrophil chemotactic activity from human umbilical vein endothelial cells (HUVEC).

Tryptase, the major product of human mast cell activation, is a potent stimulus of vascular leakage and neutrophil accumulation in vivo in animal studies, but the mechanisms of action remain unclear. Using HUVEC cultures we have sought to investigate the potential of tryptase to alter monolayer permeability or induce the release of neutrophil chemotactic activity. Tryptase (1-100 mU/ml) failed to alter the permeability of endothelial cell monolayers as assessed by albumin flux over 1 h. However, supernatants from endothelial cells treated with tryptase (1-50 mU/ml) for a 24-h period induced neutrophil migration across Transwell filters, with maximal migration observed at 10 mU/ml tryptase. Pretreatment of tryptase with the protease inhibitor leupeptin abolished the chemotactic activity, indicating a dependence on the catalytic site. Moreover, this effect was abolished by addition of an IL-8 neutralizing antibody, suggesting that IL-8 release makes an important contribution to the chemotactic activity. The interaction of mast cell tryptase with endothelial cells could be important in stimulating the ingress of neutrophils following mast cell activation in inflammatory disease.

The role of mast cell tryptase in regulating endothelial cell proliferation, cytokine release, and adhesion molecule expression: tryptase induces expression of mRNA for IL-1 beta and IL-8 and stimulates the selective release of IL-8 from human umbilical vein endothelial cells.

Mast cells are found frequently in close proximity to blood vessels, and endothelial cells are likely to be exposed to high concentrations of their granule mediators. We have investigated the proinflammatory actions of the major mast cell product tryptase on HUVEC. Addition of purified tryptase was found to stimulate thymidine incorporation, but induced little alteration in cell numbers, suggesting it is not a growth factor for HUVEC. Expression of ICAM-1, VCAM-1, and E-selectin was not altered following incubation with tryptase, but the potent granulocyte chemoattractant IL-8 was released in a dose-dependent fashion in response to physiologically relevant concentrations, with maximal levels in supernatants after 24 h. The actions of tryptase on HUVEC were inhibited by heat inactivation of the enzyme, or by preincubating with the protease inhibitors leupeptin or benzamidine, suggesting a requirement for an intact catalytic site. Reverse-transcription PCR analysis indicated up-regulation of mRNA for IL-8 as well as for IL-1 beta in response to tryptase or TNF-alpha. However, tryptase was a more selective stimulus than TNF-alpha and did not induce increased expression of mRNA for granulocyte-macrophage CSF or stimulate the release of this cytokine. Leukocyte accumulation in response to tryptase may be mediated in part through the selective secretion of IL-8 from endothelial cells.

Development and characterization of a monoclonal antibody specific for human basophils and the identification of a unique secretory product of basophil activation.

Despite increasing evidence that basophils can infiltrate into inflamed tissues during allergic reactions, determination of the extent of infiltration and elucidation of their role in allergic disease has been frustrated by the lack of reliable means for detecting this cell type in tissues. In the present study, we report on a new monoclonal antibody specific for basophils and on the initial characterization of the antigen it recognizes. Basophils were isolated from peripheral blood by Percoll density gradient centrifugation and a positive-selection immunomagnetic procedure and injected into mice to produce monoclonal antibodies. A hybridoma clone, designated BB1, secreted antibody of the IgG2a isotype; this antibody bound selectively to basophils on immunocytochemistry but did not react with any other cell type or tissue structure, although it did stain a proportion of cells from the basophilic cell line KU812F. In sections of mixed populations of peripheral blood cells, similar numbers of cells stained with Alcian blue dye and BB1 over a wide range of basophil purity. BB1 antibody was effective in identifying basophils in sections of mixed cells or in tissues after fixation with ethanol, Carnoy's solution, or formalin. Staining of basophils with BB1 gave a granular appearance, although flow cytometry indicated that some antigen was also present on the surface of the cell. Activation of these cells with anti-IgE antibody or with the calcium ionophore A23187 provoked release of the antigen in parallel with that of histamine. BB1 antibody did not, by itself, stimulate histamine release. The molecular mass of the antigen was determined on Hedrick-Smith gels to be 124+/-11 kd. This new monoclonal antibody will be a valuable experimental tool in future studies, allowing the reliable detection of basophils in tissues of patients with allergic and chronic inflammatory disease; in addition, the antigen it identifies has potential as a unique marker of basophil activation.

Reduction in diet breadth results in sequestration of plant chemicals and increases efficacy of chemical defense in a generalist grasshopper.

The lubber grasshopper,Romalea guttata, is a generalist feeding on a broad diet of many herbaceous plant species and has a metathoracic defensive secretion normally containing phenolics and quinones synthesized by the insect. When insects were reared on a restricted diet of wild onion, they sequestered sulfur volatiles from the plant into their defensive secretions. These compounds were not detected by gas chromatography-mass spectroscopy in secretions of insects on an artificial diet or a natural, generalist diet of 26 plants that included wild onion as a component, nor were they present in secretions from field-collected insects. Defensive secretions of insects reared on wild onion were significantly more deterrent, by as much as an order of magnitude, to two species of ant predators than secretions from insects on either of the other two diets, despite a reduction in the concentration of autogenous defensive chemicals in secretions of insects on the onion diet. Sequestration of plant chemicals that increased defensive efficacy occurred when diet breadth was reduced. We suggest that this occurs because under conditions of specialization, plant secondary metabolites are more likely to be ingested and bioaccumulated in sufficient concentrations to have biological activity against predators. What we define as casual bioaccumulation of bioactive plant chemicals following dietary specialization may lead to evolution of sequestered defense syndromes in insects, and this process may not necessarily require specific adaptation to or coevolution with a toxic host plant.

A polymorphic protease-activated receptor 2 (PAR2) displaying reduced sensitivity to trypsin and differential responses to PAR agonists.

Protease-activated receptor 2 (PAR2) is a trypsin-activated member of a family of G-protein-coupled PARs. We have identified a polymorphic form of human PAR2 (PAR(2)F240S) characterized by a phenylalanine to serine mutation at residue 240 within extracellular loop 2, with allelic frequencies of 0.916 (Phe(240)) and 0.084 (Ser(240)) for the wild-type and mutant alleles, respectively. Elevations in intracellular calcium were measured in permanently transfected cell lines expressing the receptors. PAR(2)F240S displayed a significant reduction in sensitivity toward trypsin ( approximately 3.7-fold) and the PAR2-activating peptides, SLIGKV-NH(2) ( approximately 2.5-fold) and SLIGRL-NH(2) ( approximately 2.8-fold), but an increased sensitivity toward the selective PAR2 agonist, trans-cinnamoyl-LIGRLO-NH(2) ( approximately 4-fold). Increased sensitivity was also observed toward the selective PAR-1 agonist, TFLLR-NH(2) ( approximately 7-fold), but not to other PAR-1 agonists tested. Furthermore, we found that TLIGRL-NH(2) and a PAR4-derived peptide, trans-cinnamoyl-YPGKF-NH(2), were selective PAR(2)F240S agonists. By introducing the F240S mutation into rat PAR2, we observed shifts in agonist potencies that mirrored the human PAR(2)F240S, suggesting that Phe(240) is involved in determining agonist specificity of PAR2. Finally, differences in receptor signaling were paralleled in a cell growth assay. We suggest that the distinct pharmacological profile induced by this polymorphism will have important implications for the design of PAR-targeted agonists/antagonists and may contribute to, or be predictive of, an inflammatory disease.

Mass, charge, and subcellular localization of a unique secretory product identified by the basophil-specific antibody BB1.

BACKGROUND: BB1 is a basophil-specific mAb (Lab Invest 1999;79:27-38). The identity of the corresponding antigen has not been determined, but it gives a granular appearance on staining and is secreted on activation of basophils. OBJECTIVE: We sought to further characterize the basophilspecific antigen identified by BB1. METHODS: Intracellular localization was determined by flow cytometry and by immunogold labeling and electron microscopy. Physical chemical properties were investigated by gel filtration chromatography and preparative isoelectric focusing. RESULTS: In flow cytometry, permeabilization of cells increased immunofluorescence 100-fold, confirming the predominantly intracellular localization of the antigen. It was further localized to the secretory granules by immunoelectron microscopy. Double labeling with a CD63-specific antibody demonstrated selective binding of BB1 to the granule matrix. Gel filtration chromatography indicated that the antigen is secreted as a complex of approximately 5 x 10(6) d, which was well resolved from the 210-kd supramolecular complex containing tryptase. The antigen was degraded by pronase. Isoelectric focusing indicated a highly basic protein with an isoelectric point of 9.6. CONCLUSION: With its granule localization, release on cell activation, and unique properties, the antigen identified by BB1 could be a novel mediator of allergic disease. We propose the name basogranulin for this novel basophil-specific protein.

Genetically defined therapy of inherited long-QT syndrome. Correction of abnormal repolarization by potassium.

BACKGROUND: Many members of families with inherited long-QT (LQT) syndrome have mutations in HERG, a gene encoding a cardiac potassium channel that is modulated by extracellular potassium. We hypothesized that an increase in serum potassium would normalize repolarization in these patients. METHODS AND RESULTS: We studied seven subjects with chromosome 7-linked LQT syndrome and five normal control subjects. Repolarization was measured by ECG and body surface potential mapping during sinus rhythm, exercise, and atrial pacing, before and after serum potassium increase. Potassium administration improved repolarization in the LQT syndrome. At baseline, LQT subjects differed from control subjects: resting corrected QT interval (QTc, 627 +/- 90 versus 425 +/- 25 ms, P = .0007), QTc dispersion (133 +/- 62 versus 36 +/- 9 ms, P = .009), QT/RR slope (0.35 +/- 0.08 versus 0.24 +/- 0.07, P = .04), and global root-mean-square QT interval (RMS-QTc; 525 +/- 68 versus 393 +/- 22, P = .002). All LQT subjects had biphasic or notched T waves. After administration of potassium, the LQT group had a 24% reduction in resting QTc interval (from 617 +/- 92 to 469 +/- 23 ms, P = .004) compared with a 4% reduction among control subjects (from 425 +/- 25 to 410 +/- 45 ms, P > .05). The reduction was significantly greater in LQT subjects (P = .018). QT dispersion became normal in LQT subjects and did not change in control subjects. The slope of the relation between QT interval and cycle length (QT/RR slope) decreased toward normal. T-wave morphology improved in six of seven LQT subjects. The LQT group had a greater reduction in RMS-QTc than control subjects (P = .04). CONCLUSIONS: An increase in serum potassium corrects abnormalities of repolarization duration, T-wave morphology, QT/ RR slope, and QT dispersion in patients with chromosome 7-linked LQT.

Mast cell tryptase as a mediator of hyperresponsiveness in human isolated bronchi.

BACKGROUND: Although the role of mediators and cytokines produced by mast cells is well established in asthmatic bronchial inflammation, the contribution of mast cell-derived proteases to the development of hyperresponsiveness remains unclear. There have been reports indicating that tryptase alters the mechanical activity of animal airway smooth muscle or spontaneously sensitized human isolated airways. OBJECTIVE: The aim of this study was to analyse the effect of purified mast cell tryptase on non-sensitized human isolated bronchi. METHODS: Both central and peripheral bronchi, dissected from lung specimens obtained at thoracotomy, were studied in terms of both mechanical activity i.e. isometric contraction in response to a variety of agonists and distribution of inflammatory cells i.e. immunohistochemistry. RESULTS: In both proximal and distal bronchi, the reactivity to histamine was significantly increased by a previous incubation in the presence of 1 microg/mL of tryptase (increase in maximal force, DeltaFmax was 12.1 +/- 3.8%, and 8.8 +/- 3.1%, respectively). This effect of tryptase on histamine-induced contraction was completely abrogated in the presence of the protease inhibitor benzamidine (100 micromol/L). Histological examination of specimens exposed to tryptase demonstrated an increase in mast cell number within the subepithelial tissue whereas mast cell numbers in the epithelial layer concomittently decreased. CONCLUSION: These results indicate that human mast cell tryptase alters the contractile response of non-sensitized human isolated bronchi and that this alteration is accompanied by a change in the mast cell distribution within the airway wall.

Trichinosis with ventilatory failure and persistent myocarditis.

Life-threatening infections with Trichinella spiralis are rare in countries that have adopted laws requiring cooking of raw garbage fed to pigs. Thus such infections may pose a diagnostic dilemma for clinicians unfamiliar with their presentation. We report a case of imported trichinosis in a Mexican national who developed respiratory failure, myocarditis, and sinus arrest. The patient recovered uneventfully after the administration of benzimidazole and corticosteroid drugs, although a pacemaker was required to maintain normal cardiac rhythm. Symptomatic myocarditis is a rare complication of trichinosis that is often associated with increased morbidity and mortality. This report illustrates and reviews important features of the epidemiology, clinical presentation, and management of trichinosis.

RANTES, macrophage-inhibitory protein 1alpha, and the eosinophil product major basic protein are released into upper respiratory secretions during virus-induced asthma exacerbations in children.

The presence of cytokines and the toxic eosinophil granule product major basic protein (MBP) was investigated in nasal aspirates from children with naturally occurring virus-induced asthma exacerbations and compared with levels in nasal aspirates taken from the same children when asymptomatic. Increased levels of MBP accompanied by increased levels of the chemokines RANTES and macrophage-inhibitory protein 1alpha were observed in nasal aspirates from children during the virus-induced exacerbations. Granulocyte-macrophage colony-stimulating factor was mostly undetectable in samples obtained during both symptomatic and asymptomatic periods. Interleukin-5 levels were low, but tended to increase in samples from symptomatic children. These data confirm that the eosinophil product MBP and the eosinophil chemoattractant chemokines RANTES and macrophage-inhibitory protein 1alpha are increased in upper respiratory viral infections associated with asthma exacerbations and suggest an important role for these chemokines in regulating eosinophil influx and activation. These chemokines may represent targets for therapeutic intervention in virus-induced asthma exacerbations.