Muscarinic hyperresponsiveness of antigen-sensitized feline airway smooth muscle in vitro

OBJECTIVE: To determine the effect of in vivo antigen sensitization (Ascaris suum) of cats on tracheal smooth muscle (TSM) and bronchial smooth muscle (BSM) muscarinic reactivity in vitro. ANIMALS: Healthy domestic shorthair cats of either sex. PROCEDURE: Cats were sensitized and were long-term antigen (or sham) challenge exposed for 6 weeks by aerosolization with soluble Ascaris suum. Tracheal and BSM preparations were obtained and stimulated in vitro by electrical field stimulation (EFS), acetylcholine (ACh, a muscarinic agonist), and physostigmine (an AChase inhibitor). Responses were compared with responses of comparable tissues from sham antigen challenge-exposed cats. RESULTS: Tracheal and BSM from sensitized, compared with sham-sensitized (control), cats had greater isometric contraction (expressed as percentage of the response observed for isotonic, 63 mM KCl-elicited contraction [% KCl]) in response to endogenous (EFS) and exogenous muscarinic receptor activation (ACh). Contractions in response to EFS by TSM from control cats were 74% KCl vs 97% KCl for antigen-sensitized TSM (P < 0.04). Muscarinic responses were augmented comparably by in vivo sensitization; TSM from control cats contracted to 190% KCl vs 230% KCl (P < 0.03) for TSM from immune-sensitized cats. Physostigmine augmented responses of all tissues to ACh so that TSM from control (290% KCl) and antigen-sensitized (257% KCl) cats were similar. Responses of BSM from antigen-sensitized cats had similar augmentation of contractile response to EFS and ACh. CONCLUSIONS: Long-term in vivo antigen sensitization increases numbers of muscarinic receptors on airway smooth muscle or decreases the availability or activity of AChase in cats. CLINICAL RELEVANCE: Modulation of muscarinic receptors may be useful for treatment of asthmatic cats with in vivo airway hyperreactivity.     

Carbamoyl phosphate synthetase: a tunnel runs through it

BACKGROUND: Sensitivity to specific allergens and increased sensitivity to common spasmogens are characteristic features of allergic asthma and are also features demonstrated by tissues passively sensitized with serum from atopic donors, displaying high levels of IgE. It is evident that the specific response to allergen is related to circulating levels of allergen-specific IgE, but it is unclear whether the histamine hypersensitivity is also related to this immunoglobulin. OBJECTIVE: The objective was to deplete IgE in the serum of a donor with high levels of total and allergen-specific IgE and compare specific-allergen sensitivity and sensitivity to histamine in tissues passively sensitized with either the whole serum or the IgE-depleted serum. METHODS: Serum from a Dermatophagoides farinae-sensitive asthmatic (total IgE = 1047 U/mL, D. farinae-specific IgE > 17.5 U/mL) was subjected to an immunomagnetic separation technique to reduce the levels of IgE (total and specific) to below 10 U/mL. Bronchial tissue from six non-atopic donors was then passively sensitized overnight with either the whole serum or IgE-depleted serum and D. farinae and histamine sensitivity were evaluated the next day using standard organ bath techniques. RESULTS: Passive sensitization with the whole serum resulted in the development of sensitivity to D. farinae and increased sensitivity to histamine (750+/-169 mg contraction to 10 U/mL D. farinae, histamine pEC50 5.64+/-0.16 and max. 813+/-109 mg in sensitized vs 37+/-34 mg contraction to 10 U/mL D. farinae histamine pEC50 5.05+/-0.23 and max. 490+/-84 in non-sensitized tissues, P>0.05). Incubation with IgE-depleted serum still produced histamine hypersensitivity (histamine pEC50 5.57+/-0.16 and max. 737+/-70 mg P>0.05), but no significant response to allergen was detected (20+/-13 mg contraction to 10 U/mL D. farinae). CONCLUSION: These results demonstrate, that increased reactivity to histamine and airway contraction to allergen induced by passive sensitization, occur through independent mechanisms and that, unlike allergen-sensitivity, histamine hypersensitivity is caused by a serum factor other than IgE.     

IgE-induced passive sensitization of human isolated bronchi and lung mast cells

Passive sensitization of human isolated lung with serum from atopic asthmatic patients provides an opportunity to study the link between airway hyper-responsiveness and the allergic process. To directly demonstrate the role of immunoglobulin E (IgE) in the effect of the atopic serum, we have compared the effect of passively sensitizing both human bronchi and isolated lung mast cells with either serum from atopic asthmatic patients or human monoclonal IgE. Peripheral bronchi ( < 5 mm in internal diameter) were dissected out from human lung obtained at thoractomy and isometric contraction was studied in response to a variety of immunological stimuli according to the sensitization protocol. Mast cells were also isolated from human lung and histamine release was measured under similar experimental conditions. A contractile response was elicited by either the specific antigen or anti-IgE (0.6-600 ng.mL-1) but not anti-immunoglobulin G (IgG) 0.2-20 micrograms.mL-1) in airways sensitized with atopic serum (total IgE concentration of approximately 1,000 international units (IU).mL-1). The maximal contractile response to anti-IgE was 75 +/- 22% of the response to 1 mM acetylcholine. Similarly, anti-IgE released histamine from isolated lung mast cells sensitized with atopic serum up to 22.4 +/- 2% of total histamine measured within mast cells. When isolated airways or mast cells were sensitized with human monoclonal IgE (1,000 IU.mL-1), response to anti-IgE in terms of contractile response or histamine release, respectively, were not significantly different from those obtained following passive sensitization with atopic serum. Finally, the bronchial contractile response to anti-IgE depended not only on the concentration of anti-IgE but also on that of IgE (300-2,000 IU.mL-1) used to sensitize the airways. These results indicate that the effect of antigen or anti-IgE in peripheral bronchi passively sensitized with atopic serum is mimicked when sensitization is carried out directly with human monoclonal IgE.     

Identification of peptide fragments chemically cross-linked in cytochrome c oxidase from thermophilic Bacillus PS3

It has been suggested that differential histamine-releasing activity of an IgE-dependent histamine-releasing factor (HRF), which has recently been cloned, is related to carbohydrate difference in the IgE molecule. Lectins are able to recognize specific glycoforms and might therefore be useful in characterizing the proposed heterogeneity of IgE molecules. As one test of this hypothesis, we examined the histamine release potency of several well-characterized lectins on basophils passively sensitized with serum containing IgE molecules that support HRF-induced histamine release (IgE+) or serum that does not support release by this stimulus (IgE-). Histamine release was induced by challenging basophils with different concentrations of concanavalin A, Lens culinaris (LcH), and Pisum sativum (PSA). Dose-response curves revealed that LcH caused 30% histamine release at 2 micrograms/ml with IgE+ sensitized cells, whereas the same release with IgE- cells required sixfold higher concentrations. Similar values for PSA showed a sevenfold difference. With concanavalin A, the selectivity was reversed in that it was fourfold more active on IgE- -sensitized cells. Another pair of sera (IgE+ vs IgE-) revealed the same result for concanavalin A, but no difference occurred in LcH and PSA induced release between the IgE+ - and IgE- -sensitized cells. These contrasting findings with different pairs of IgE+ -and IgE- -containing sera indicated that the lectins LcH and PSA are not able to discriminate between IgE+ -and IgE- -sensitized cells as does HRF and therefore cannot be used to further define the proposed heterogeneity of IgE. this conclusion was supported by experiments in which basophil preparations from donors possessing natural sensitivity or insensitivity to HRF (having IgE+ or IgE- on their surface) were examined fro their response to these lectins. No difference was found in the sensitivity of the cells to challenge with LcH or PSA, and the response to concanavalin A was the opposite of that found when passively sensitized cells were used (30% histamine release at 0.9 vs 3.5 micrograms/ml for IgE+ vs IgE- donors, respectively). We conclude that oligosaccharide-specific lectins do not differentiate between HRF-reactive and HRF-nonreactive IgE molecules on basophils.     

Persistent airway hyperresponsiveness and histologic alterations after chronic antigen challenge in cats

We studied the effect of chronic immune sensitization on the airway reactivity and associated cytologic and histologic alterations in initially nonatopic cats, a species that spontaneously develops idiopathic asthma. Seven cats were sensitized by intramuscular injection of Ascaris suum antigen (AA) for 4 wk, and four other cats served as sham controls. Airway sensitization was demonstrated by an increased response to nebulized AA in sensitized animals (RL = 45.9 +/- 6.1 cm H2O/L/s, versus a baseline response of 24.7 +/- 1.5 cm H2O/L/s, p < 0.01), and hyperresponsiveness was demonstrated by an increased response to acetylcholine (ACh)-challenge 24 h after AA (approximately 1.0 log decrease in PD200, p < 0.01). The number of eosinophils in the sensitized animals' bronchoalveolar lavage (BAL) fluid increased 12-fold (p < 0.01 versus control) in response to AA challenge; 32 +/- 5% of the BAL eosinophils had a specific density < 1.050, versus 8 +/- 2% prior to AA challenge (p < 0.05). There was no change in airway reactivity, eosinophil recovery, or density in the control group 24 h after sham challenge with saline. The same seven sensitized cats further received nebulized AA three times weekly for 4 to 6 wk, after which BAL samples were again obtained and ACh dose-response curves generated 72 h after the final administration of nebulized AA. Airway hyperresponsiveness increased (approximately 1.5 log decrease in PD200, p < 0.001) and the number of eosinophils recovered in BAL fluid was increased 11-fold (p < 0.05). Necropsy specimens demonstrated bronchoconstriction in AA-challenged animals but not controls; luminal narrowing was accompanied by: (1) a 29.0 +/- 0.34% increase in smooth-muscle thickness (p < 0.05); (2) goblet-cell and submucosal-gland hypertrophy and hyperplasia; and (3) epithelial erosion and eosinophilic infiltration. We demonstrate in nonhuman species persistent airway hyperreactivity associated with a complete constellation of histologic changes in epithelium, smooth muscle, and mucus glands, and cytologic changes in BAL fluid, all induced by immune sensitization. Our data suggest that chronic immune sensitization per se could be a salient factor in causing many of the changes associated with chronic bronchial asthma.     

Norepinephrine accelerates HIV replication via protein kinase A-dependent effects on cytokine production

In vivo, IgE production is related to bronchial hyperresponsiveness and, in vitro, passive sensitization of human airways with asthmatic serum containing a high concentration of IgE enhances the contractile response to a variety of agonists. However, cell types implicated in this IgE sensitization are not fully determined. The aim of this study was to determine IgE-bearing cells during passive sensitization with special reference to mast cells. Peripheral bronchi were dissected out from 10 lung specimens obtained at thoracotomy and processed into glycolmethacrylate resin. Sections, each 2 microm thick, were passively sensitized by incubation for 2 h at 37 degrees C in either buffer supplemented with monoclonal IgE or asthmatic serum with a high concentration of IgE (> or = 1,000 IU/ml). Immunohistochemistry was performed using monoclonal antibodies directed against the epsilon chain, and markers of the various IgE-bearing cells (e.g., AA1, antichymase). The number of IgE-bearing cells was significantly higher in passively sensitized specimens as compared with nonsensitized specimens (6.63 +/- 1.71 versus 4.29 +/- 1.35/mm2; p = 0.013, n = 10). Mast cells represented 65% of IgE-bearing cells, 41.6 and 23.4% for TC and T subtypes, respectively. These results indicate that mast cell is the main cell type involved in IgE-induced passive sensitization. The involvement of mast cell-derived tryptase in the mechanisms of IgE-related hyperresponsiveness should be further examined.     

Allergen challenge of passively sensitized human bronchi alters M2 and beta2 receptor function

We investigated whether antigen challenge may alter M2 and beta2 receptor function in isolated passively sensitized human bronchi. Bronchial rings (2-4 mm internal diameter) were obtained from 12 patients. Passive sensitization was induced by serum containing high IgE levels to Dermatophagoides pteronyssinus and Dermatophagoides farinae. Rings from six patients were used to study M2 receptor function by incubating with cumulatively increasing concentrations (10(-8) M to 10(-4) M) of pilocarpine and applying electric field stimulation (EFS) at 24 Hz. The rings from the other six patients were used to study beta2 receptor function by precontracting with carbachol 10(-6) M and adding cumulatively salbutamol (10(-9) M to 10(-4) M). Additional rings from these patients were used to determine whether dysfunction occurred distal to cAMP production by precontracting with carbachol 10(-6) M and adding cumulatively theophylline (10(-9) M to 10(-4) M). The attenuation of EFS-induced force by pilocarpine and the relaxation of carbachol-precontracted rings by salbutamol were less in challenged than in control and sensitized rings. No difference between challenged, sensitized, and control rings was observed with theophylline. We conclude that allergen challenge in passively sensitized isolated human bronchial rings may result in M2 and beta2 receptor dysfunction without involving mechanisms distal to cAMP formation. It appears that products from inflammatory cells recruited from blood are not necessary for this receptor dysfunction.     

Prognostic evaluation of cardiogenic shock by the severity

The aim of this study was to assess the total IgE levels in the supernatants and cellular components of colostrum from atopic and nonatopic mothers. Immunoglobulin E protein was detected in 34/39 milk samples, with a median level of 0.3 microgram/l. In 13 mothers, IgE protein was also detected in the cellular fraction of colostrum, with a median level of 0.13 microgram/l. Of the total IgE content in breast milk, 5-12% was transported intracellularly. The total IgE antibody levels were similar in both milk supernatants and cells from atopic and nonatopic mothers. There was a strong relationship between total IgE antibody levels in serum and in breast milk (r = 1.0, P < 0.001), suggesting that IgE antibodies were passively transported from blood into breast milk. The levels of total IgE in human milk are probably too low to have a significant effect on the regulation of the IgE antibody levels in the neonate.     

In vitro effects of the pyrethroid S-bioallethrin on lymphocytes and basophils from atopic and nonatopic subjects

Synthetic pyrethroids are increasingly used as insecticides and marketed as having relatively low human toxicity. The aim of this study was to examine the in vitro effects of the synthetic pyrethroid S-bioallethrin on human blood lymphocytes and basophils in atopic individuals and nonatopic control subjects. S-bioallethrin caused inhibition of lymphocyte proliferation after a 72-h culture period in a concentration-dependent manner. The inhibition of the lymphocyte proliferation by S-bioallethrin at the concentration 6.5 microM correlated well with the total serum IgE values (r = -0.89, P < 0.001). Samples from atopic subjects were more sensitive to this inhibition than those from nonatopic volunteers. The regulatory interleukin-4/interferon-gamma (JL-4/IFN-gamma) balance showed a significant difference between atopic and nonatopic subjects after a short-term culture period (24 h) in the presence of the same concentration range of S-bioallethrin (P < 0.001). Additionally, IFN-gamma secretion was consistently lower in cells from the atopic donors. Furthermore, S-bioallethrin induced histamine release from human basophils in a concentration-dependent manner. Although the effect was small compared to histamine liberators such as N-formyl-Met-Leu-Phe and anti-IgE, the response to S-bioallethrin was significantly different in atopic donors from nonatopic (P = 0.0431). These findings are the first demonstration of the immunotoxicologic properties of the synthetic pyrethroid S-bioallethrin by this combined in vitro approach with human lymphocytes and basophils. Further studies will investigate the responses of lymphocytes from patients who are sensitive to these agents.     

Investigation into the role of cyclooxygenase products in the bradykinin response on isolated human myometrium and umbilical artery

The aim of this study was to investigate the response to bradykinin (BK) on human myometrium and umbilical artery with respect to cyclo-oxygenase (CO) products. Dose/concentration response curves to BK were performed +/-2.79 microM indomethacin. On human myometrium the response to BK (0.001-50 nmol) was biphasic and consisted of a dose-related increase in myometrial tension which was followed by a period of inhibition of myogenic activity. In tissues from P donors the presence of indomethacin had no significant effect on the excitatory response, but the inhibitory component of the response was reduced. In tissues from NP donors indomethacin significantly enhanced the BK effect at higher doses and the inhibitory component of the response was reduced. On the HUA cumulative addition of BK (1-1000 nM) resulted in dose dependent constriction with desensitisation at the highest dose (EC50 = 38 nM). The presence of indomethacin had no significant effect on BK response on HUA. These findings suggest that CO products contribute significantly to response to BK on the human myometrium but not on HUA and that different CO products are produced by P and NP tissue.     

Treatment of patients with acute lymphoblastic leukemia with bulky extramedullary disease and T-cell phenotype or other poor prognostic features: randomized controlled trial from the Children's Cancer Group

BACKGROUND: Children with acute lymphoblastic leukemia with multiple poor prognostic factors and who have a lymphomatous mass at diagnosis, whether of T- or non-T-immunophenotype, are at increased risk of short term remission and extramedullary recurrence, and are in need of better therapies. METHODS: Six hundred and ninety-four eligible patients ranging in age from 1-20 years were entered on the study. Sixty-five percent of the patients had T-cell immunophenotype. Of these, 678 were randomized to one of four regimens: Regimen A: Berlin-Frankfurt-Munster (BFM) 76/79; Regimen B: LSA2-L2 with cranial irradiation; Regimen C: LSA2-L2 without cranial irradiation; and Regimen D: the New York (NY) regimen. RESULTS: Complete remission was induced in 97% of patients. The overall event free survival (EFS) +/- the standard deviation was 60 +/- 4% 6 years after diagnosis, in contrast to 36 +/- 6% in a comparable historic group. The EFS of the 371 T-cell patients was 62 +/- 7%. EFS was best on the NY (67 +/- 7%) and the BFM (67 +/- 6%) arms. These were significantly better than the EFS on the 2 LSA-L2 regimens, with an EFS of 53 +/- 8% (Regimen B) and 42 +/- 11% (Regimen C) (P = 0.03 and 0.0003 for NY vs. Regimen B and NY vs. Regimen C; P = 0.01 and 0.0001 for BFM vs. Regimen B and BFM vs. Regimen C). Regimen C had a 3-fold greater central nervous system (CNS) recurrence rate than the identical chemotherapy Regimen B (16 +/- 5% vs. 6 +/- 4%; P = 0.02), although the difference in overall EFS did not reach the required level for significance. Testicular recurrence varied from 2-8% in comparison with 20% in the historic group. EFS was not influenced by age, gender, CNS disease at diagnosis, morphology, or immunophenotype. In addition to treatment regimen and early response rate, initial leukocyte count, hemoglobin level, liver, spleen, and lymph node enlargement, and the presence of a mediastinal mass had univariate prognostic influence on EFS. In multivariate analysis, only the kinetics of response, leukocyte count (unfavorably, P < 0.0001), and mediastinal mass status (favorably, P = 0.01) were prognostic. CONCLUSIONS: The adverse prognostic implications of lymphomatous ALL can be minimized by the NY and BFM regimens. Cranial irradiation resulted in better CNS disease control when added to the LSA2-L2 regimen, but did not improve the overall disease free survival. With improved systemic chemotherapy, there was no excess of lymph node, testicular, or other local recurrence without prophylactic irradiation to sites of initial bulk disease or to the testes.     

Expression of protein kinase C gene family members is temporally and spatially regulated during neural development in vitro

The dog mastocytoma BR cell line provides us with a permanent source of canine mast cells, allowing a characterization of secretory mediators that exert important effects in canine allergic and nonallergic diseases and in physiological processes. We studied the ultrastructural characteristics and histamine releasing activity after immunological and non-immunological stimuli of the dog mastocytoma BR cell line, and compared the cell line to normal skin mast cells enzymatically isolated from healthy dogs. The histamine content of BR cells was 0.04 +/- 0.002 pg/cell, approximately 100-fold less than that found in canine skin mast cells. Non-immunologic stimuli induced similar concentration-dependent histamine release from skin mast cells and BR cells: 29.3 +/- 0.9% vs. 12.7 +/- 0.7% (calcium ionophore A23187), 23.3 +/- 0.7% vs. 18.8 +/- 0.7% (substance P) and 12.5 +/- 0.3% vs. 12.1 +/- 0.9% (compound 48/80), respectively. Immunologic stimulation, however, was only effective on canine skin mast cells, causing 30.9 +/- 1.7%, 27.7 +/- 0.6% and 12.2 +/- 0.9% histamine release in response to anti-canine IgE, concanavalin A, and antigen Asc S 1, respectively. The absence of functional IgE receptors in BR cells was confirmed by the lack of response to anti-IgE and antigen Asc S 1 following passive sensitization with dog atopic serum and dog antigen sensitized serum. We conclude that BR cells are able to release histamine after non-immunologic stimulation in a similar manner to canine skin mast cells, but that there are morphological and functional differences possibly due to different states of maturity or differentiation. For this reason the study of the highly homogeneous BR cells could offer insights into dog mast cell biology in contexts where freshly isolated cells cannot be used because of low purity and recovery.     

In vitro passive sensitization of the ureter as a basis for the study of noninfectious ureteral inflammation

PURPOSE: To develop an in vitro model of passive sensitization for the ureter for the study of noninfectious ureteral inflammation. MATERIALS AND METHODS: Human ureteral tissues were obtained from excess segments of ureters from patients undergoing donor nephrectomy. Following excision, ureters were placed in physiologic salt solution (PSS) and passively sensitized by incubating with ragweed serum from allergic donor (1 ml. serum: 4 ml. PSS) for 20 hours at room temperature. Ureteral segments were incubated with PSS only and served as non-sensitized controls (n = 4). After sensitization, excess serum was removed by serial washing with PSS without serum. Ureteral strips were then suspended in vitro for determination of tissue contraction. Contractile responses and histamine release were measured. Tissues were then exposed to antigen. To investigate the role of inflammatory mediators in tissue contraction, 4 groups of 8 sensitized ureteral segments were incubated for 1 hour with the following substances: a H1 histamine receptor antagonist (pyrilamine), an inhibitor of prostaglandin synthesis (indomethacin), an inhibitor of leukotriene synthesis (A-64077), and a control substance (DMSO). Following incubation, the tissues were exposed to antigen, and contraction and histamine release were determined. RESULTS: Sensitized ureteral segments (n = 8) responded to antigen with contraction (30% BaCl maximum; p <0.01) and histamine release (205/ng./gm. tissue) within the first 5 minutes of superfusion. Non-sensitized control segments (n = 4) did not respond. Both indomethacin and pyrilamine reduced (7-10% of BaCl maximum; p <0.05) the contractile response of sensitized ureter to antigen, whereas A-64077 did not. Analysis of the superfusate for histamine indicates that indomethacin reduced histamine release (150 ng./gm.) whereas A-64077 and pyrilamine did not (p <0.05). CONCLUSION: We have demonstrated that ureteral segments can be passively sensitized and that subsequent antigen challenge stimulates contraction and histamine release. Our findings suggest that contraction of ureteral tissue and histamine release may be utilized as an inherent bioassay indicating the activity of inflammatory mediators. In addition, these results suggest that both prostaglandins and histamine, but apparently not leukotrienes, participate in the early inflammatory response to antigen challenge of the sensitized ureter.     

Effects of load and tone on the mechanics of isolated human bronchial smooth muscle

Isotonic and isometric properties of nine human bronchial smooth muscles were studied under various loading and tone conditions. Freshly dissected bronchial strips were electrically stimulated successively at baseline, after precontraction with 10(-7) M methacholine (MCh), and after relaxation with 10(-5) M albuterol (Alb). Resting tension, i.e., preload determining optimal initial length (Lo) at baseline, was held constant. Compared with baseline, MCh decreased muscle length to 93 +/- 1% Lo (P < 0.001) before any electrical stimulation, whereas Alb increased it to 111 +/- 3% Lo (P < 0.01). MCh significantly decreased maximum unloaded shortening velocity (0.045 +/- 0.007 vs. 0.059 +/- 0.007 Lo/s), maximal extent of muscle shortening (8.4 +/- 1.2 vs. 13.9 +/- 2.4% Lo), and peak isometric tension (6.1 +/- 0.8 vs. 7.2 +/- 1.0 mN/mm2). Alb restored all these contractile indexes to baseline values. These findings suggest that MCh reversibly increased the number of active actomyosin cross bridges under resting conditions, limiting further muscle shortening and active tension development. After the electrically induced contraction, muscles showed a transient phase of decrease in tension below preload. This decrease in tension was unaffected by afterload levels but was significantly increased by MCh and reduced by Alb. These findings suggest that the cross bridges activated before, but not during, the electrically elicited contraction may modulate the phase of decrease in tension below preload, reflecting the active part of resting tension.     

The scimitar syndrome: clinical spectrum and surgical treatment

The stretch-induced myogenic response (MR) of large-capacitance pulmonary arteries were studied in normal and pulmonary hypertensive fetuses as well as normal newborn and adult sheep. Pulmonary hypertension in the fetus was induced by ligation of the ductus arteriosus for 12 d. The MR was obtained by stretching the vessel segments in vitro from their resting diameter (no load) to the diameter at which the muscle fibers were at optimal length (Lo), and the response was measured as a percentage of force obtained after supramaximal electrical stimulation (Po). In five control and four pulmonary hypertensive fetuses, the MR was also obtained after a stretch of 140% of Lo. The pulmonary hypertensive fetal arteries had a lower stress (1.3 +/- 0.4 versus 4.0 +/- 0.5 mN/mm2; p < 0.001) and shortening capacity compared with the fetal control (5.1 +/- 1.6 versus 9.9 +/- 0.8% of Lo; p < 0.01). The MR was observed in 21% of the control and 30% of the experimental fetuses, and it was of greater magnitude in the latter (14.8 +/- 1.9 of Po versus 34.3 +/- 2.5%, respectively; p < 0.01). When stretched to 140% of Lo, the MR was also greater in the experimental (514 +/- 148% of Po) than the control fetuses (142 +/- 68; p < 0.05). Postnatally, the MR was present in 67% of the newborn and 15% of the adult pulmonary artery segments, and the response was greatest in the newborn (23.1 +/- 4.2% of Po) compared with the adult (2.3 +/- 0.8; p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Correct blue-light regulation of pea Lhcb genes in an Arabidopsis background

The phasic contraction of the isolated guinea pig vas deferens induced by adenosine 5'-triphosphate (ATP) 1mM was significantly augmented by acidification of bathing solution induced by administration of hydrochloric acid (HCL) 10mM (pH = 6.87 +/- 0.015 (n = 5); mean +/- S.E.), while the tonic contraction induced by norepinephrine (NE) 10microM was significantly depressed by HCl 10mM. The contractile response to ATP 1mM was markedly potentiated in the presence of NE 10 microM. The potentiated contractile response to ATP 1mM in the presence of NE 10microM was significantly augmented by HCl 1mM or 10mM. The potentiating ratio of the contraction induced by ATP 1mM in the presence of NE 10microM to that induced by ATP 1mM alone was almost unaffected by the administration of HCl. Electrical field stimulation (EFS) produced a biphasic contractile response of the isolated guinea pig vas deferens; viz. the first rapid phasic contractile response and the second slow maintained tonic contractile response. The phasic contractile response to EFS was significantly augmented by HCl 10mM. These results may indicate that acidification of the medium potentiates the neurochemical transmission between the nerve terminals and the smooth muscle of vas deferens via sensitization of P2X (presumably P2X1 or P2X2) receptors existing in the smooth muscle.     

Evidence that tachykinins are the main NANC excitatory neurotransmitters in the guinea-pig common bile duct

Application of electrical field stimulation (EFS; trains of 10 Hz, 0.25 ms pulse width, supramaximal voltage for 60 s) to the guinea-pig isolated common bile duct pretreated with atropine (1 microM), produced a slowly-developing contraction ('on' response) followed by a quick phasic 'off' contraction ('off peak' response) and a tonic response ('off late' response), averaging 16+/-2, 73+/-3 and 20+/-4% of the maximal contraction to KCl (80 mM), n=20 each, respectively. Tetrodotoxin (1 microM; 15 min before) abolished the overall response to EFS (n 8). Neither in vitro capsaicin pretreatment (10 microM for 15 min), nor guanethidine (3 microM, 60 min before) affected the excitatory response to EFS (n 5 each), showing that neither primary sensory neurons, nor sympathetic nerves were involved. Nomega-nitro-L-arginine (L-NOARG, 100 microM, 60 min before) or naloxone (10 microM, 30 min before) significantly enhanced the 'on' response (294+/-56 and 205+/-25% increase, respectively; n=6-8, P<0.01) to EFS. The combined administration of L-NOARG and naloxone produced additive enhancing effects (655+/-90% increase of the 'on' component, n = 6, P<0.05). The tachykinin NK2 receptor-selective antagonist MEN 11420 (1 microM) almost abolished both the 'on' and 'off late' responses (P<0.01: n=5 each) to EFS, and reduced the 'off-peak' contraction by 55+/-8% (n=5, P<0.01). The subsequent administration of the tachykinin NK1 receptor-selective antagonist GR 82334 (1 microM) and of the tachykinin NK3 receptor-selective antagonist SR 142801 (30 nM), in the presence of MEN 11420 (1 microM), did not produce any further inhibition of the response to EFS (P>0.05; n=5 each). At 3 microM, GR 82334 significantly reduced (by 68+/-9%, P<0.05, n=6) the 'on' response to EFS. The contractile 'off peak' response to EFS observed in the presence of both MEN 11420 and GR 82334 (3 microM each) was abolished (P<0.01; n=6) by the administration of the P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM). PPADS (30 microM) selectively blocked (75+/-9 and 50+/-7% inhibition, n = 4 each) the contractile responses produced by 100 and 300 microM ATP. Tachykinin-containing nerve fibres were detected by using immunohistochemical techniques in all parts of the bile duct, being distributed to the muscle layer and lamina propria of mucosa. In the terminal part of the duct (ampulla) some labelled ganglion cells were observed. In conclusion, this study shows that in the guinea-pig terminal biliary tract tachykinins, released from intrinsic neuronal elements, are the main NANC excitatory neurotransmitters, which act by stimulating tachykinin NK2 (and possibly NK1) receptors. ATP is also involved as excitatory neurotransmitter. Nitric oxide and opioids act as inhibitory mediators/modulators in this preparation.     

Studies on the mechanisms involved in antigen-evoked pleural inflammation in rats: contribution of IgE and complement

Immunoglobulin E (IgE) has been shown to play a critical role in the allergic late-phase reaction, which is marked by intense leukocyte infiltration and edema. In this study we assessed the allergic pleural inflammation triggered by intrapleural (i.pl.) challenge in sensitized rats. We examined pleural effluent from actively sensitized rats following anti-IgE monoclonal antibody (mAb) (MARE-1) provocation for protein exudation, neutrophil as well as eosinophil accumulation. Inflammatory changes triggered by antigen after passive sensitization with IgE mAb was also assessed for comparison. Total serum level of IgE was found to be about threefold increased 7-8 days post-active sensitization, remaining augmented for at least 30 days. Increased levels of peritoneal leukocyte-bound IgE and serum IgE with specificity to ovalbumin were also detected. Nevertheless, the anti-IgE challenge in 14-day actively sensitized was shown to be a weak stimulus of neutrophil and eosinophil accumulation, despite being able to cause intense protein extravasation. Similarly, antigen challenge of IgE-passively sensitized rats caused protein leakage that was comparable to that induced by anti-IgE mAb in actively sensitized rats but led to a much lower neutrophil/eosinophil infiltration. Also, blockade of complement with recombinant human soluble C receptor-1 (sCR1) treatment prevented actively sensitized rats from reacting to antigen with neutrophil and eosinophil recruitment without modifying protein extravasation. These data suggest that IgE and complement-mediated mechanisms probably account for the exudation and leukocyte infiltration that is characteristic of the pleural inflammatory response observed in actively sensitized rats.     

ALL R-87 protocol in the treatment of children with acute lymphoblastic leukaemia in early bone marrow relapse

Seventy-three children with acute lymphoblastic leukaemia (ALL) in first bone marrow (BM) relapse, occurring within 30 months from complete remission (CR), were enrolled in an Italian cooperative study (ALL R-87 protocol). This treatment programme consisted of an induction phase with intermediate-dose cytarabine (IDARA-C) plus idarubicin (IDA) and prednisone (PDN), followed by a multidrug consolidation therapy and bone marrow transplant (BMT). 55/73 children achieved CR (75.3%); 15 (20.5%) failed to respond and three (4.2%) died during induction. The response rate was significantly higher for children with a first CR duration > or = 12 months (P=0.0005) and for those with a white blood cell (WBC) count at relapse < 20 x 10(9)/l (P=0.004). The estimated disease-free survival (DFS +/- SE) at 82 months was 0.18 +/- 0.05 for all responders, and 0.70 +/- 0.14 for allotransplanted patients versus 0.05 +/- 0.05 for those autografted (P=0.001). The estimated probabilities of survival +/- SE and event-free survival (EFS +/- SE) at 83 months were 0.16 +/- 0.07 and 0.13 +/- 0.04, respectively. for all enrolled children. Univariate analysis showed that age < 10 years at initial diagnosis and B-lineage immunophenotype favourably influenced both DFS (P=0.001) and EFS probabilities (P=0.0014 and P=0.012, respectively), whereas a first CR duration > or = 12 months and a WBC count at relapse < 20 x 10(9)/l were associated only with a better EFS rate (P=0.026 and P=0.004, respectively). Our results show the efficacy of the IDA plus IDARA-C schedule used in the ALL R-87 protocol in high-risk relapsed ALL children. Allogeneic BMT proved effective for patients with an HLA sibling donor. In a multivariate analysis, age > or = 10 years at initial diagnosis (P=0.016) and WBC count at relapse > or = 20 x 10(9)/l (P=0.048) were independently associated with a worse disease outcome.     

Olive pollen allergy: searching for immunodominant T-cell epitopes on the Ole e 1 molecule

BACKGROUND: The amino-acid and nucleotide sequence of Ole e 1 (the major antigen of olive pollen) has been described and the IgE antibody response to this major allergen was associated with DR7/DQ2 antigens. With this previous data we try to define the T-cell epitopes implicated in Ole e 1 reactivity. OBJECTIVES: To study the recognition of T cells (derived from allergic and non-allergic Ole e 1 patients) to Ole e 1 synthetic peptides in order to define immunodominant T-cell epitopes. METHODS: We have compared the proliferative response of the peripheral blood mononuclear cells from Ole e 1 sensitized patients vs. non-sensitized controls, induced by 14 Ole e 1 synthetic peptides. Thirty subjects were classified in two groups: group 1 (non-responders against Ole e 1, n=16) and group 2 (Ole e 1 responders, n=14), according to their clinical parameters and the presence or not in their sera of the significant Ole e 1 IgE antibody levels. RESULTS: Our results shown that it is possible to find T cells reactive to Ole e 1 peptides in patients with and without significant levels of Ole e 1 IgE antibodies. However, the percentage of response was higher in patients with IgE antibodies 71.4% vs 25%), and the recognition profile was different: the control group showed a broad reactivity pattern, in contrast, the response by the 'Ole e 1 responders' group was mainly directed against three peptides of the carboxi-terminal region, peptides 10 (91-102), 12 (109-120) and 13 (119-130), with a response frequency of 35.7, 28.5 and 28.5%, respectively. By direct and inhibition test no antibody response was found against the synthetic peptides. CONCLUSIONS: Our data suggest that the regions between 91 and 102 and 109-130 aminoacids on the Ole e 1 molecule are immunodominant T-cell epitopes. These epitopes are not recognized by IgE antibodies.