Expression of HCLCA1 in cystic fibrosis lungs is associated with mucus overproduction.

Mucus overproduction is typical in cystic fibrosis (CF) airway disease. The human calcium-activated chloride channel, hCLCA1, has been reported to be upregulated by interleukin (IL)-9 and to regulate the expression of mucins. Therefore, the expression of IL-9, IL-9 receptor (IL-9R) and hCLCA1 between the lungs of CF patients and healthy control subjects was compared. Endoscopic biopsy samples of bronchial mucosa from 10 CF patients and six control subjects were stained with periodic acid-Schiff. IL-9, IL-9R and hCLCA1 expression was determined by immunocytochemistry. Expression of hCLCA1 mRNA was also determined by in situ hybridisation. The present study found significant increases in IL-9, IL-9R and hCLCA1 immunoreactivity, hCLCA1 mRNA expression, and numbers of mucus-producing cells in the mucosa of CF patients compared to control subjects. Positive correlations were found between IL-9R-positive-cells with IL-9-positive cells and hCLCA1-positive cells, and between PAS-positive cells with hCLCA1-positive cells and IL-9R-positive cells. Expression of hCLCA1 mRNA was colocalised with IL-9R expression and PAS-positive staining in epithelial cells. Increased expression of interleukin-9 and interleukin-9 receptor, as well as an upregulation of the human calcium-activated chloride channel, hCLCA1, in mucus-producing epithelium of cystic fibrosis patients, support the hypothesis that interleukin-9 contributes to mucus overproduction in cystic fibrosis airway disease.

A phase I and pharmacokinetic study of squalamine, a novel antiangiogenic agent, in patients with advanced cancers.

PURPOSE: A Phase I study of squalamine, a novel antiangiogenic agent originally isolated from the dogfish shark Squalus acanthias, was conducted in patients with advanced cancers to: (a) determine the maximum tolerated dose (MTD), dose-limiting toxicity (DLT) and pharmacokinetics of squalamine lactate when given as a 120-h continuous i.v. infusion every two weeks; and (b) to obtain information on prolonged (>120-h) continuous i.v. infusions in patients who have tolerated 120-h infusions. EXPERIMENTAL DESIGN: A rapid dose escalation scheme was used that permitted intrapatient dose escalation. Three or more patients were treated at each dose, of which at least one patient started treatment de novo at that dose. Once DLT was encountered, the dose was decreased by one dose level, and the duration of infusion was prolonged from 10 up to 30 days in 5-day increments. RESULTS: Nineteen patients were treated at eight squalamine dose levels; the number of patients/dose level who received 120-h infusions were [expressed as dose in mg/m(2)/day (number of patients initiated de novo at that dose/total number of patients treated at that dose)]: 6 (3/3), 12 (3/6), 24 (1/5), 48 (2/6), 96 (4/10), 192 (2/6), 384 (3/8), and 538 (1/5). DLT was encountered at 384 mg/m(2)/day (1/3 de novo patients, 5/8 total patients) and 538 mg/m(2)/day (1/1 de novo patients, 4/5 total patients) and consisted of hepatotoxicity, characterized by grade 3 transaminase elevations that resolved 3-11 days after ceasing squalamine infusion. Three patients did not experience hepatotoxicity when first treated at 384 mg/m(2)/day but developed DLT at the same dose when de-escalated from 538 mg/m(2)/day. Other toxicities included grade 1-3 fatigue, grade 1-2 nausea, anorexia, and neuromuscular symptoms. The maximum duration of continuous i.v. infusion was 20 days at a dose rate of 192 mg/m(2)/day in one patient without adverse effects. Pharmacokinetic calculations revealed a linear relationship between area under the curve or Cmax and squalamine dose rate up to 384 mg/m(2)/day, with a prolonged terminal squalamine persistence in patient plasma (median t(1/2) = 18 h; range, 8-48 h). Transient tumor responses were observed in a patient with synovial cell sarcoma and a patient with breast carcinoma with cutaneous metastases. CONCLUSIONS: The best tolerated dose rate of squalamine when administered as a 120-h continuous i.v. infusion was 192 mg/m(2)/day; however, patients without prior exposure to squalamine appeared to tolerate a dose rate of 384 mg/m(2)/day without DLT. On the basis of preclinical evidence of synergy with cytotoxic agents and demonstration of human safety from this trial, additional clinical trials have been initiated with squalamine in combination with chemotherapy for patients with late stage lung cancer and ovarian cancer.

Squalamine treatment of human tumors in nu/nu mice enhances platinum-based chemotherapies.

Squalamine, an antiangiogenic aminosterol, is presently undergoing Phase II clinical trials in cancer patients. To broaden our understanding of the clinical potential for squalamine, this agent was evaluated in nu/nu mouse xenograft models using the chemoresistant MV-522 human non-small cell lung carcinoma and the SD human neuroblastoma lines. Squalamine was studied alone and in combination with either cisplatin or paclitaxel plus carboplatin. Squalamine alone produced a modest MV-522 tumor growth inhibition (TGI) and yielded a TGI with cisplatin that was better than cisplatin alone. Squalamine also significantly enhanced the activity of paclitaxel/carboplatin combination therapy in the MV-522 tumor model. Squalamine similarly improved the effectiveness of cisplatin in producing TGI when screened against the SD human neuroblastoma xenograft. Xenograft tumor shrinkage was seen for the MV-522 tumor in combination treatments including squalamine, whereas no tumor shrinkage was seen when squalamine was omitted from the treatment regimen. To gain a greater understanding of the mechanism by which squalamine inhibited tumor growth in the xenograft studies, in vitro experiments were carried out with vascular endothelial growth factor-stimulated human umbilical vein endothelial cells in culture exposed to squalamine. Squalamine treatment was found to retard two cellular events necessary for angiogenesis, inducing disorganization of F-actin stress fibers and causing a concomitant reduction of detectable cell the surface molecular endothelial cadherin (VE-cadherin). We propose that the augmentation by squalamine of cytotoxicity from platinum-based therapies is attributable to interference by squalamine with the ability of stimuli to promote endothelial cell movement and cell-cell communication necessary for growth of new blood vessels in xenografts after chemotherapeutic injury to the tumor.

Interleukin-9 receptor expression in asthmatic airways In vivo.

Inflammation of the airway wall is a defining feature in asthma and is likely the cause of the hyperreactivity and variable airflow limitation found in asthma. Immune response biased toward production of Th2 cytokines has been proposed as a mechanism in the pathogenesis of airway inflammation in asthma. The Th2 cytokine interleukin-9 (IL-9) is one candidate gene for asthma on the basis of position cloning and animal models of airway inflammation. To determine whether IL-9 is involved in the chronic inflammation of the asthmatic airway, we investigated the expression of IL-9 and the IL-9 specific receptor chain in asthmatic airways compared with healthy airways. IL-9 and IL-9 receptor expression in airway epithelial cells and bronchoalveolar lavage cells obtained at bronchoscopy of healthy (n = 9) and mild intermittent asthmatic individuals (n = 7) were studied by Northern analyses and reverse-transcription polymerase chain reaction technique. Primary and transformed human airway epithelial cells were also evaluated for IL-9 specific receptor chain expression in vitro. IL-9 was not detected in airways of healthy or mild asthmatic individuals. In contrast, IL-9 specific receptor chain expression was found in asthmatic airway samples but not in healthy controls. In vitro, airway epithelial cells did not express IL-9 specific receptor chain until stimulation with interferon gamma. Our results support that IL-9 may play a role in the mechanism leading to chronic airway inflammation and asthma.

IL-9 and its receptor in allergic and nonallergic lung disease: increased expression in asthma.

BACKGROUND: Bronchial asthma is a chronic inflammatory disease associated with genetic components. Recently IL-9 has been reported as a candidate gene for asthma and to be associated with bronchial hyperresponsiveness and elevated levels of total serum IgE. OBJECTIVE: To investigate the contribution of IL-9 to the pathogenesis of asthma, we examined the expression of IL-9 and its receptor (IL-9R) in bronchial tissue from subjects with atopic asthma (n = 10), chronic bronchitis (n = 11), and sarcoidosis (n = 9) and from atopic (n = 7) and nonatopic (n = 10) healthy control subjects. METHODS: Bronchial biopsy specimens were examined for the presence of IL-9 and IL-9R protein and messenger RNA (mRNA) by immunocytochemistry and in situ hybridization, respectively. To phenotype the cells expressing IL-9 in asthmatic tissue, combined in situ hybridization and immunocytochemistry was also performed. RESULTS: There was a highly significant difference (P <.001) in the expression of IL-9 mRNA in asthmatic airways (20.6 +/- 4.0 cells/mm of basement membrane) compared with chronic bronchitis (5.6 +/- 4.4), sarcoidosis (2.5 +/- 1.8), atopic control subjects (7.7 +/- 2.2), and healthy control subjects (2.7 +/- 2.3). The number of IL-9 immunoreactive cells was also greater in asthmatic patients compared with the other groups (P <.05). Although the level of IL-9R mRNA expression did not differ in any of the groups (P >.05), IL-9R immunoreactivity was significantly higher in asthmatic compared with control subjects. Furthermore, IL-9 mRNA expression levels were also significantly correlated with FEV(1) (P <.05) and the airway responsiveness to methacholine producing a 20% fall in FEV(1) (P <. 01). The cells expressing IL-9 mRNA in asthmatic tissue were CD3(+) lymphocytes (68%), major basic protein(+) eosinophils (16%), and elastase(+) neutrophils (8%). CONCLUSION: The results of this study demonstrate the potential of IL-9 to be a marker for atopic asthma and furthermore suggest an important role for this cytokine in the pathophysiologic mechanisms of this disease.

In vitro susceptibility to pexiganan of bacteria isolated from infected diabetic foot ulcers.

During two clinical trials involving the treatment of 835 outpatients with infected diabetic foot ulcers, 2515 bacterial isolates, including 2337 aerobes and 178 anaerobes, were grown from cultures of the ulcers. The in vitro susceptibility of these isolates was determined to pexiganan, a peptide anti-infective evaluated in these clinical trials, and to other classes of antibiotics. Pexiganan demonstrated broad spectrum antimicrobial activity against Gram-positive and Gram-negative aerobes and anaerobes. The MIC90 values for the most common species among 1735 Gram-positive aerobes isolated, such as Staphylococcus aureus, coagulase-negative staphylococci, Group A streptococci, and Group B streptococci, were 16 micrograms/mL or less. Of 602 Gram-negative aerobes tested, the MIC90 values for pexiganan were 16 micrograms/mL or less for Acinetobacter, Pseudomonas, Stenotrophomonas, Citrobacter, Enterobacter, Escherichia, Klebsiella, and Flavobacterium species. Pexiganan had a MIC90 of 4 to 16 micrograms/mL against the anaerobic isolates of Bacteroides, Peptostreptococcus, Clostridium, and Prevotella species. Importantly, pexiganan did not exhibit cross-resistance with other commonly used antibiotics, including beta-lactams, quinolones, macrolides, and lincosamides. The broad spectrum in vitro antimicrobial activity of pexiganan against clinical isolates from infected diabetic foot ulcers supports its potential as a local therapy for infected diabetic foot ulcers.

IL-9 induces chemokine expression in lung epithelial cells and baseline airway eosinophilia in transgenic mice.

Recent data have identified IL-9 as a key cytokine in determining susceptibility to asthma. These data are supported by the finding that allergen-exposed IL-9-transgenic mice exhibit many features that are characteristic of human asthma (airway eosinophilia, elevated serum IgE and bronchial hyperresponsiveness) as compared to the background strain. A striking feature of these animals is a robust peribronchial and perivascular eosinophilia after allergen challenge, suggesting that IL-9 is a potent factor in regulating this process. In an attempt to gain insights into the molecular mechanism governing IL-9 modulation of lung eosinophilia, we investigated the ability of this cytokine to induce the expression of CC-type chemokines in the lung because of their effect on stimulating eosinophil chemotaxis. Here we show that IL-9-transgenic mice in contrast to their congenic controls exhibit baseline lung eosinophilia that is associated with the up-regulation of CC-chemokine expression in the airway. This effect appears to be through a direct action of IL-9 because the addition of recombinant IL-9 to primary epithelial cultures and cell lines induced the expression of these chemokines in vitro. These data support a mechanism for IL-9 in regulating the expression of eosinophil chemotactic factors in lung epithelial cells.

IL-9 pathway in asthma: new therapeutic targets for allergic inflammatory disorders.

BACKGROUND: Asthma is a complex heritable inflammatory disorder of the airways associated with clinical signs of allergic inflammation and bronchial hyperresponsiveness (BHR). The incidence of asthma continues to rise in industrialized countries despite advances in the identification of cellular and molecular mediators that are associated with the disease. Because of its importance in human health, additional research and alternative therapeutic strategies are justified to create more effective treatments for this debilitating disease. OBJECTIVE: Studies use recombinant inbred mice to demonstrate that BHR in mouse models of asthma is associated with a genetic alteration at the IL-9 locus, where IL-9 expression in lung is strongly associated with bronchial responsiveness. We have investigated the ability of intratracheal instilled IL-9 to induce asthmatic-like responses in naive C57BL/6 (B6) mice, which express very low levels of IL-9. METHODS: IL-9 or vehicle was intratracheal instilled in naive B6 mice for 10 days. Mice were analyzed for effects on BHR, lung eosinophilia, and serum total IgE levels. RESULTS: Phenotypic effects of B6 mice instilled with IL-9 were increased eosinophils in the bronchoalveolar lavage and significantly elevated serum total IgE. Moreover, IL-9 was found to induce IL-5Ralpha in vivo and in vitro, suggesting a potential mechanism for the novel actions described for IL-9 on eosinophils. CONCLUSION: Increased levels of IL-9 in the airway of naive B6 mice induced lung eosinophilia and serum total IgE levels, which are 2 clinical features of asthma. These data support a central role for the IL-9 pathway in the complex pathogenesis of allergic inflammation.

In vitro antibacterial properties of pexiganan, an analog of magainin.

Pexiganan, a 22-amino-acid antimicrobial peptide, is an analog of the magainin peptides isolated from the skin of the African clawed frog. Pexiganan exhibited in vitro broad-spectrum antibacterial activity when it was tested against 3,109 clinical isolates of gram-positive and gram-negative, anaerobic and aerobic bacteria. The pexiganan MIC at which 90% of isolates are inhibited (MIC90) was 32 micrograms/ml or less for Staphylococcus spp., Streptococcus spp., Enterococcus faecium, Corynebacterium spp., Pseudomonas spp., Acinetobacter spp., Stenotrophomonas spp., certain species of the family Enterobacteriaceae, Bacteroides spp., Peptostreptococcus spp., and Propionibacterium spp. Comparison of the MICs and minimum bactericidal concentrations (MBCs) of pexiganan for 143 isolates representing 32 species demonstrated that for 92% of the isolates tested, MBCs were the same or within 1 twofold difference of the MICs, consistent with a bactericidal mechanism of action. Killing curve analysis showed that pexiganan killed Pseudomonas aeruginosa rapidly, with 10(6) organisms/ml eliminated within 20 min of treatment with 16 micrograms of pexiganan per ml. No evidence of cross-resistance to a number of other antibiotic classes was observed, as determined by the equivalence of the MIC50s and the MIC90s of pexiganan for strains resistant to oxacillin, cefazolin, cefoxitin, imipenem, ofloxacin, ciprofloxacin, gentamicin, and clindamicin versus those for strains susceptible to these antimicrobial agents. Attempts to generate resistance in several bacterial species through repeated passage with subinhibitory concentrations of pexiganan were unsuccessful. In conclusion, pexiganan exhibits properties in vitro which make it an attractive candidate for development as a topical antimicrobial agent.

Interleukin-9 promotes allergen-induced eosinophilic inflammation and airway hyperresponsiveness in transgenic mice.

Human atopic asthma is a complex heritable inflammatory disorder of the airways associated with clinical signs of allergic inflammation and airway hyperresponsiveness. Recent studies demonstrate that the degree of airway responsiveness is strongly associated with interleukin (IL)-9 expression in murine lung. To investigate the contribution of IL-9 to airway hyperresponsiveness, and to explore directly its relationship to airway inflammation, we studied transgenic mice overexpressing IL-9. In this report we show that IL-9 transgenic mice (FVB/N-TG5), in comparison with FVB/NJ mice, display significantly enhanced eosinophilic airway inflammation, elevated serum total immunoglobulin E, and airway hyperresponsiveness following lung challenge with a natural antigen (Aspergillus fumigatus). These data support a central role for IL-9 in the complex pathogenesis of allergic inflammation.

Asthma and bronchial hyperresponsiveness linked to the XY long arm pseudoautosomal region.

We examined the long arm XY pseudoautosomal region for linkage to asthma, serum IgE, and bronchial hyperresponsiveness. In 57 Caucasian families multipoint nonparametric analyses provide evidence for linkage between DXYS154 and bronchial hyperresponsiveness (P = 0.000057) or asthma (P = 0.00065). This genomic region is approximately 320 kb in size and contains the interleukin-9 receptor gene. These results suggest that a gene controlling asthma and bronchial hyperresponsiveness maybe located in this region and that the interleukin-9 receptor is a potential candidate.

Molecular analysis of human interleukin-9 receptor transcripts in peripheral blood mononuclear cells. Identification of a splice variant encoding for a nonfunctional cell surface receptor.

Genetic studies on mouse models of asthma have identified interleukin-9 (IL9) as a determining factor in controlling bronchial hyperresponsiveness, a hallmark of the disease. Recently, the human IL9 receptor (hIL9R) gene locus has also been implicated in determining susceptibility to bronchial hyperresponsiveness and asthma. In order to evaluate the structure and function of the encoded product, we analyzed receptor transcripts derived from peripheral blood mononuclear cells of 50 unrelated donors. Sequence analysis of the entire coding region identified a splice variant that contains an in frame deletion of a single residue at codon 173 (DeltaQ). This variant could be permanently expressed in a cytokine-dependent murine T-cell line but lacked the ability to induce proliferation in response to human IL9. In situ analyses of cells expressing the wild-type and DeltaQ receptors found both forms to be expressed at the cell surface, but the DeltaQ receptor was unable to bind hIL9 and could not be recognized by N-terminal specific antibodies. These findings demonstrate that hIL9RDeltaQ presents an altered structure and function and suggests its potential role in down-regulating IL9 signaling in effector cells and associated biological processes.

Interleukin 9: a candidate gene for asthma.

Asthma is a complex heritable inflammatory disorder of the airways associated with clinical signs of atopy and bronchial hyperresponsiveness. Recent studies localized a major gene for asthma to chromosome 5q31-q33 in humans. Thus, this segment of the genome represents a candidate region for genes that determine susceptibility to bronchial hyperresponsiveness and atopy in animal models. Homologs of candidate genes on human chromosome 5q31-q33 are found in four regions in the mouse genome, two on chromosome 18, and one each on chromosomes 11 and 13. We assessed bronchial responsiveness as a quantitative trait in mice and found it linked to chromosome 13. Interleukin 9 (IL-9) is located in the linked region and was analyzed as a gene candidate. The expression of IL-9 was markedly reduced in bronchial hyporesponsive mice, and the level of expression was determined by sequences within the qualitative trait locus (QTL). These data suggest a role for IL-9 in the complex pathogenesis of bronchial hyperresponsiveness as a risk factor for asthma.

Linkage analysis of susceptibility to ozone-induced lung inflammation in inbred mice.

Exposures to the common air pollutant ozone (O3) cause decrements in pulmonary function and induce airway inflammation that is characterized by infiltration of polymorphonuclear neutrophils (PMNs; refs 1-4). Because of the impact that O3 may have on public health, it is critical to identify susceptibility factors. Highly reproducible, significant inter-individual variations in human pulmonary function responses to O3 support the hypothesis that genetic background is an important determinant. Initial analysis of PMN responses to O3 exposure in segregant populations derived from inflammation-prone (susceptible) C57BL/6J (B6) and inflammation-resistant C3H/HeJ (C3) inbred mice indicated that susceptibility was controlled by a locus we termed Inf2 (ref. 7). Subsequent analyses with recombinant inbred strains suggested that a more complex interaction of genes is involved. In this report, we identify a quantitative trait locus (QTL) for O3 susceptibility on chromosome 17. Candidate genes for the locus include Tnf, the gene encoding the pro-inflammatory cytokine tumour necrosis factor-alpha (Tnf). Antibody neutralization of the protein product of this putative candidate gene significantly protected against O3 injury in susceptible mice. These results strongly support linkage of O3 susceptibility to a QTL on chromosome 17 and Tnf as a candidate gene.

Genetic modeling of susceptibility to nitrogen dioxide-induced lung injury in mice.

We investigated the mode of inheritance of susceptibility to nitrogen dioxide (NO2)-induced lung injury in inbred mice. Susceptible C57BL/6J (B6) and resistant C3H/HeJ (C3) mice, as well as F1, F2, and backcross (BX) populations derived from them, were exposed to 15 parts per million NO2 for 3 h. Six hours after exposure, animals were lavaged, and differential cell counts and cell viability (cytotoxicity) were measured. Statistically significant (P < 0.05) differences in numbers of lavageable macrophages, epithelial cells, and dead cells were found between inbred strains. Distributions of cellular responses in F1 progeny overlapped both progenitors, and mean responses were intermediate. In C3:BX progeny, ranges of responses to NO2 closely resembled C3 mice, and means were not significantly different between populations. Ranges of cellular responses to NO2 in B6:BX and intercross progeny overlapped both progenitors; mean responses of both populations were intermediate to progenitors. Segregation analyses tested goodness of fit of phenotyping data with various inheritance models, and the highest likelihood for each cell response to NO2 was for the hypothesis two-unlinked loci general. We conclude that there are likely two major unlinked genes that account for differential susceptibility to acute NO2 exposure. The chromosomal location of the genes is not known.

Genetic susceptibility to asthma--bronchial hyperresponsiveness coinherited with a major gene for atopy.

BACKGROUND: Bronchial hyperresponsiveness, a risk factor for asthma, consists of a heightened bronchoconstrictor response to a variety of stimuli. The condition has a heritable component and is closely related to serum IgE levels and airway inflammation. The basis for these relations is unknown, as is the mechanism of genetic susceptibility to bronchial hyperresponsiveness. We attempted to define the interrelation between atopy and bronchial hyperresponsiveness and to investigate the chromosomal location of this component of asthma. METHODS: We studied 303 children and grandchildren of 84 probands with asthma selected from a homogeneous population in the Netherlands. Ventilatory function, bronchial responsiveness to histamine, and serum total IgE were measured. The association between the last two variables was evaluated. Using analyses involving pairs of siblings, we tested for linkage between bronchial hyperresponsiveness and genetic markers on chromosome 5q31-q33, previously shown to be linked to a genetic locus regulating serum total IgE levels. RESULTS: Serum total IgE levels were strongly correlated (r = 0.65, P < 0.01) in pairs of siblings concordant for bronchial hyperresponsiveness (defined as a > or = 20 percent decrease in the forced expiratory volume in one second produced by histamine [threshold dose, < or = 16 mg per milliliter]), suggesting that these traits are coinherited. However, bronchial hyperresponsiveness was not correlated with serum IgE levels (r = 0.04, P > 0.10). Analyses of pairs of siblings showed linkage of bronchial hyperresponsiveness with several genetic markers on chromosome 5q, including D5S436 (P < 0.001 for a histamine threshold value of < or = 16 mg per milliliter). CONCLUSIONS: This study demonstrates that a trait for an elevated level of serum total IgE is coinherited with a trait for bronchial hyperresponsiveness and that a gene governing bronchial hyperresponsiveness is located near a major locus that regulates serum IgE levels on chromosome 5q. These findings are consistent with the existence of one or more genes on chromosome 5q31-q33 causing susceptibility to asthma.

Evidence for two unlinked loci regulating total serum IgE levels.

Studies investigating the genetic control of total serum IgE levels are of major importance in understanding basic pathophysiologic mechanisms in atopy and asthma, since IgE levels predict onset and correlate with the clinical expression of these disorders. Previous analysis of data from 92 families, ascertained through a parent with asthma, showed evidence for recessive inheritance of high IgE levels with linkage to chromosome 5q. Since there was significant residual familial correlation in the one-locus segregation analysis, two-locus segregation and linkage analyses were performed. Segregation analyses provided evidence for a second major locus unlinked to the locus on 5q. Utilization of this two-locus model corroborates the previous evidence for linkage between this trait and markers on 5q31-q33. The LODs for the most informative marker D5S436 increased from 3.00 at 10% recombination to 4.67 at 9% recombination, when the two-locus model was used. Additional linkage studies are needed to map this second locus. These results demonstrate the importance of performing multilocus segregation and linkage analyses for quantitative traits that are related to the phenotype of a complex disorder. This approach has given further insight into the genetics of allergy and asthma by providing evidence for a two-locus model.