Hermann Lindemann

Reduction of the Total IgE Level by Omalizumab in Children and Adolescents

Background: Current data from clinical studies show that patients with severe allergic asthma experience a significant improvement from omalizumab. The early and late allergic reactions are inhibited by formation of complexes with free circulating immunoglobulin E (IgE), independent of which antigen activates the allergic cascade. The dosage of omalizumab depends on body weight and IgE level, yet no parameter has been established to guide dosage changes during therapy. The aim of this study was to investigate the value of the determination of total IgE by ADVIA Centaur assay to monitor the therapy progress. Patients and Methods: Nine patients, 8 to 17 years of age, received therapy with omalizumab due to severe allergic bronchial asthma. In addition, the patients had pronounced rhinoconjunctivitis, food allergy, insect sting allergy, and/or neurodermitis. The total IgE in the serum (Sandwich-Immunoassay ADVIA Centaur) was measured in the patients once monthly before each omalizumab injection as a potential progress parameter. Results: Six months after the beginning of therapy with omalizumab, a significant decrease of the total IgE concentration was found, in comparison to the baseline values (p < 0.01). In all patients, the tolerability of omalizumab was very good; there was a reduction in the frequency of the asthma exacerbations and rescue medications. The dosage of inhaled glucocorticoids could be lowered. All patients reported a clearly improved quality of life. Conclusions: The increase of the total IgE concentrations after administration of omalizumab described in the literature could not be confirmed. The value of total serum IgE as a progress parameter should be investigated in controlled studies with regard to sensitivity and specificity of the respective assays. The establishment of a test procedure for therapeutic monitoring appears urgently necessary, so that the appropriate dosage of omalizumab is applied in children and adolescents. Patients receiving omalizumab therapy should be closely monitored.

Nitric oxide has no beneficial effects on ion transport defects in cystic fibrosis human nasal epithelium

Abstract. Nitric oxide (NO) has been reported to activate Cl– secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) and inhibit epithelial Na+ absorption mediated by amiloride-sensitive epithelial Na+ channels (ENaC). These ion transport systems are defective in cystic fibrosis (CF): Cl– secretion by CFTR is impaired and Na+ absorption by ENaC is dramatically increased. By activating CFTR and depressing ENaC, NO is a potentially beneficial therapeutic agent for ion transport defects in human CF respiratory epithelia. To assess the effects of NO on human respiratory epithelial cells, the NO donors sodium nitroprusside (SNP) and spermine NONOate were applied to primary cultured nasal cells, surgically obtained from non-CF and CF patients. Measurements of transepithelial short-circuit current (ISC) showed that NO has no inhibitory potency against amiloride-sensitive nasal ENaC (nENaC) or amiloride-insensitive Na+-absorbing mechanisms in non-CF and CF epithelia. Furthermore, NO had no stimulatory effect on Cl– secretion by CFTR or any other Cl– conductance pathway in either tissue. Although NO elevated the intracellular Ca2+ concentration, we did not detect any activation of Ca2+-dependent Cl– channels. These results demonstrate that NO has no beneficial effect on CF epithelial cells of the upper airways.

Minor role of Cl- secretion in non-cystic fibrosis and cystic fibrosis human nasal epithelium.

Na⁺ and Cl^– currents were studied in primary cultures of human nasal epithelium derived from non-cystic fibrosis (non-CF) and cystic fibrosis (CF) patients. We found that Na⁺ absorption dominates transepithelial transport and the Na⁺ current contains an amiloride-sensitive and amiloride-insensitive component. In non-CF tissue both components contribute about equally to the entire short-circuit current (I_SC), whereas in CF tissues the major part of the current is amiloride-sensitive. Na⁺ removal reduced I_SC to values close to zero. Several Cl^– channel blockers were used to identify the remaining tiny Na⁺-independent current. Under unstimulated, physiological conditions in the presence of Cl^– on both sides and amiloride on the apical side of the epithelium diphenylamine-2-carboxic acid (DPC), 4,4′-diisothiocyanatostilbene-2,2′- disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) failed to induce clearcut inhibition of I_SC. cAMP as well as ATP did not affect I_SC either in CF or in non-CF epithelia. Reduction of apical Cl^– increased I_SC and depolarized transepithelial potential; however, the observed increase was insensitive to DIDS, DPC and NPPB. From these data we conclude that Cl^– conductances in primary cultures of human nasal epithelium derived from CF patients as well as from non-CF patients are present only in low numbers or do not contribute significantly to transepithelial ion transport.

CYSTIC FIBROSIS AND NON-CYSTIC-FIBROSIS HUMAN NASAL EPITHELIUM SHOW ANALOGOUS NA+ ABSORPTION AND REVERSIBLE BLOCK BY PHENAMIL

Abstract Transepithelial short-circuit current (ISC), potential (VT) and resistance (RT) of confluent monolayers of human nasal epithelium cultured from patients with and without cystic fibrosis (CF) were measured. In our Ussing chamber experiments with monolayers derived from non-CF and CF patients neither ISC (non-CF: 14.1 ± 1.0 μA/cm2, n = 77; CF: 16.7 ± 1.5 μA/cm2, n = 42), nor RT (non-CF: 288 ± 15 Ω· cm2; CF: 325 ± 20 Ω· cm2) showed any significant differences, only VT showed moderate but significant different values (non-CF: –3.6 ± 0.4 mV; CF: –5.6 ± 0.7 mV, respectively). Total ISC in CF cells was nearly completely inhibited by amiloride (92 ± 9.6%), while in non-CF tissue amiloride-insensitive conductances mediated a considerable amount of the ISC (36.3 ± 6.1%), indicating a lower activity of amiloride-sensitive Na+ conductances in non-CF cells. In both tissues the amiloride-sensitive ISC could also be blocked by the amiloride analogues benzamil, phenamil and 5-(N-ethyl-N-isopropyl)2’,4’-amiloride (EIPA) with different affinities. However, amiloride had a significant lower affinity in CF tissue (half-maximal blocker concentration, K1/2 = 586 ± 59 nM) compared with non-CF tissue (K1/2 = 294 ± 22 nM). Astonishingly, phenamil, a blocker which irreversibly blocks all epithelial Na+ channels hitherto described, inhibited the Na+ conductances of human nasal epithelium in a completely reversible way, but nevertheless with high affinity (non-CF: K1/2 = 12.5 ± 1.2 nM; CF: K1/2 = 17.1 ± 1.1 nM). Even in high doses none of these blockers had any effect on intracellular Ca2+ concentration as measured with Fura-2. From these findings, we conclude that the epithelial Na+ conductances of human CF nasal epithelium show modified regulation or are functionally different from those of other tissues.

Specific inhibition of epithelial Na + channels by antisense oligonucleotides for the treatment of Na + hyperabsorption in cystic fibrosis

Cystic fibrosis (CF) respiratory epithelia are characterized by a defect Cl− secretion and an increased Na+ absorption through epithelial Na+ channels (ENaC). The present study aimed to find an effective inhibitor of human ENaC with respect to replacing amiloride therapy for CF patients. Therefore, we developed specific antisense oligonucleotides (AON) that efficiently suppress Na+ hyperabsorption by inhibiting the expression of the α‐ENaC subunit.

Capnovolumetry: a new tool for lung function testing in children with asthma.

In capnovolumetry, the expiratory CO2 concentration of exhaled air is plotted against the volume and thereby allows to determine functional dead space volumes. This method might offer additional information in lung function testing in children and adolescents with bronchial asthma. We aimed at determining whether a bronchospasmolysis (BSL) effect in the lower airways could also be detected by capnovolumetry as reflected by changes in the functional threshold dead space volumes (VDT). In 47 patients (aged 4–16 years) with a mild persistent bronchial asthma, VDT were determined before and after bronchodilation prior to starting therapy with inhaled steroids and after 6 months of treatment. Additionally, spirometry and body plethysmography were performed in all patients. There were significantly higher VDT values after BSL before and after 6 months of therapy (P<0·0001). VDT values before BSL were tendatively higher after 6 months of therapy compared with baseline values (P = 0·07). VDT values correlated with parameters derived from conventional pulmonary function testing, i.e. vital capacity, forced expiratory volume in 1 s (FEV1), and maximum expiratory flow (MEF50). As VDT values particularly reflect the volumes of the lower bronchi this method may provide supplementary information to conventional lung function tests which are based on breathing mechanics. This seems to be especially helpful in situations where body plethysmography is not available or cooperation in forced expiration manoeuvres is insufficient.

Ionentransporte über die Nasen- und Nasennebenhöhlenschleimhaut bei Mukoviszidose und chronischer Sinusitis

ZusammenfassungMukoviszidose [zystische Fibrose (CF)] ist die häufigste Erbkrankheit in der kaukasischen Bevölkerung. Sie wird hervorgerufen durch Mutation eines Gens, welches für ein membranständiges Transportprotein kodiert, den „cystic fibrosis transmembrane conductance regulator” (CFTR). Dieser CFTR ist in der apikalen Membran von Epithelzellen lokalisiert und fungiert dort als cAMP-aktivierbarer Cl–-Kanal. Neuere Untersuchungen haben ergeben, daß der CFTR mehr als ein normaler Cl–-Kanal ist und wahrscheinlich in die Regulation einer Reihe weiterer Transportsysteme involviert ist. In dieser Studie zeigen wir, daß der CFTR in seiner Rolle als Cl–-Kanal nur eine untergeordnete Rolle in primärkultivierten menschlichen Nasenepithelzellen von CF- und Nicht-CF-Patienten spielt. Dieses Ergebnis ist besonders für das Nasenepithel von Nicht-CF-Patienten erstaunlich, da hier der CFTR korrekt eingebaut wird. In beiden Geweben spielt also eine Cl–-Sekretion verglichen mit der Na+-Absorption eine vergleichsweise geringe Rolle. Wir bestätigen mit dieser Untersuchung unsere früheren Beobachtungen, daß im menschlichen Nasenepithel Na+-Absorption der dominierende Ionentransportprozeß ist und eine Cl–-Sekretion weder in CF- noch in Nicht- CF-Geweben in relevantem Maße vorhanden ist. Zudem zeigen wir, daß weder cAMP noch ATP irgendeine Cl–-Sekretion in CF- oder Nicht-CF-Nasenepithelzellen stimulieren können. Desweiteren ergaben sich Hinweise darauf, daß sich die epithelialen Na+-Kanäle (ENaC) im Nasenepithel, welche für einen Teil der Na+-Absorption zuständig sind, von den epithelialen Na+-Kanälen in anderen Geweben unterscheiden. Diese Unterschiede zwischen den Na+-Kanälen im menschlichen Nasenepithel und den „klassischen” epithelialen Na+-Kanälen bestehen einerseits in ihrer fehlenden Aktivierbarkeit durch den intrazellulären Botenstoff cAMP und das Steroidhormon Aldosteron. Wir zeigen weiterhin, daß menschliche nasale Na+-Kanäle durch Cl–-Kanal-Blocker gehemmt werden können und eine andere Pharmakologie auf gebräuchliche Na+-Kanal-Blocker aufweisen.SummaryCystic fibrosis (CF) is the most commonly inherited disease in Caucasians and is caused by a mutation in the gene encoding a membrane transport protein. This cystic fibrosis transmembrane conductance regulator (CFTR) is thought to be an apical Cl– channel activated by intracellular cAMP. Most recent findings suggest that CFTR is more than a pure Cl– channel and might be involved in the regulation of other transport systems. In the present study we show that CFTR as a Cl– channel plays only a minor role in primary cultured human nasal epithelium derived from non-CF and CF patients. These findings are especially of interest for non-CF human nasal epithelia in which CFTR is correctly inserted. In both tissues Cl– secretion is negligible as compared with Na+ absorption. We confirm and expand our previous observations that Na+ absorption in human nasal epithelium is the dominant ion transport process and that Cl– secretion is detectable in both CF and non-CF tissue. Moreover, we show that cAMP and ATP were not able to stimulate any silent Cl– channels in CF or non-CF human nasal epithelial cells. We further give evidence that in human nasal CF and non-CF epithelium Na+ absorption is mediated by epithelial Na+ channels (ENaC) that are either different from those of other epithelia or which exhibit altered regulation. These differences between Na+ channels of human nasal epithelium and ”classical” epithelial Na+ channels include lack of activation by the intracellular second messenger cAMP and the steroid hormone aldosterone. We show further that human nasal Na+ channels are inhibited by Cl–-channel blockers and exhibit a different pharmacology towards common Na+ channel blockers.

Antisense oligonucleotides for therapy of cystic fibrosis. Inhibition of sodium absorption mediated by ENaC in nasal epithelial cells

BACKGROUND The genetic disease cystic fibrosis (CF) is characterised by reduced chloride secretion mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) and Na(+) hyperabsorption through amiloride-sensitive epithelial sodium channels (ENaC). Mutations in CFTR cause the accumulation of thick mucus and dysfunction of mucociliary clearance in the respiratory tract. MATERIAL AND METHODS In this project it was investigated whether Na(+) hyperabsorption is inhibited by the use of antisense oligonucleotides (AON). For functional analyses monolayers of human non-CF and CF nasal epithelial cells were measured in modified Ussing chambers. To analyse the AON effects on the protein level Western blotting analyses were carried out. RESULTS AON transfection significantly inhibits Na(+) absorption via ENaC in non-CF and CF cells. Furthermore, Western blot analyses demonstrate a suppression of the ENaC protein in AON transfected human non-CF cells. CONCLUSION The inhibition of ENaC associated Na(+) absorption by specific AON could offer a new perspective for the regulation of the Na(+) hyperabsorption in CF patients.ZusammenfassungHintergrundDer Erbkrankheit Mukoviszidose („cystic fibrosis“, CF) liegt neben einer reduzierten Chloridsekretion über den „cystic fibrosis transmembrane conductance regulator“ (CFTR) auch eine Na+-Hyperabsorption über den amiloridsensitiven epithelialen Na+-Kanal (ENaC) zugrunde. Mutationen des CFTR sind ursächlich für die Bildung eines zähflüssigen Mukus und eine gestörte mukoziliäre Reinigung im respiratorischen Trakt.Material und MethodenInnerhalb dieses Projekts sollte die Inhibierung der Na+-Hyperabsorption durch Antisense-Oligonukleotide (AON) erreicht werden. Für funktionale Untersuchungen wurden Monolayer humaner Nicht-CF- und CF-Nasenepithelzellen mittels Ussing-Kammer-Messungen analysiert. Zur Charakterisierung der AON-Wirkung auf Proteinebene wurden Western-Blot-Analysen durchgeführt.ErgebnisseDie AON-Transfektion verringerte die Na+-Absorption über den ENaC in Nicht-CF- und in CF-Zellen deutlich. Außerdem zeigten Western-Blot-Analysen eine Reduzierung des ENaC-Proteins in den mit AON-transfizierten Nicht-CF-Zellen.SchlussfolgerungDie Reduktion der ENaC-vermittelten Na+-Absorption mit spezifischen AON eröffnet eine neue Perspektive, die krankhafte Na+-Hyperabsorption bei CF-Patienten gezielt zu regulieren.AbstractBackgroundThe genetic disease cystic fibrosis (CF) is characterised by reduced chloride secretion mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) and Na+ hyperabsorption through amiloride-sensitive epithelial sodium channels (ENaC). Mutations in CFTR cause the accumulation of thick mucus and dysfunction of mucociliary clearance in the respiratory tract.Material and MethodsIn this project it was investigated whether Na+ hyperabsorption is inhibited by the use of antisense oligonucleotides (AON). For functional analyses monolayers of human non-CF and CF nasal epithelial cells were measured in modified Ussing chambers. To analyse the AON effects on the protein level Western blotting analyses were carried out.ResultsAON transfection significantly inhibits Na+ absorption via ENaC in non-CF and CF cells. Furthermore, Western blot analyses demonstrate a suppression of the ENaC protein in AON transfected human non-CF cells.ConclusionThe inhibition of ENaC associated Na+ absorption by specific AON could offer a new perspective for the regulation of the Na+ hyperabsorption in CF patients.

Reduction of the total immunoglobulin E level by omalizumab in children and adolescents.

We would like to comment on the recently published Letter to the Editor by R. Slavin and P. Jimenez in the Journal of Asthma (1, 2). Omalizumab is a successfully implemented supplementary therapy f...

Antisense-Oligonukleotide zur Therapie der Mukoviszidose

BACKGROUND The genetic disease cystic fibrosis (CF) is characterised by reduced chloride secretion mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) and Na(+) hyperabsorption through amiloride-sensitive epithelial sodium channels (ENaC). Mutations in CFTR cause the accumulation of thick mucus and dysfunction of mucociliary clearance in the respiratory tract. MATERIAL AND METHODS In this project it was investigated whether Na(+) hyperabsorption is inhibited by the use of antisense oligonucleotides (AON). For functional analyses monolayers of human non-CF and CF nasal epithelial cells were measured in modified Ussing chambers. To analyse the AON effects on the protein level Western blotting analyses were carried out. RESULTS AON transfection significantly inhibits Na(+) absorption via ENaC in non-CF and CF cells. Furthermore, Western blot analyses demonstrate a suppression of the ENaC protein in AON transfected human non-CF cells. CONCLUSION The inhibition of ENaC associated Na(+) absorption by specific AON could offer a new perspective for the regulation of the Na(+) hyperabsorption in CF patients.ZusammenfassungHintergrundDer Erbkrankheit Mukoviszidose („cystic fibrosis“, CF) liegt neben einer reduzierten Chloridsekretion über den „cystic fibrosis transmembrane conductance regulator“ (CFTR) auch eine Na+-Hyperabsorption über den amiloridsensitiven epithelialen Na+-Kanal (ENaC) zugrunde. Mutationen des CFTR sind ursächlich für die Bildung eines zähflüssigen Mukus und eine gestörte mukoziliäre Reinigung im respiratorischen Trakt.Material und MethodenInnerhalb dieses Projekts sollte die Inhibierung der Na+-Hyperabsorption durch Antisense-Oligonukleotide (AON) erreicht werden. Für funktionale Untersuchungen wurden Monolayer humaner Nicht-CF- und CF-Nasenepithelzellen mittels Ussing-Kammer-Messungen analysiert. Zur Charakterisierung der AON-Wirkung auf Proteinebene wurden Western-Blot-Analysen durchgeführt.ErgebnisseDie AON-Transfektion verringerte die Na+-Absorption über den ENaC in Nicht-CF- und in CF-Zellen deutlich. Außerdem zeigten Western-Blot-Analysen eine Reduzierung des ENaC-Proteins in den mit AON-transfizierten Nicht-CF-Zellen.SchlussfolgerungDie Reduktion der ENaC-vermittelten Na+-Absorption mit spezifischen AON eröffnet eine neue Perspektive, die krankhafte Na+-Hyperabsorption bei CF-Patienten gezielt zu regulieren.AbstractBackgroundThe genetic disease cystic fibrosis (CF) is characterised by reduced chloride secretion mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) and Na+ hyperabsorption through amiloride-sensitive epithelial sodium channels (ENaC). Mutations in CFTR cause the accumulation of thick mucus and dysfunction of mucociliary clearance in the respiratory tract.Material and MethodsIn this project it was investigated whether Na+ hyperabsorption is inhibited by the use of antisense oligonucleotides (AON). For functional analyses monolayers of human non-CF and CF nasal epithelial cells were measured in modified Ussing chambers. To analyse the AON effects on the protein level Western blotting analyses were carried out.ResultsAON transfection significantly inhibits Na+ absorption via ENaC in non-CF and CF cells. Furthermore, Western blot analyses demonstrate a suppression of the ENaC protein in AON transfected human non-CF cells.ConclusionThe inhibition of ENaC associated Na+ absorption by specific AON could offer a new perspective for the regulation of the Na+ hyperabsorption in CF patients.