Amitriptyline inhibits bronchoconstriction and directly promotes dilatation of the airways.

INTRODUCTION: The standard therapy for bronchial asthma consists of combinations of acute (short-acting ß2-sympathomimetics) and, depending on the severity of disease, additional long-term treatment (including inhaled glucocorticoids, long-acting ß2-sympathomimetics, anticholinergics, anti-IL-4R antibodies). The antidepressant amitriptyline has been identified as a relevant down-regulator of immunological TH2-phenotype in asthma, acting-at least partially-through inhibition of acid sphingomyelinase (ASM), an enzyme involved in sphingolipid metabolism. Here, we investigated the non-immunological role of amitriptyline on acute bronchoconstriction, a main feature of airway hyperresponsiveness in asthmatic disease. METHODS: After stimulation of precision cut lung slices (PCLS) from mice (wildtype and ASM-knockout), rats, guinea pigs and human lungs with mediators of bronchoconstriction (endogenous and exogenous acetylcholine, methacholine, serotonin, endothelin, histamine, thromboxane-receptor agonist U46619 and leukotriene LTD4, airway area was monitored in the absence of or with rising concentrations of amitriptyline. Airway dilatation was also investigated in rat PCLS by prior contraction induced by methacholine. As bronchodilators for maximal relaxation, we used IBMX (PDE inhibitor) and salbutamol (ß2-adrenergic agonist) and compared these effects with the impact of amitriptyline treatment. Isolated perfused lungs (IPL) of wildtype mice were treated with amitriptyline, administered via the vascular system (perfusate) or intratracheally as an inhalation. To this end, amitriptyline was nebulized via pariboy in-vivo and mice were ventilated with the flexiVent setup immediately after inhalation of amitriptyline with monitoring of lung function. RESULTS: Our results show amitriptyline to be a potential inhibitor of bronchoconstriction, induced by exogenous or endogenous (EFS) acetylcholine, serotonin and histamine, in PCLS from various species. The effects of endothelin, thromboxane and leukotrienes could not be blocked. In acute bronchoconstriction, amitriptyline seems to act ASM-independent, because ASM-deficiency (Smdp1-/-) did not change the effect of acetylcholine on airway contraction. Systemic as well as inhaled amitriptyline ameliorated the resistance of IPL after acetylcholine provocation. With the flexiVent setup, we demonstrated that the acetylcholine-induced rise in central and tissue resistance was much more marked in untreated animals than in amitriptyline-treated ones. Additionally, we provide clear evidence that amitriptyline dilatates pre-contracted airways as effectively as a combination of typical bronchodilators such as IBMX and salbutamol. CONCLUSION: Amitriptyline is a drug of high potential, which inhibits acute bronchoconstriction and induces bronchodilatation in pre-contracted airways. It could be one of the first therapeutic agents in asthmatic disease to have powerful effects on the TH2-allergic phenotype and on acute airway hyperresponsiveness with bronchoconstriction, especially when inhaled.

Corrigendum: Evaluation of lung function in a German single center cohort of young patients with sickle cell disease using EIT and standard techniques.

[This corrects the article DOI: 10.3389/fmed.2023.1100180.].

Evaluation of lung function in a German single center cohort of young patients with sickle cell disease using EIT and standard techniques.

Background and objective: Sickle cell disease (SCD) is a very common autosomal recessive hemoglobinopathy leading to multiple pulmonary complications that are closely associated with mortality. The pathophysiology of chronic pulmonary involvement is not yet fully understood and no specific therapies are available. Methods: The aim of this cross-sectional study was to characterize the lung function of children and young adolescents with SCD in a German single-center cohort and to extend conventional lung function testing by the use of a new imaging method. We performed spirometry and body plethysmography in 35 children and young adults with hemoglobin SS, SC, S/ß-thalassemia as well as 50 controls. These data were compared with clinical characteristics and typical laboratory parameters of hemolysis and disease activity in SCD. To identify lung inhomogeneities, for example due to atelectasis, hyperinflation, air trapping or vascular occlusions, we used the promising new method of electrical impedance tomography (EIT) and calculated global inhomogeneity indices. Results: Lung function of patients with SCD was significantly reduced compared to that of healthy controls. When the result was found to be pathological, the most commonly observed type of breathing disorder was classified as restrictive. Laboratory parameters showed typical features of SCD including decreased levels of hemoglobin and hematocrit and elevated levels of leucocytes, platelets, lactate dehydrogenase and total bilirubin. However, there was no correlation between blood values and reduced lung function. Electrical impedance tomography (EIT) revealed no abnormalities in SCD patients compared to healthy controls. In particular, we were unable to demonstrate any regional inhomogeneities in lung ventilation. Conclusion: In our study, SCD patients showed impaired lung function, with a relevant percentage of patients suffering from restrictive breathing disorder. Signs of obstruction could not be detected. Electrical impedance tomography (EIT) measurements revealed no unevenness that would suggest air entrapment, blockage of blood vessels, excessive inflation, obstruction, or other forms of lung disease. Additionally, the reduction in lung function observed in SCD patients was not related to the disease severity or laboratory test results.

Comparing SARS-CoV-2 variants among children and adolescents in Germany: relative risk of COVID-19-related hospitalization, ICU admission and mortality.

PURPOSE: SARS-CoV-2 infections cause COVID-19 and have a wide spectrum of morbidity. Severe disease courses among children are rare. To date, data on the variability of morbidity in relation to variant of concern (VOC) in children has been sparse and inconclusive. We compare the clinical severity of SARS-CoV-2 infection among children and adolescents in Germany during the Wildtype and Alpha combined, Delta and Omicron phases of the COVID-19 pandemic. METHODS: Comparing risk of COVID-19-related hospitalization, intensive care unit (ICU) admission and death due to COVID-19 in children and adolescents, we used: (1) a multi-center seroprevalence study (SARS-CoV-2-KIDS study); (2) a nationwide registry of pediatric patients hospitalized with SARS-CoV-2 infections; and (3) compulsory national reporting for RT-PCR-confirmed SARS-CoV-2 infections in Germany. RESULTS: During the Delta predominant phase, risk of COVID-19-related hospitalization among all SARS-CoV-2 seropositive children was 3.35, ICU admission 1.19 and fatality 0.09 per 10,000; hence about halved for hospitalization and ICU admission and unchanged for deaths as compared to the Wildtype- and Alpha-dominant period. The relative risk for COVID-19-related hospitalization and ICU admission compared to the alpha period decreased during Delta [0.60 (95% CI 0.54; 0.67) and 0.51 (95% CI 0.42; 0.61)] and Omicron [0.27 (95% CI 0.24; 0.30) and 0.06 (95% CI 0.05; 0.08)] period except for the < 5-year-olds. The rate of case fatalities decreased slightly during Delta, and substantially during Omicron phase. CONCLUSION: Morbidity caused by SARS-CoV-2 infections among children and adolescents in Germany decreased over the course of the COVID-19 pandemic, as different VOCs) emerged.

Association of SARS-CoV-2 Seropositivity With Myalgic Encephalomyelitis and/or Chronic Fatigue Syndrome Among Children and Adolescents in Germany.

Importance: During the COVID-19 pandemic, a reduction in quality of life and physical and mental health among children and adolescents has been reported that may be associated with SARS-CoV-2 infection and/or containment measures. Objective: To assess the association of SARS-CoV-2 seropositivity with symptoms that may be related to myalgic encephalomyelitis and/or chronic fatigue syndrome (ME/CFS) among children and adolescents. Design, Setting, and Participants: This substudy of the cross-sectional SARS-CoV-2 seroprevalence surveys in Germany (SARS-CoV-2 KIDS) was performed in 9 pediatric hospitals from May 1 to October 31, 2021. Pediatric patients were recruited during an inpatient or outpatient visit regardless of the purpose of the visit. Parental questionnaires and serum samples were collected during clinically indicated blood draws. The parental questionnaire on demographic and clinical information was extended by items according to the DePaul Symptom Questionnaire, a pediatric screening tool for ME/CFS in epidemiological studies in patients aged 5 to 17 years. Exposures: Seropositivity was determined by SARS-CoV-2 IgG antibodies in serum samples using enzyme-linked immunosorbent assays. Main Outcomes and Measures: Key symptoms of ME/CFS were evaluated separately or as clustered ME/CFS symptoms according to the DePaul Symptom Questionnaire, including fatigue. Results: Among 634 participants (294 male [46.4%] and 340 female [53.6%]; median age, 11.5 [IQR, 8-14] years), 198 (31.2%) reported clustered ME/CFS symptoms, including 40 of 100 SARS-CoV-2-seropositive (40.0%) and 158 of 534 SARS-CoV-2-seronegative (29.6%) children and adolescents. After adjustment for sex, age group, and preexisting disease, the risk ratio for reporting clustered ME/CFS symptoms decreased from 1.35 (95% CI, 1.03-1.78) to 1.18 (95% CI, 0.90-1.53) and for substantial fatigue from 2.45 (95% CI, 1.24-4.84) to 2.08 (95% CI, 1.05-4.13). Confinement to children and adolescents with unknown previous SARS-CoV-2 infection status (n = 610) yielded lower adjusted risks for all symptoms except joint pain ME/CFS-related symptoms. The adjusted risk ratio was 1.08 (95% CI, 0.80-1.46) for reporting clustered ME/CFS symptoms and 1.43 (95% CI, 0.63-3.23) for fatigue. Conclusions and Relevance: These findings suggest that the risk of ME/CFS in children and adolescents owing to SARS-CoV-2 infection may be very small. Recall bias may contribute to risk estimates of long COVID-19 symptoms in children. Extensive lockdowns must be considered as an alternative explanation for complex unspecific symptoms during the COVID-19 pandemic.

Platelet-derived growth factor (PDGF)-BB regulates the airway tone via activation of MAP2K, thromboxane, actin polymerisation and Ca2+-sensitisation.

BACKGROUND: PDGFR-inhibition by the tyrosine kinase inhibitor (TKI) nintedanib attenuates the progress of idiopathic pulmonary fibrosis (IPF). However, the effects of PDGF-BB on the airway tone are almost unknown. We studied this issue and the mechanisms beyond, using isolated perfused lungs (IPL) of guinea pigs (GPs) and precision-cut lung slices (PCLS) of GPs and humans. METHODS: IPL: PDGF-BB was perfused after or without pre-treatment with the TKI imatinib (perfused/nebulised) and its effects on the tidal volume (TV), the dynamic compliance (Cdyn) and the resistance were studied. PCLS (GP): The bronchoconstrictive effects of PDGF-BB and the mechanisms beyond were evaluated. PCLS (human): The bronchoconstrictive effects of PDGF-BB and the bronchorelaxant effects of imatinib were studied. All changes of the airway tone were measured by videomicroscopy and indicated as changes of the initial airway area. RESULTS: PCLS (GP/human): PDGF-BB lead to a contraction of airways. IPL: PDGF-BB decreased TV and Cdyn, whereas the resistance did not increase significantly. In both models, inhibition of PDGFR-(ß) (imatinib/SU6668) prevented the bronchoconstrictive effect of PDGF-BB. The mechanisms beyond PDGF-BB-induced bronchoconstriction include activation of MAP2K and TP-receptors, actin polymerisation and Ca2+-sensitisation, whereas the increase of Ca2+ itself and the activation of EP1-4-receptors were not of relevance. In addition, imatinib relaxed pre-constricted human airways. CONCLUSIONS: PDGFR regulates the airway tone. In PCLS from GPs, this regulatory mechanism depends on the ß-subunit. Hence, PDGFR-inhibition may not only represent a target to improve chronic airway disease such as IPF, but may also provide acute bronchodilation in asthma. Since asthma therapy uses topical application. This is even more relevant, as nebulisation of imatinib also appears to be effective.

Cross-sectional seroprevalence surveys of SARS-CoV-2 antibodies in children in Germany, June 2020 to May 2021.

The rate of SARS-CoV-2 infections in children remains unclear due to many asymptomatic cases. We present a study of cross-sectional seroprevalence surveys of anti-SARS-CoV-2 IgG in 10,358 children recruited in paediatric hospitals across Germany from June 2020 to May 2021. Seropositivity increased from 2.0% (95% CI 1.6, 2.5) to 10.8% (95% CI 8.7, 12.9) in March 2021 with little change up to May 2021. Rates increased by migrant background (2.8%, 4.4% and 7.8% for no, one and two parents born outside Germany). Children under three were initially 3.6 (95% CI 2.3, 5.7) times more likely to be seropositive with levels equalising later. The ratio of seropositive cases per recalled infection decreased from 8.6 to 2.8. Since seropositivity exceeds the rate of recalled infections considerably, serologic testing may provide a more valid estimate of infections, which is required to assess both the spread and the risk for severe outcomes of SARS-CoV-2 infections.

Levosimendan reduces segmental pulmonary vascular resistance in isolated perfused rat lungs and relaxes human pulmonary vessels.

INTRODUCTION: Levosimendan is approved for acute heart failure. Within this context, pulmonary hypertension represents a frequent co-morbidity. Hence, the effects of levosimendan on segmental pulmonary vascular resistance (PVR) are relevant. So far, this issue has been not studied. Beyond that the relaxant effects of levosimendan in human pulmonary vessel are unknown. We addressed these topics in rats' isolated perfused lungs (IPL) and human precision-cut lung slices (PCLS). MATERIAL AND METHODS: In IPL, levosimendan (10 µM) was perfused in untreated and endothelin-1 pre-contracted lungs. The pulmonary arterial pressure (PPA) was continuously recorded and the capillary pressure (Pcap) was determined by the double-occlusion method. Thereafter, segmental PVR, expressed as precapillary (Rpre) and postcapillary resistance (Rpost) and PVR were calculated. Human PCLS were prepared from patients undergoing lobectomy. Levosimendan-induced relaxation was studied in naïve and endothelin-1 pre-contracted PAs and PVs. In endothelin-1 pre-contracted PAs, the role of K+-channels was studied by inhibition of KATP-channels (glibenclamide), BKCa2+-channels (iberiotoxin) and Kv-channels (4-aminopyridine). All changes of the vascular tone were measured by videomicroscopy. In addition, the increase of cAMP/GMP due to levosimendan was measured by ELISA. RESULTS: Levosimendan did not relax untreated lungs or naïve PAs and PVs. In IPL, levosimendan attenuated the endothelin-1 induced increase of PPA, PVR, Rpre and Rpost. In human PCLS, levosimendan relaxed pre-contracted PAs or PVs to 137% or 127%, respectively. In pre-contracted PAs, the relaxant effect of levosimendan was reduced, if KATP- and Kv-channels were inhibited. Further, levosimendan increased cGMP in PAs/PVs, but cAMP only in PVs. DISCUSSION: Levosimendan reduces rats' segmental PVR and relaxes human PAs or PVs, if the pulmonary vascular tone is enhanced by endothelin-1. Regarding levosimendan-induced relaxation, the activation of KATP- and Kv-channels is of impact, as well as the formation of cAMP and cGMP. In conclusion, our results suggest that levosimendan improves pulmonary haemodynamics, if PVR is increased as it is the case in pulmonary hypertension.

Acid sphingomyelinase regulates TH 2 cytokine release and bronchial asthma.

BACKGROUND: Allergic diseases and especially allergic asthma are widespread diseases with high prevalence in childhood, but also in adults. Acid sphingomyelinase (ASM) is a key regulator of the sphingolipid pathway. Previous studies defined the association of ASM with the pathogenesis of TH 1-directed lung diseases like cystic fibrosis and acute lung injury. Here, we define the role of ASM in TH 2-regulated allergic bronchial asthma. METHODS: To determine the role of Asm under baseline conditions, wild-type (WT) and Asm-/- mice were ventilated with a flexiVent setup and bronchial hyperresponsiveness was determined using acetylcholine. Flow cytometry and cytokine measurements in bronchoalveolar lavage fluid and lung tissue were followed by in vitro TH 2 differentiations with cells from WT and Asm-/- mice and blockade of Asm with amitriptyline. As proof of principle, we conducted an ovalbumin-induced model of asthma in WT- and Asm-/-  mice. RESULTS: At baseline, Asm-/- mice showed better lung mechanics, but unaltered bronchial hyperresponsiveness. Higher numbers of Asm-/- T cells in bronchoalveolar lavage fluid released lower levels of IL-4 and IL-5, and these results were paralleled by decreased production of typical TH 2 cytokines in Asm-/- T lymphocytes in vitro. This phenotype could be imitated by incubation of T cells with amitriptyline. In the ovalbumin asthma model, Asm-/- animals were protected from high disease activity and showed better lung functions and lower levels of eosinophils and TH 2 cytokines. CONCLUSION: Asm deficiency could induce higher numbers of TH 2 cells in the lung, but those cells release decreased TH 2 cytokine levels. Hereby, Asm-/- animals are protected from bronchial asthma, which possibly offers novel therapeutic strategies, for example, with ASM blockade.

Initiation of LPS-induced pulmonary dysfunction and its recovery occur independent of T cells.

BACKGROUND: The acute respiratory distress syndrome (ARDS) is a serious disease in critically ill patients that is characterized by pulmonary dysfunctions, hypoxemia and significant mortality. Patients with immunodeficiency (e.g. SCID with T and B cell deficiency) are particularly susceptible to the development of severe ARDS. However, the role of T cells on pulmonary dysfunctions in immune-competent patients with ARDS is only incompletely understood. METHODS: Wild-type (wt) and RAG2-/- mice (lymphocyte deficient) received intratracheal instillations of LPS (4 mg/kg) or saline. On day 1, 4 and 10 lung mechanics and bronchial hyperresponsiveness towards acetylcholine were measured with the flexiVent ventilation set-up. The bronchoalveolar lavage fluid (BALF) was examined for leukocytes (FACS analysis) and pro-inflammatory cytokines (ELISA). RESULTS: In wt mice, lung mechanics, body weight and body temperature deteriorated in the LPS-group during the early phase (up to d4); these alterations were accompanied by increased leukocyte numbers and inflammatory cytokine levels in the BALF. During the late phase (day 10), both lung mechanics and the cell/cytokine homeostasis recovered in LPS-treated wt mice. RAG2-/- mice experienced changes in body weight, lung mechanics, BAL neutrophil numbers, BAL inflammatory cytokines levels that were comparable to wt mice. CONCLUSION: Following LPS instillation, lung mechanics deteriorate within the first 4 days and recover towards day 10. This response is not altered by the lack of T lymphocytes suggesting that T cells play only a minor role for the initiation, propagation or recovery of LPS-induced lung dysfunctions or function of T lymphocytes can be compensated by other immune cells, such as alveolar macrophages.

Nrf2 Is a Central Regulator of Metabolic Reprogramming of Myeloid-Derived Suppressor Cells in Steady State and Sepsis.

Arising in inflammatory conditions, myeloid-derived suppressor cells (MDSCs) are constantly confronted with intracellular and extracellular reactive oxygen species molecules and oxidative stress. Generating mice with a constitutive activation of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) we show a pivotal role of the antioxidant stress defense for development of these immune-modulatory cells. These mice are characterized by a massive increase of splenic CD11b+Gr-1+ cells, which exhibit typical suppressive characteristics of MDSCs. Whole transcriptome analysis revealed Nrf2-dependent activation of cell cycle and metabolic pathways, which resemble pathways in CD11b+Gr-1+ MDSCs expanded by in vivo LPS exposure. Constitutive Nrf2 activation thereby regulates activation and balance between glycolysis and mitochondrial metabolism and hence expansion of highly suppressive MDSCs, which mediate protection in LPS-induced sepsis. Our study establishes Nrf2 as key regulator of MDSCs and acquired tolerance against LPS-induced sepsis.

PDGF-BB regulates the pulmonary vascular tone: impact of prostaglandins, calcium, MAPK- and PI3K/AKT/mTOR signalling and actin polymerisation in pulmonary veins of guinea pigs.

BACKGROUND: Platelet-derived growth factor (PDGF)-BB and its receptor PDGFR are highly expressed in pulmonary hypertension (PH) and mediate proliferation. Recently, we showed that PDGF-BB contracts pulmonary veins (PVs) and that this contraction is prevented by inhibition of PDGFR-ß (imatinib/SU6668). Here, we studied PDGF-BB-induced contraction and downstream-signalling in isolated perfused lungs (IPL) and precision-cut lung slices (PCLS) of guinea pigs (GPs). METHODS: In IPLs, PDGF-BB was perfused after or without pre-treatment with imatinib (perfused/nebulised), the effects on the pulmonary arterial pressure (PPA), the left atrial pressure (PLA) and the capillary pressure (Pcap) were studied and the precapillary (Rpre) and postcapillary resistance (Rpost) were calculated. Perfusate samples were analysed (ELISA) to detect the PDGF-BB-induced release of prostaglandin metabolites (TXA2/PGI2). In PCLS, the contractile effect of PDGF-BB was evaluated in pulmonary arteries (PAs) and PVs. In PVs, PDGF-BB-induced contraction was studied after inhibition of PDGFR-α/ß, L-Type Ca2+-channels, ROCK/PKC, prostaglandin receptors, MAP2K, p38-MAPK, PI3K-α/γ, AKT/PKB, actin polymerisation, adenyl cyclase and NO. Changes of the vascular tone were measured by videomicroscopy. In PVs, intracellular cAMP was measured by ELISA. RESULTS: In IPLs, PDGF-BB increased PPA, Pcap and Rpost. In contrast, PDGF-BB had no effect if lungs were pre-treated with imatinib (perfused/nebulised). In PCLS, PDGF-BB significantly contracted PVs/PAs which was blocked by the PDGFR-ß antagonist SU6668. In PVs, inhibition of actin polymerisation and inhibition of L-Type Ca2+-channels reduced PDGF-BB-induced contraction, whereas inhibition of ROCK/PKC had no effect. Blocking of EP1/3- and TP-receptors or inhibition of MAP2K-, p38-MAPK-, PI3K-α/γ- and AKT/PKB-signalling prevented PDGF-BB-induced contraction, whereas inhibition of EP4 only slightly reduced it. Accordingly, PDGF-BB increased TXA2 in the perfusate, whereas PGI2 was increased in all groups after 120 min and inhibition of IP-receptors did not enhance PDGF-BB-induced contraction. Moreover, PDGF-BB increased cAMP in PVs and inhibition of adenyl cyclase enhanced PDGF-BB-induced contraction, whereas inhibition of NO-formation only slightly increased it. CONCLUSIONS: PDGF-BB/PDGFR regulates the pulmonary vascular tone by the generation of prostaglandins, the increase of calcium, the activation of MAPK- or PI3K/AKT/mTOR signalling and actin remodelling. More insights in PDGF-BB downstream-signalling may contribute to develop new therapeutics for PH.

CD11c.DTR mice develop a fatal fulminant myocarditis after local or systemic treatment with diphtheria toxin.

To assess the role of alveolar macrophages (AMs) during a pulmonary Aspergillus fumigatus infection AMs were depleted by intratracheal application of diphtheria toxin (DTX) to transgenic CD11c.DTR mice prior to fungal infection. Unexpectedly, all CD11c.DTR mice treated with DTX died within 4-5 days, whether being infected with A. fumigatus or not. Despite measurable impact of DTX on lung functional parameters, these constrictions could not explain the high mortality rate. Instead, DTX-treated CD11c.DTR animals developed fulminant myocarditis (FM) characterized by massive leukocyte infiltration and myocardial cell destruction, including central parts of the heart's stimulus transmission system. In fact, standard limb lead ECG recordings of diseased but not healthy mice showed a "Brugada"-like pattern with an abnormally high ST segment pointing to enhanced susceptibility for potential lethal arrhythmias. While CD11c.DTR mice are extensively used for the characterization of CD11c(+) cells, including dendritic cells, several studies have already mentioned adverse side effects following DTX treatment. Our results demonstrate that this limitation is based on severe myocarditis but not on the expected lung constrictions, and has to be taken into consideration if this animal model is used. Based on these properties, however, the CD11c.DTR mouse might serve as useful animal model for FM.

The cAMP response element modulator (CREM) regulates TH2 mediated inflammation.

A characteristic feature of allergic diseases is the appearance of a subset of CD4+ cells known as TH2 cells, which is controlled by transcriptional and epigenetic mechanisms. We aimed to analyze the role of CREM, a known transcriptional activator of T cells, with regard to TH2 responses and allergic diseases in men and mice. Here we demonstrate that T cells of asthmatic children and PBMCs of adults with atopy express lower mRNA levels of the transcription factor CREM compared to cells from healthy controls. CREM deficiency in murine T cells results in enhanced TH2 effector cytokines in vitro and in vivo and CREM-/- mice demonstrate stronger airway hyperresponsiveness in an OVA-induced asthma model. Mechanistically, both direct CREM binding to the IL-4 and IL-13 promoter as well as a decreased IL-2 dependent STAT5 activation suppress the TH2 response. Accordingly, mice selectively overexpressing CREMα in T cells display decreased TH2 type cytokines in vivo and in vitro, and are protected in an asthma model. Thus, we provide evidence that CREM is a negative regulator of the TH2 response and determines the outcome of allergic asthma.

Milrinone relaxes pulmonary veins in guinea pigs and humans.

INTRODUCTION: The phosphodiesterase-III inhibitor milrinone improves ventricular contractility, relaxes pulmonary arteries and reduces right ventricular afterload. Thus, it is used to treat heart failure and pulmonary hypertension (PH). However, its action on pulmonary veins (PVs) is not defined, although particularly PH due to left heart disease primarily affects the pulmonary venous bed. We examined milrinone-induced relaxation in PVs from guinea pigs (GPs) and humans. MATERIAL AND METHODS: Precision-cut lung slices (PCLS) were prepared from GPs or from patients undergoing lobectomy. Milrinone-induced relaxation was studied by videomicroscopy in naïve PVs and in PVs pre-constricted with the ETA-receptor agonist BP0104. Baseline luminal area was defined as 100%. Intracellular cAMP was measured by ELISA and milrinone-induced changes of segmental vascular resistances were studied in the GP isolated perfused lung (IPL). RESULTS: In the IPL (GP), milrinone (10 µM) lowered the postcapillary resistance of pre-constricted vessels. In PCLS (GP), milrinone relaxed naïve and pre-constricted PVs (120%) and this relaxation was attenuated by inhibition of protein kinase G (KT 5823), adenyl cyclase (SQ 22536) and protein kinase A (KT 5720), but not by inhibition of NO-synthesis (L-NAME). In addition, milrinone-induced relaxation was dependent on the activation of K ATP-, BK Ca (2+)- and Kv-channels. Human PVs also relaxed to milrinone (121%), however only if pre-constricted. DISCUSSION: Milrinone relaxes PVs from GPs and humans. In GPs, milrinone-induced relaxation is based on K ATP-, BK Ca (2+)- and Kv-channel-activation and on cAMP/PKA/PKG. The relaxant properties of milrinone on PVs lead to reduced postcapillary resistance and hydrostatic pressures. Hence they alleviate pulmonary edema and suggest beneficial effects of milrinone in PH due to left heart disease.

Levosimendan Relaxes Pulmonary Arteries and Veins in Precision-Cut Lung Slices - The Role of KATP-Channels, cAMP and cGMP.

INTRODUCTION: Levosimendan is approved for left heart failure and is also used in right heart failure to reduce right ventricular afterload. Despite the fact that pulmonary arteries (PAs) and pulmonary veins (PVs) contribute to cardiac load, their responses to levosimendan are largely unknown. MATERIALS AND METHODS: Levosimendan-induced vasorelaxation of PAs and PVs was studied in precision-cut lung slices from guinea pigs by videomicroscopy; baseline luminal area was defined as 100%. Intracellular cAMP- and cGMP-levels were measured by ELISA and NO end products were determined by the Griess reaction. RESULTS: Levosimendan relaxed control PVs (116%) and those pre-constricted with an endothelinA-receptor agonist (119%). PAs were only relaxed if pre-constricted (115%). Inhibition of KATP-channels (glibenclamide), adenyl cyclase (SQ 22536) and protein kinase G (KT 5823) largely attenuated the levosimendan-induced relaxation in control PVs, as well as in pre-constricted PAs and PVs. Inhibition of BKCa (2+)-channels (iberiotoxin) and Kv-channels (4-aminopyridine) only contributed to the relaxant effect of levosimendan in pre-constricted PAs. In both PAs and PVs, levosimendan increased intracellular cAMP- and cGMP-levels, whereas NO end products remained unchanged. Notably, basal NO-levels were higher in PVs. The KATP-channel activator levcromakalim relaxed PAs dependent on cAMP/PKA/PKG and increased cAMP-levels in PAs. DISCUSSION: Levosimendan initiates complex and divergent signaling pathways in PAs and PVs. Levosimendan relaxes PAs and PVs primarily via KATP-channels and cAMP/cGMP; in PAs, BKCa (2+)- and Kv-channels are also involved. Our findings with levcromakalim do further suggest that in PAs the activation of KATP-channels leads to the production of cAMP/PKA/PKG. In conclusion, these results suggest that levosimendan might reduce right ventricular afterload by relaxation of PAs as well as pulmonary hydrostatic pressure and pulmonary edema by relaxation of PVs.

Overexpression of CREMα in T cells aggravates lipopolysaccharide-induced acute lung injury.

Transcription factor cAMP response element modulator (CREM)α contributes to various cellular and molecular abnormalities in T cells, including increased IL-17 and decreased IL-2 expression. For development of acute lung injury (ALI), the invasion and regulation of immune cells are highly important, but the role of T cells remains unclear. In this study, we show that CREMα is upregulated in LPS-induced ALI. During the early phase of ALI (day 1), T cell-specific CREMα overexpression enhances the numbers of T cells and expression of TNF-α in bronchoalveolar lavage fluid and deteriorates lung functions. On day 3 of ALI, CREMα transgenic mice present a stronger inflammatory response with higher levels of TNF-α, IL-6, and IL-17 correlating with increased numbers of T cells and neutrophils in bronchoalveolar lavage fluid, whereas expression of Foxp3 and IL-2 and numbers of regulatory T cells are decreased. These changes result in restricted lung function in CREMα transgenic mice. Finally, an adoptive transfer of CREM(-/-) CD4(+) T cells, but not of wild-type T cells into RAG-1(-/-) mice results in ameliorated disease levels. Thus, levels of CREM in T cells determine the outcome of ALI, and CREMα transgenic animals represent a model in which proinflammatory T cells aggravate ALI in different phases of the disease. Given the fact that patients with autoimmune diseases like systemic lupus erythematosus show higher levels of CREMα and an increased susceptibility toward infectious complications, our finding is of potential clinical significance and may enable new therapeutic strategies.

Dual role of macrophage migration inhibitory factor (MIF) in human breast cancer.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine and mediator of acute and chronic inflammatory diseases. MIF is overexpressed in various tumours and has been suggested as a molecular link between chronic inflammation and cancer. MIF overexpression is observed in breast cancer but its causal role in the development of this tumour entity is unclear. METHODS: MIF levels in breast cancer cell lines were determined by ELISA and Western blot. CD74 was measured by Western blot, fluorescence microscopy and flow cytometry. Cell proliferation was studied by BrdU incorporation, cell adhesion by Matrigel adhesion assay, and cell invasion by migration assay through Matrigel-coated filters using the Transwell system. MIF expression in primary human breast cancers was measured by tissue microarray and a semi-quantitative immunoreactivity score (IRS) and comparison with histopathological parameters and patient outcome data. RESULTS: MIF was abundantly expressed in the non-invasive breast cancer cell lines MDA-MB-468 and ZR-75-1, but not in invasive MDA-MB-231 cells, which in turn expressed higher levels of the MIF-receptor CD74. Stimulation with exogenous MIF led to a dramatic upregulation of MIF secretion (50-fold) in MDA-MB-231 cells. Autocrine MIF promoted tumour cell proliferation, as indicated by blockade of MIF or CD74 in MDA-MB-231 and MDA-MB-468, and MDA-MB-231 invasiveness was enhanced by exogenous MIF. We correlated the expression of MIF with histopathological parameters and patient outcome data, using a tissue microarray of 175 primary invasive breast cancers and 35 normal control tissues. MIF was upregulated in breast cancer versus normal tissue (median IRS = 8 versus 6). MIF expression showed positive correlations with progesterone (p = 0.006) and estrogen (p = 0.028) receptor expression, markers of a favourable prognosis and a negative correlation to tumour size (p = 0.007). In line with these data, disease-specific overall (OS) as well as recurrence-free (RFS) survival was significantly improved in breast cancer patients with abundant cytosolic MIF expression compared to MIF low expressers (5-year OS = 67% versus 50%, p = 0.0019; 5-year RFS = 52% versus 36%, p = 0.0327). CONCLUSION: We conclude that intracellular expression of MIF in breast cancer cells is beneficial, whereas extracellular MIF may play a pro-oncogenic role in promoting breast cancer cell-stroma interactions.