PD-L1 expression in patients with non-small-cell lung cancer is associated with sex and genetic alterations: A retrospective study within the Caucasian population.

BACKGROUND: Programmed cell death-ligand 1 (PD-L1) expression is a well-established biomarker for predicting responses to immune checkpoint inhibitors and certain targeted therapies. As a result, treatment strategies for patients vary based on their PD-L1 expression status. Understanding the clinical features of patients with distinct PD-L1 levels is crucial for personalized treatment approaches. METHODS: Demographic and clinicopathological characteristics of 227 patients (54% male, mean age 67 ± 9.9 years) newly diagnosed with non-small-cell lung cancer (NSCLC) between April 2020 and December 2022 were retrospectively compared among three groups based on the PD-L1 expression: PD-L1 Tumor Proportion Score (TPS) negative, 1-50%, and ≥50%. Logistic regression analysis was performed to evaluate predictors for high PD-L1 expression ≥50%. RESULTS: PD-L1 expression levels were distributed as follows: negative in 29% of patients, between 1% and 50% in 41%, and greater than 50% (high) in 29%. In comparison to negative PD-L1 expression, low and high PD-L1 expression was associated with female sex (32.9% vs. 52.7% vs. 50.7%, p = 0.031), with the absence of epidermal growth factor receptor (EGFR) mutations (83.6% vs. 91.1% vs. 98.1% p = 0.029), and with the absence of ERBB2 (HER2) tyrosine kinase mutations (90.9% vs. 100% vs. 98.1% p = 0.007), respectively. Age, smoking status, histological subtype, and disease stage showed no significant differences among the three patient groups. In the univariate logistic regression, EGFR mutation appeared to be the only predictor for PD-L1 expression, although it did not reach statistical significance (p = 0.06). CONCLUSION: Although sex and genomic alterations are associated with PD-L1 expression in patients with NSCLC, no clinical characteristics seem to predict PD-L1 expression significantly.

Evaluation of PD-1 T lymphocytes in bronchoalveolar lavage in lung cancer compared to benign lung diseases.

The expression of the programmed cell death protein 1 (PD-1) has been shown to be markedly increased in tumor-infiltrating lymphocytes. However, the proportion of PD-1 + T cells in the bronchoalveolar lavage (BAL) of lung cancer patients has not been sufficiently evaluated so far. In this prospective study, the proportion of PD-1 + CD4 + as well as PD-1 + CD8 + T cells in BAL samples, isolated from patients with lung cancer, asthma or interstitial lung disease (ILD), were determined via flow cytometry and compared for differences. Bronchoalveolar lavage was performed in 34 patients (14 patients with lung cancer, 10 patients with asthma, 10 patients with ILD). The highest median proportion of PD-1 + CD4 + or PD-1 + CD8 + T cells were found in patients with ILD (83.1% [IQR 72.1; 87.5] and 73.8% [IQR 60.3; 86.3]) followed by patients with lung cancer (66.4% [IQR 59; 69] and 77.1% [IQR 35.8; 82.3]) and patients with asthma (61.3% [IQR 57.4; 70.5] and 57.3% [IQR 46; 65]). Thereby, the difference in the proportion of PD-1 + CD3 + CD4 + BAL cells between ILD patients and asthmatics was significantly different (p = 0.04). The proportion of PD-1 + CD4 + and PD-1 + CD8 + T cells in the BAL of patients with lung cancer did not differ significantly to patients with benign lung diseases. The highest proportion was observed in ILD patients suggesting further research to evaluate the role of the PD-1/PD-L1 pathway in ILD patients.

Correlation of PD-L1 expression on tumour cells between diagnostic biopsies and surgical specimens of lung cancer in real life with respect to biopsy techniques and neoadjuvant treatment.

PURPOSES: Programmed death-ligand 1 (PD-L1) testing is performed mainly on biopsy specimens in patients with advanced lung cancer. It is questionable whether the small amount of tissue analysed in biopsies may represent the true PD-L1 expression of a tumour. METHODS: In this retrospective study, PD-L1 expression on tumour cells derived from bronchoscopy brush cytology, endobronchial ultrasound guided transbronchial needle aspiration (EBUS-TBNA), endobronchial biopsy, transbronchial biopsy (TBB) and computed tomography (CT)-guided transthoracic biopsy was compared to the PD-L1 expression of the corresponding surgical resection in lung cancer patients with regard to neoadjuvant treatment in-between. RESULTS: A quantitative comparison between the diagnostic biopsy of the primary tumour with corresponding resected surgical specimens in a total of 113 lung cancer patients (60% male, mean age 65 ± 9 years) revealed a statistically significant correlation of PD-L1 expression on tumour cells (r = 0.58, p< 0.001), for patients without neoadjuvant treatment in-between and for patients who underwent neoadjuvant treatment (both p < 0.001). Using a cut-off value of ≥ 50% PD-L1 TPS for comparing the biopsy samples and resected specimens, the concordance rate was 78% with a Cohen's Kappa of 0.45. CONCLUSION: A statistically significant concordance for PD-L1 expression on tumour cells between biopsies from primary lung tumour and resected specimen was found, but of uncertain clinical accuracy. The use of a cut-off value of ≥ 50% PD-L1 TPS resulted only in a moderate agreement. Therefore, the interpretation of the PD-L1 determined form biopsy specimens status should only be considered with caution for treatment decisionsQuery.

Bronchodilator Reversibility in the GAN Severe Asthma Cohort.

BACKGROUND AND OBJECTIVE: Positive bronchodilator reversibility (BDR) is a diagnostic criterion for asthma. However, patients with asthma may exhibit a negative BDR response. Aim: To describe the frequency of positive and Negative BDR response in patients with severe asthma and study associations with phenotypic characteristics. METHODS: A positive BDR response was defined as an increase in FEV1 >200 mL and >12% upon testing with a short-acting ß-agonist. RESULTS: BDR data were available for 793 of the 2013 patients included in the German Asthma Net (GAN) severe asthma registry. Of these, 250 (31.5%) had a positive BDR response and 543 (68.5%) a egative BDR response. Comorbidities significantly associated with a negative response were gastroesophageal reflux disease (GERD) (28.0% vs 40.0%, P<.01) and eosinophilic granulomatosis with polyangiitis (0.4% vs 3.0%; P<.05), while smoking history (active: 2.8% vs 2.2%; ex: 40.0% vs 41.7%) and comorbid chronic obstructive pulmonary disease (COPD) (5.2% vs 7.2%) were similar in both groups. Patients with a positive BDR response had worse asthma control (median Asthma Control Questionnaire 5 score, 3.4 vs 3.0, P<.05), more frequently reported dyspnea at rest (26.8% vs 16.4%, P<.001) and chest tightness (36.4% vs 26.2%, P<.001), and had more severe airway obstruction at baseline (FEV1% predicted, 56 vs 64, P<.001) and higher fractional exhaled nitric oxide (FeNO) levels (41 vs 33 ppb, P<0.05). There were no differences in diffusion capacity of the lung for carbon monoxide, single breath (% pred, 70% vs 71%). Multivariate linear regression analysis identified an association between positive BDR response and lower baseline FEV1% (P<.001) and chest tightness (P<.05) and a negative association between BDR and GERD (P<.05). CONCLUSION: In this real-life setting, most patients with severe asthma had a negative BDR response. Interestingly, this was not associated with smoking history or COPD, but with lower FeNO and presence of GERD.

Bronchodilator Reversibility in the GAN Severe Asthma Cohort

Background: Positive bronchodilator reversibility (BDR) is a diagnostic criterion for asthma. However, patients with asthma may exhibit a negative BDR response. Aim: To describe the frequency of positive and negative BDR response in patients with severe asthma and study associations with phenotypic characteristics. Methods: A positive BDR response was defined as an increase in FEV1 >200 mL and >12% upon testing with a short-acting ß-agonist. Results: BDR data were available for 793 of the 2013 patients included in the German Asthma Net (GAN) severe asthma registry. Of these, 250 (31.5%) had a positive BDR response and 543 (68.5%) a negative BDR response. Comorbidities significantly associated with a negative response were gastroesophageal reflux disease (GERD) (28.0% vs 40.0%, P<.01) and eosinophilic granulomatosis with polyangiitis (0.4% vs 3.0%; P<.05), while smoking history (active: 2.8% vs 2.2%; ex: 40.0% vs 41.7%) and comorbid chronic obstructive pulmonary disease (COPD) (5.2% vs 7.2%) were similar in both groups. Patients with a positive BDR response had worse asthma control (median Asthma Control Questionnaire 5 score, 3.4 vs 3.0, P<.05), more frequently reported dyspnea at rest (26.8% vs 16.4%, P<.001) and chest tightness (36.4% vs 26.2%, P<.001), and had more severe airway obstruction at baseline (FEV1% predicted, 56 vs 64, P<.001) and higher fractional exhaled nitric oxide (FeNO) levels (41 vs 33 ppb, P<0.05). There were no differences in diffusion capacity of the lung for carbon monoxide, single breath (% pred, 70% vs 71%). Multivariate linear regression analysis identified an association between positive BDR response and lower baseline FEV1% (P<.001) and chest tightness (P<.05) and a negative association between BDR and GERD (P<.05). Conclusion: In this real-life setting, most patients with severe asthma had a negative BDR response. Interestingly, this was not associated with smoking history or COPD, but with lower FeNO and presence of GERD. (AU)

Antecedentes: La reversibilidad broncodilatadora (RB) positiva es un criterio diagnóstico para el asma. Sin embargo, los pacientes con asma pueden presentar una prueba RB negativa. Objetivos: Describir la frecuencia de RB positivas y negativas en pacientes con asma grave y sus asociaciones con características fenotípicas. Métodos: La RB positiva se definió como un aumento del FEV1 > 200 ml y > 12% tras la inhalación de un agonista beta de acción corta (SABA). Resultados: De 2013 pacientes incluidos en el registro de asma grave del German Asthma Net (GAN), 793 tenían datos sobre RB. De estos, 250 (31,5%) tuvieron una prueba RB positiva y 543 (68,5%) negativa. Las comorbilidades significativamente asociadas con RB negativa fueron el reflujo gastroesofágico (ERGE) (28,0% frente a 40,0%, p<0,01) y EGPA (0,4% frente a 3,0%; p<0,05), mientras que el antecedente de tabaquismo (activo: 2,8% frente a 2,2%; exfumador: 40,0% vs. 41,7%) y la comorbilidad de la EPOC (5,2% vs. 7,2%) fueron similares en ambos grupos. Los pacientes con RB positiva tenían peor control del asma (mediana ACQ-5 3,4 vs. 3,0, p<0,05), más disnea en reposo (26,8% vs. 16,4%, p<0,001) y mayor opresión torácica (36,4% vs. 26,2%, p<0,001), además presentaban una obstrucción de las vías respiratorias más grave al inicio del estudio (FEV1% pred: 56 frente a 64, p<0,001) y niveles más altos de FeNO (41 frente a 33 ppb, p<0,05), mientras que la capacidad de difusión fue similar (DLCO-SB% pred. 70% vs. 71%). El análisis de regresión lineal multivariable identificó una asociación de FEV1% basal inferior (p<0,001) y opresión torácica (p<0,05) con RB positiva y ERGE (p<0,05) con RB negativa. Conclusión: En este entorno en vida real, la mayoría de los pacientes con asma grave tuvieron una RB negativa. Curiosamente, esto no se asoció con antecedentes de tabaquismo o EPOC, sino con FeNO más bajo y presencia de ERGE. (AU)

[COVID-19 Vaccination in Asthma Patients Treated with Biologicals - Statement of the Austrian Society of Pneumology and German Respiratory Society]. / COVID-19-Impfungen bei Biologika-Therapie von Asthma bronchiale.

Patients with asthma should be vaccinated against COVID-19. This includes patients with severe asthma. Treatment with a biological for asthma is no contra-indication for vaccination against COVID-19.

[Statement of the Austrian Society of Pneumology (ASP)]. / Stellungnahme der Österreichischen Gesellschaft für Pneumologie (ÖGP): Management von Patienten mit SARS-CoV-2-Infektionen und von Patienten mit chronischen Lungenerkrankungen während der COVID-19-Pandemie (Stand 27.04.2020).

The COVID-19 pandemic is currently a challenge worldwide. In Austria, a crisis within the health care system has so far been avoided. The treatment of patients with community-acquired pneumonia (CAP), including SARS-CoV­2 infections, should continue to be based on evidence-based CAP guidelines during the pandemic. However, COVID-19-specific adjustments are useful. The treatment of patients with chronic lung diseases must be adapted during the pandemic, but must still be guaranteed.

Lipopolysaccharide (LPS) induced pulmonary neutrophil recruitment and platelet activation is mediated via the P2Y1 and P2Y14 receptors in mice.

Platelet activation occurs during host defence and in various inflammatory disorders. In animal models of infection and inflammation, experimental depletion of platelets leads to significantly reduced leukocyte recruitment and impaired clearance of pathogens from the lung. It is now appreciated that purinergic receptor activation is required for leukocyte activation, motility and adhesion, and platelet interactions with leukocytes can be modulated by purinergic stimulation of platelets. Here, we have investigated the role of platelet P2Y1, P2Y12, P2Y14, and P2X1 receptors on leukocyte recruitment and chemotaxis. Mice were administered either vehicle controls or selective P2Y1, P2Y12, P2Y14, or P2X1 antagonists intravenously before intranasal administration of lipopolysaccharide (LPS) to investigate the effect of these drugs on pulmonary leukocyte recruitment, peripheral platelet counts, bleeding times, and ex vivo platelet aggregation. Separately, platelets were incubated with P2Y1, P2Y12, P2X1 antagonists, or P2Y14 agonists to assess effects on platelet-induced neutrophil chemotaxis in vitro. Pulmonary neutrophil recruitment induced by intranasal LPS administration was inhibited in mice administered either with P2Y1 or P2Y14 antagonists, but not with P2Y12 or P2X1 antagonists. Furthermore, the administration of either a P2Y1 or a P2Y14 antagonist reversed the incidence of peripheral thrombocytopaenia associated with LPS exposure. Bleeding times were significantly increased in mice administered P2Y1, P2Y12, or P2X1 antagonists, whilst ex vivo platelet aggregation to ADP was significantly reduced. These haemostatic responses remained unaltered following antagonism of P2Y14. In vitro chemotaxis assays revealed direct antagonism of platelet P2Y1, but not P2Y12 or P2X1 receptors suppressed platelet-dependent neutrophil motility towards Macrophage derived chemokine (MDC, CCL22). Furthermore, the stimulation of platelets with selective P2Y14 agonists (UDP-glucose, MRS2690) resulted in significant platelet-dependent neutrophil chemotaxis. These results reveal a role for P2Y1 and P2Y14 activation of platelets following exposure to LPS, whilst haemostatic indices were unaffected by inhibition of platelet function with the P2Y14 antagonist in response to LPS.

MicroRNA-155 modulates P2R signaling and Th2 priming of dendritic cells during allergic airway inflammation in mice.

BACKGROUND: Dendritic cells (DCs) are the professional antigen-presenting cells (APCs) in the lung. They are known to be key players in the induction and maintenance of allergic asthma by cross-linking innate and adaptive immune responses. MicroRNAs (miRNAs) are known to influence cell fate and function by translational suppression or induction of messenger RNA (mRNA) degradation. miR-155 has been shown to be a crucial regulator of the immune system. However, its function in the pathogenesis of allergic airway inflammation (AAI) is not completely elucidated yet. METHODS: Wild type (WT) and miR-155-deficient (miR-155(-/-) ) mice were used in ovalbumin (OVA) and house dust mite (HDM) models of AAI. Adoptive transfer of sensitized DCs to the lungs, migration, and T-cell priming assays were used to investigate the functional relevance of miR-155 in DCs. RESULTS: miR-155(-/-) mice showed reduced eosinophilic airway inflammation compared to WT mice in both models of AAI. Furthermore, miR-155(-/-) DCs showed limited Th2 priming capacity and failed to induce airway inflammation in allergen-exposed WT mice. miR-155 deficiency on DCs was also associated with impaired purinergic receptor signaling, as miR-155(-/-) DCs showed reduced chemotaxis and IL-1beta secretion upon stimulation with ATP, probably due to direct targeting of ectonucleoside triphosphate diphosphohydrolases (ENTPD) by miR-155. CONCLUSIONS: miR-155 deficiency alleviates AAI by diminishing Th2 priming capacity and ATP-/P2R-induced activation of DCs in mice, suggesting this miRNA as a potential therapeutic target of AAI.

Apoptosis induced by the fungal pathogen gliotoxin requires a triple phosphorylation of Bim by JNK.

We previously reported that gliotoxin (GT), the major virulence factor of the mold Aspergillus fumigatus causing invasive aspergillosis (IA) in immunocompromised patients, induces apoptosis in a Bak-dependent manner. The signaling pathway leading to Bak activation and subsequent mitochondrial outer membrane permeabilization (MOMP) is elusive. Here, we show that GT and the supernatant of A. fumigatus (but not its GT-defective mutant) activate the JNK pathway and require a co-operative JNK-mediated BimEL phosphorylation at three sites (S100, T112 and S114) to induce apoptosis in mouse fibroblasts, human bronchial and mouse alveolar epithelial cells. Cells (i) treated with the JNK inhibitor SP600125, (ii) deleted or knocked down for JNK1/2 or Bim or (iii) carrying the BimEL triple phosphomutant S100A/T112A/S114A instead of wild-type BimEL are similarly resistant to GT-induced apoptosis. Triple-phosphorylated BimEL is more stable, redistributes from a cytoskeletal to a membrane fraction, better interacts with Bcl-2 and Bcl-xL and more effectively activates Bak than the unphosphorylated mutant. These data indicate that JNK-mediated BimEL phosphorylation at S100, T112 and S114 constitutes a novel regulatory mechanism to activate Bim in response to apoptotic stimuli.

Contrast ultrasound for the quantification of deep vein thrombosis in living mice: effects of enoxaparin and P2Y12 receptor inhibition.

BACKGROUND/OBJECTIVES: We examined the applicability of contrast-enhanced ultrasound (CEUS) for imaging of murine deep vein thrombosis (DVT) and measured the effects of enoxaparin, ticagrelor and P2Y(12) receptor deficiency in vivo. METHODS: Deep vein thrombosis was induced by exposure to ferric chloride or ligation of the infrarenal vena cava of C57BL/6 mice after pretreatment with enoxaparin, ticagrelor or vehicle and in P2Y(12-/-) mice. Initial thrombus growth was visualized by intravital microscopy. Thrombi were weighed and examined by immunohistochemistry. CEUS was performed with a standard ultrasound system (Vivid 7, GE Healthcare) in the open abdominal cavity after injection of stabilized sulphur hexafluoride microbubbles. RESULTS: Incubation with ferric chloride resulted in non-occluding platelet-containing thrombus growth within 15-25 min. Sham-operated mice, enoxaparin- and ticagrelor-pretreated wild-type and P2Y(12-/-) mice developed only small thrombi. After injection of the contrast agent, growing thrombi were delineated clearly as negative contrast on CEUS. Thrombus size on CEUS after 25 min was significantly smaller in enoxaparin- (0.3 ± 0.1 mm(2)) and ticagrelor-treated (0.5 ± 0.1 mm(2)) wild-type and in P2Y(12-/-) mice (0.4 ± 0.1 mm(2)) as compared with vehicle-treated wild-type mice (2.0 ± 0.3 mm(2)) in the maximal sagittal plane (P < 0.001, n = 5-10). CEUS-derived thrombus size correlated linearly with thrombus weight and also reflected the extent of ligation-induced DVT. CONCLUSIONS: Contrast-enhanced ultrasound allowed the real-time quantification of DVT in living mice. Genetic and pharmacologic antithrombotic interventions were well reflected by CEUS and suggested an important role of the platelet P2Y(12) receptor in early DVT formation.

Attenuated allergic airway inflammation in Cd39 null mice.

BACKGROUND: Extracellular Adenosine-5'-Triphosphate (ATP) is known to accumulate in the lung, following allergen challenge, and contributes via activation of purinergic receptors on dendritic cells (DC), to the development of allergic airway inflammation (AAI). Extracellular ATP levels in the airways are normally tightly regulated by CD39. This ectonucleotidase is highly expressed by DC purified from skin (Langerhans cells) and bone marrow, and has been shown to modulate DC adaptive/haptenic immune responses. In this study, we have evaluated the impact of Cd39 deletion and associated perturbation of purinergic signaling in AAI. METHODS: Standard ovalbumin (OVA)-alum and house dust mite (HDM) bone marrow-derived DC (BMDC)-dependent models of AAI were used to study effects of Cd39. Migration assays, time lapse microscopy, and T-cell priming assays were further used to determine functional relevance of Cd39 expression on BMDC in the setting of immune and Th2-mediated responses in these models. RESULTS: Cd39(-/-) mice exhibited marked increases in BALF ATP levels but paradoxically exhibited limited AAI in both OVA-alum and HDM models. These pathophysiological abnormalities were associated with decreased myeloid DC activation and chemotaxis toward ATP, and were linked to purinergic receptor desensitization responses. Further, Cd39(-/-) DCs exhibited limited capacity to both prime Th2 responses and form stable immune synaptic interactions with OVA-transgenic naïve T cells. CONCLUSIONS: Cd39-deficient DCs exhibit limited capacity to induce Th2 immunity in a DC-driven model of AAI in vivo. Our data demonstrate a role of CD39 and perturbed purinergic signaling in models of AAI.

The purinergic receptor P2Y2 receptor mediates chemotaxis of dendritic cells and eosinophils in allergic lung inflammation.

BACKGROUND: Extracellular ATP contributes to the pathogenesis of asthma via signalling at purinergic receptors. However, the precise purinergic receptors subtypes mediating the pro-asthmatic effects of ATP have not been identified, yet. METHODS: In vivo studies were performed using the OVA-alum model. Functional expression of the P2Y(2) purinergic receptor subtype on human monocyte-derived dendritic cells and eosinophils was investigated using real-time PCR, migration assays, and production of reactive oxygen species. RESULTS: Compared to wild-type animals P2Y(2) -/- mice showed reduced allergic airway inflammation which can be explained by defective migration of blood myeloid DCs towards ATP in vitro and in vivo, whereas the influence of ATP on maturation and cytokine production was not changed. Additionally, ATP failed to induce migration of bone marrow-derived eosinophils from P2Y(2) R-deficient animals. The relevance of our findings for humans was confirmed in functional studies with human monocyte-derived DCs and eosinophils. Interestingly, stimulation of human DCs derived from allergic individuals with house dust mite allergen induced functional up-regulation of the P2Y(2) R subtype. Furthermore, eosinophils isolated from asthmatic individuals expressed higher levels of P2Y(2) R compared to healthy controls. This was of functional relevance as these eosinophils were more sensitive to ATP-induced migration and production of reactive oxygen metabolites. CONCLUSIONS: In summary, P2Y(2) R appears to be involved in asthmatic airway inflammation by mediating ATP-triggered migration of mDCs and eosinophils, as well as reactive oxygen species production. Together our data suggest that targeting P2Y(2) R might be a therapeutic option for the treatment of asthma.

Iloprost has potent anti-inflammatory properties on human monocyte-derived dendritic cells.

BACKGROUND: The stable prostaglandin I2 analogue (iloprost) iloprost has been shown to inhibit allergic airway inflammation in mice by modulating the function of myeloid dendritic cells (DCs). OBJECTIVE: The aim of the current study was to investigate the biological activity of iloprost on human monocyte-derived DCs. METHODS: I prostanoid (IP) receptor expression was analysed by RT-PCR. Cytokine secretion by DCs and CD4+ T cells was measured by ELISA. The expression of the transcription factor FoxP3 after co-culture of DCs with CD4+ CD45RA+ T cells was analysed by flow cytometry. RESULTS: Human monocyte-derived DCs were found to express mRNA specific for the PGI2 receptor IP, and stimulation with iloprost resulted in increased cyclic AMP levels in both immature DCs (iDCs) and mature DCs (mDCs). Moreover, iloprost dose dependently inhibited the secretion of TNF-alpha, IL-6, IL-8 and IL-12p70 in mDCs, while it enhanced IL-10 production. Changes in cytokine secretion were paralleled by an altered T-cell priming capacity of DCs: in co-culture experiments of iloprost-treated mDC and naïve CD45RA+ T cells, an induction of regulatory T cells could be observed, as demonstrated by increased intracellular FoxP3 expression and IL-10 production. Additionally, iloprost inhibited the MIP-3beta-induced migration of mDCs. CONCLUSION: In summary, our results provide evidence that iloprost profoundly affects the function of human myeloid DCs. Therefore, iloprost might also be a new therapeutical option for the treatment of asthma in humans.

Local administration of uridine suppresses the cardinal features of asthmatic airway inflammation.

BACKGROUND: The immuno-modulatory properties of nucleotides such as adenosine or inosine, have been described extensively. Recently, the nucleoside uridine and its analogue 4-thiouridine have gained attention for their protective role in acute lung inflammation. OBJECTIVE: In this study, we investigated the influence of uridine on asthmatic airway inflammation. METHODS: We used the classical ovalbumin (OVA)-alum model, as well as a model of house dust mite-(HDM)-induced airway inflammation. The degree of inflammation was determined by bronchoalveolar lavage (BAL), histology, and measurement of bronchial hyperresponsiveness. RESULTS: Intratracheal treatment of OVA-sensitized animals with uridine before allergen challenge resulted in a reduction in total BAL cells and BAL eosinophils. This was accompanied by reduced tissue infiltration and diminished production of T helper type 2-cytokines by mediastinal lymph node cells. Additionally, mice treated with uridine developed less bronchial hyperresponsiveness. Uridine was also effective in reducing airway inflammation in HDM-induced asthma. The protective effects of uridine were independent of myeloid dendritic cell (mDC) function, because in vitro pre-treatment of allergen-pulsed DCs with uridine did not alter the degree of inflammation. However, uridine inhibited the release of pro-inflammatory mediators in vivo and by cultured lung epithelial cells, suggesting an effect on lung structural cells. CONCLUSION: In summary, we were able to show that uridine inhibits the classical features of asthmatic airway inflammation. As uridine supplementation is well tolerated in humans, it might be a new therapeutic approach for the treatment of bronchial asthma.

Compliance in sleep apnoea therapy: influence of home care support and pressure mode.

Continuous positive airway pressure (CPAP) is an effective treatment for obstructive sleep apnoea syndrome (OSAS) but therapy adherence is often low. The hypothesis that CPAP-adherence and clinical outcomes can be improved by either using an autoadjusting-CPAP (APAP) device or an intensive support was tested. A controlled parallel group study was performed with 100 newly diagnosed OSAS patients, randomised into 4 groups (n = 25 each): standard or intensive support plus either APAP or CPAP. Intensive support included education and monthly home visits for 6 months. Clinical outcome was monitored by polysomnography at CPAP initiation and, after 3 and 9 months, compliance data were downloaded from the CPAP devices. After 9 months, intensively supported patients returned for follow-up in 88 versus 68% in the standard-support-group. Daily usage (mean+/-sem 5.7+/-0.2 for intensive support versus 4.6+/-0.4 h for standard support), percentage of days used (80.4+/-2.8 versus 57.0+/-5.9%) and proportion of individual sleep time (80.6+/-3.2 versus 64.9+/-6.2%) were also higher. There was no significant difference between APAP or CPAP, (daily usage 5.2+/-0.4 versus 5.1+/-0.3 h, percentage of days 67.9+/-5.0 versus 69.2+/-4.9%, proportion of sleep time 72.5+/-5.0% versus 72.1+/-5.2%, for APAP and CPAP) but retention rate was higher with CPAP. In summary, intensive support after continuous positive airway pressure initiation, rather than the application of autoadjusting-continuous positive airway pressure, increased therapy adherence.

Modulation of P2X7 receptor functions by polymyxin B: crucial role of the hydrophobic tail of the antibiotic molecule.

BACKGROUND AND PURPOSE: P2X7 is a membrane receptor for extracellular ATP which is highly expressed in dendritic cells, macrophages and microglia where it mediates pro-inflammatory responses. The antibiotic polymyxin B, which binds to and neutralizes the toxic residue of bacterial lipopolysaccharide, greatly amplifies cellular responses mediated by the P2X7 receptor. However, the molecular mechanism involved is so far unknown. EXPERIMENTAL APPROACH: We investigated the effects of polymyxin B and polymyxin B nonapeptide (PMBN) which is the deacylated amino derivative of polymyxin B lacking the N-terminal fatty amino acid 6-methylheptanoic/octanoic-Dab residue, in human macrophages and HEK293 cells stably expressing the human P2X7 receptor (HEK293-hP2X7). Differences between the two antibiotics were assessed by monitoring the following: nucleotide-induced cytoplasmic free Ca2+ concentration changes, plasma membrane permeability changes, lactate dehydrogenase activity, cell morphology changes. Western blot and microscopic analyses of P2X7GFP-expressing cells were also performed. KEY RESULTS: In contrast to polymyxin B, the polymyxin B nonapeptide was unable to potentiate: a) the ATP-induced Ca2+ increase, b) pore formation and consequently ATP-mediated plasma membrane permeabilization; c) ATP-dependent cytotoxicity. Moreover, in contrast to polymyxin B, polymyxin B nonapeptide did not affect aggregation of the P2X7 receptor subunits and it did not potentiate P2X7-dependent cell fusion. CONCLUSIONS AND IMPLICATIONS: The effects of polymyxin B depended on the presence of its N-terminal fatty amino acid 6-methylheptanoic/octanoic-Dab residue as deletion of this residue abolished polymyxin B-dependent modulation of ATP-triggered responses. These findings are important in the search for allosteric modulators of the P2X7 receptor.

Elevated expression of extracellular matrix metalloproteinase inducer (CD147) and membrane-type matrix metalloproteinases in venous leg ulcers.

BACKGROUND: Matrix metalloproteinases (MMPs) contribute to matrix remodelling in venous leg ulcers. Extracellular MMP inducer (EMMPRIN; CD147) has been reported to increase MMP expression, and membrane type 1 MMP (MT1-MMP) has been implicated in the activation of MMPs. OBJECTIVES: To examine whether and to what degree EMMPRIN, MMP-2, MT1-MMP and membrane type 2 MMP (MT2-MMP) are expressed in venous leg ulcers as well as the association with MMP activity. METHODS: EMMPRIN, MMP-2, MT1-MMP and MT2-MMP were analysed by zymography and immunohistochemistry in biopsies from healthy skin and lesional tissue from venous leg ulcers. RESULTS: Zymography provided direct evidence of increased proteolytic activity of MMP-2 in lesional skin in comparison with healthy controls. Immunostaining showed intense expression of EMMPRIN, MMP-2, MT1-MMP and MT2-MMP in dermal structures of venous leg ulcers, whereas only EMMPRIN and MMP-2 showed elevated expression in perivascular regions. Our findings indicate that venous leg ulcers are characterized by elevated expression of EMMPRIN, MMP-2, MT1-MMP and MT2-MMP. The immunohistological findings of skin alterations reflect the dynamic process of activation of soluble and membrane-bound MMPs, which may be highly induced by EMMPRIN. CONCLUSIONS: These data suggest for the first time that membrane-bound MMPs may favour enhanced turnover of the extracellular matrix and support unrestrained MMP activity in venous leg ulcers.

Expression and function of adenosine receptors in human dendritic cells.

Dendritic cells (DCs) are specialized antigen-presenting cells characterized by their ability to migrate into target sites, process antigens, and activate naive T cells. In this study, we analyzed the biological activity and intracellular signaling of adenosine by using reverse transcriptase-polymerase chain reaction assays to investigate mRNA expression of A(1), A(2a) and A(3) adenosine receptors in immature and mature human DCs. Functional experiments on adenosine stimulation showed chemotaxis, intracellular calcium transients, and actin polymerization, but no activation of adenylate cyclase in immature DCs. Experiments with receptor isotype-selective agonists and antagonists as well as pertussis toxin revealed that chemotaxis, calcium transients, and actin polymerization were mediated via G(i-) or G(0-)protein-coupled A(1) and A(3) receptors. Maturation of DCs induced by lipopolysaccharide (LPS) resulted in down-regulation of A(1) and A(3) receptor mRNAs, although A(2a) receptor mRNA was still expressed. However, in LPS-differentiated DCs, adenosine and an A(2a) receptor agonist stimulated adenylate cyclase activity, enhanced intracellular cAMP levels, and inhibited interleukin 12 (IL-12) production. These effects could be completely prevented by pretreatment with A(2) receptor antagonist. These findings strongly suggest that adenosine has important but distinct biological effects in DCs activity as a chemotaxin for immature DCs and as a modulator of IL-12 production in mature DCs. These effects can be explained by differential expression of adenosine receptor subtypes.