Acute outcome of chronic total occlusion (CTO) recanalizsation in the elderly.

OBJECTIVES: Percutaneous coronary intervention (PCI) of total chronic total occlusion (CTO) still remains a major challenge in interventional cardiology. There is only insignificant knowledge reported in the literature about age differences in CTO recanalization. We analyzed in this study the issue of the impact of age on procedural characteristics, complications and short-term outcome. METHODS: Between 2012-2016 we included 440 patients. They underwent PCI for at least one CTO. Antegrade and retrograde CTO techniques were applied. The retrograde approach was used only after failed antegrade intervention. Continuous data are presented as the mean ± standard deviation; categorical data are presented as numbers and percentages unless otherwise specified. We used Twosamplet- t-test with equal variance to test the significant differences of the variables between the two cohorts. RESULTS: Procedural success proved independently of age. There was no significant interaction between age and procedural success (p=0.5). Complication rates were low in both groups (2.7% vs. 4%; p=0,4) with no difference in statistical significance. CONCLUSIONS: Our study suggests that in an aging society patients with severe coronary artery disease and chronical total occlusions an interventional therapy should be used more intensively. It can be performed safe and feasible.

Gender-based acute outcome in percutaneous coronary intervention of chronic total coronary occlusion.

BACKGROUND: Percutaneous coronary intervention (PCI) of total chronic coronary occlusion (CTO) still remains a major challenge. Insignificant data are reported in the literature about gender differences in CTO-PCI in the era of new drug-eluting stents. In this study we analysed the impact of gender on procedural characteristics, complications and acute results. METHODS: Between 2010-2015 we included 780 consecutive patients. They underwent PCI for at least one CTO. Antegrade and retrograde CTO techniques were applied. RESULTS: Patients undergoing CTO-PCI were mainly men (84%). Male patients were younger (66.9 years ±10.6 vs. 61.1 years ±10.4; p < 0.001), more often smokers, but less frequently had a history of coronary artery disease (24.4% vs. 32.7%; p = 0.085) compared with female patients. Female patients more often had diabetes mellitus (29.6% vs. 26.7%; p = 0.55) and hypertension (82.7% vs. 80.7%; p = 0.55). There were no differences with respect to the amount of contrast fluid, fluoroscopy time and examination time as well as to the length of the stent or the number of the stents. The stent diameter was slightly smaller in women, which was not surprising because the lumen calibre tends to be smaller in women than in men (3.0 mm (2.5-3) vs. 3.0 mm (3-3.5); p < 0.001). The success rates were 81.0% in women and 80.1% in men. There was no significant interaction between gender and procedural success and complication rates. CONCLUSIONS: Our retrospective study suggests that women and men have a comparable success rate at a low complication rate after recanalisation of CTO.

Safety of sublingual immunotherapy Timothy grass tablet in subjects with allergic rhinitis with or without conjunctivitis and history of asthma.

BACKGROUND: Patients with asthma may be more susceptible to adverse events (AEs) with sublingual immunotherapy tablet (SLIT-tablet) treatment, such as severe systemic reactions and asthma-related events. Using data from eight trials of grass SLIT-tablet in subjects with allergic rhinitis with/without conjunctivitis (AR/C), AE frequencies were determined in adults and children with and without reported asthma. METHODS: Data from randomized, double-blind, placebo-controlled trials of Timothy grass SLIT-tablet MK-7243 (2800 BAU/75 000 SQ-T, Merck/ALK-Abelló) were pooled for post hoc analyses. Subjects with uncontrolled and severe asthma were excluded from the trials. Frequencies for treatment-emergent AEs (TEAEs), local allergic swelling (mouth or throat), systemic allergic reactions, and asthma-related treatment-related AEs (TRAEs) were calculated. RESULTS: Among adults (n = 3314) and children (n = 881), 24% and 31%, respectively, had reported asthma. No serious local allergic swellings or serious systemic allergic reactions occurred in subjects with asthma treated with SLIT-tablet. There was no evidence of increased TEAEs, systemic allergic reactions, or severe local allergic swellings in adults or children with asthma treated with grass SLIT-tablet versus subjects without asthma in or outside of pollen season. There were 6/120 asthma-related TRAEs assessed as severe with grass SLIT-tablet and 2/60 with placebo, without a consistent trend among subjects with and without asthma (5 and 3 events, respectively). CONCLUSIONS: In the AR/C subjects with reported well-controlled mild asthma included in these studies, grass SLIT-tablet did not increase TEAE frequency, severe local allergic swelling, or systemic allergic reactions versus subjects without asthma. There was no indication that treatment led to acute asthma worsening.

Childhood asthma is associated with mutations and gene expression differences of ORMDL genes that can interact.

BACKGROUND: Genomewide association studies identified ORMDL3 as a plausible asthma candidate gene. ORMDL proteins regulate sphingolipid metabolism and ceramide homeostasis and participate in lymphocyte activation and eosinophil recruitment. Strong sequence homology between the three ORMDL genes and ORMDL protein conservation among different species suggest that they may have shared functions. We hypothesized that if single nucleotide polymorphisms (SNPs) in ORMDL3 alter its gene expression and play a role in asthma, variants in ORMDL1 and ORMDL2 might also be associated with asthma. METHODS: Asthma associations of 44 genotyped SNPs were determined in at least 1303 subjects (651 asthmatics). ORMDL expression was evaluated in peripheral blood mononuclear cells (PBMC) from 55 subjects (eight asthmatics) before and after allergen stimulation, and in blood (n = 60, 5 asthmatics). Allele-specific cis-effects on ORMDL expression were assessed. Interactions between human ORMDL proteins were determined in living cells. RESULTS: Sixteen SNPs in all three ORMDLs were associated with asthma (14 in ORMDL3). Baseline expression of ORMDL1 (P = 1.7 × 10(-6) ) and ORMDL2 (P = 4.9 × 10(-5) ) was significantly higher in PBMC from asthmatics, while induction of ORMDLs upon stimulation was stronger in nonasthmatics. Disease-associated alleles (rs8079416, rs4795405, rs3902920) alter ORMDL3 expression. ORMDL proteins formed homo- and heterooligomers and displayed similar patterns of interaction with SERCA2 and SPT1. CONCLUSIONS: Polymorphisms in ORMDL genes are associated with asthma. Asthmatics exhibit increased ORMDL levels, suggesting that ORMDLs contribute to asthma. Formation of heterooligomers and similar interaction patterns with proteins involved in calcium homeostasis and sphingolipid metabolism could indicate shared biological roles of ORMDLs, influencing airway remodeling and hyperresponsiveness.

Fine-mapping of IgE-associated loci 1q23, 5q31, and 12q13 using 1000 Genomes Project data.

BACKGROUND: Genome-wide association studies (GWAS) repeatedly identified 1q23 (FCER1A), 5q31 (RAD50-IL13 and IL4), and 12q13 (STAT6) as major susceptibility loci influencing the regulation of total serum IgE levels. As GWAS may be insufficient to capture causal variants, we performed fine-mapping and re-genotyping of the three loci using 1000 Genomes Project datasets. METHODS: Linkage disequilibrium tagging polymorphisms and polymorphisms of putative functional relevance were genotyped by chip technology (24 polymorphisms) or MALDI-TOF-MS (40 polymorphisms) in at least 1303 German children (651 asthmatics). The effect of polymorphisms on total serum IgE, IgE percentiles, and atopic diseases was assessed, and a risk score model was applied for gene-by-gene interaction analyses. Functional effects of putative causal variants from these three loci were studied in silico. RESULTS: Associations from GWAS were confirmed and extended. For 1q23 and 5q31, the majority of associations were found with mild to moderately elevated IgE levels, while in the 12q13 locus, single-nucleotide polymorphisms (SNPs) were associated with strongly elevated IgE levels. Gene-by-gene interaction analyses suggested that the presence of mutations in all three loci increases the risk for elevated IgE up to fourfold. CONCLUSION: This fine-mapping study confirmed previous associations and identified novel associations of SNPs in 1q23, 5q31, and 12q13 with different levels of serum IgE and their concomitant contribution to IgE regulation.

A role of FCER1A and FCER2 polymorphisms in IgE regulation.

BACKGROUND: Both FCER2 and FCER1A encode subunits of IgE receptors. Variants in FCER1A were previously identified as major determinants of IgE levels in genome-wide association studies. METHODS: Here we investigated in detail whether FCER2 polymorphisms affect IgE levels alone and/or by interaction with FCER1A polymorphisms. To cover the genetic information of FCER2, 21 single-nucleotide polymorphisms (SNPs) were genotyped by Illumina HumanHap300 BeadChip (5 SNPs) and the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; 14 SNPs) in at least 1303 Caucasian children (651 asthmatics) (ISAAC II/ MAGICS population); genotypes of two SNPs were imputed. RESULTS: SNP rs3760687 showed the most consistent effect on total serum IgE levels (b [SE] = -0.38 [0.16]; P = 0.016), while FCER2 polymorphisms in general were predominantly associated with mildly-to-moderately increased IgE levels (50th and 66th percentiles). Gene-by-gene interaction analysis suggests that FCER2 polymorphism rs3760687 influences IgE levels mainly in individuals not homozygous for the risk allele of FCER1A polymorphism rs2427837, which belongs to the major IgE-determining tagging bin in the population. CONCLUSION: FCER2 polymorphism rs3760687 affects moderately elevated total serum IgE levels, especially in the absence of homozygosity for the risk allele of FCER1A SNP rs2427837.

A novel synthetic lipopeptide is allergy-protective by the induction of LPS-tolerance.

BACKGROUND: Exposure to the environment of traditional farms can protect children from some allergic disease. Due to this exposure, TLR2 expression in these children is increased. TLR2 ligands derived from gram-positive bacteria are found in the dust of these farms. OBJECTIVES: We proved whether a synthetic lipopeptide binding to the TLR1/2 heterodimer is able to protect from allergic disease in two different murine models of allergy. We also investigated the immunological mechanisms underlying the protective properties of the lipopeptide. METHODS: We synthesized a lipopeptide derived from a germination lipoprotein of Bacillus cereus (LPGerD). We evaluated the immunomodulatory activity of LPGerD in a murine model of systemic sensitization (OVA/Alum) and in a model in which mice were sensitized with OVA pulsed bone-marrow-derived dendritic cells (BMDCs) via the airways. Furthermore, the induction of LPS tolerance was studied. RESULTS: Treatment of mice with LPGerD in a mouse model of asthma led to protection against sensitization and airway inflammation. Similarly, bone-marrow-derived dendritic cells (BMDCs) pre-treated with LPGerD were not able to prime mice for allergic immune response. We observed that pre-treatment with LPGerD led to the induction of a LPS-tolerant state in BMDCs. These cells secreted markedly lower amounts of pro-inflammatory cytokines upon LPS stimulation. Furthermore, we observed an up-regulation of IRAK-M mRNA in BMDCs pre-treated with LPGerD. CONCLUSIONS AND CLINICAL RELEVANCE: Our results suggest that induction of a LPS-tolerant state in antigen-presenting cells (APCs) may contribute to the protective effect of a farming environment. TLR2 agonists similar to those appearing in cowshed dust extracts, such as our synthetic LPGerD, lead to the ignorance of the LPS stimulus, which is important for the activation of APCs to mount a Th2 immune response. This substance might be a promising candidate for allergy-preventive treatments as LPGerD had only low pro-inflammatory characteristics.

Polymorphisms in the IRF-4 gene, asthma and recurrent bronchitis in children.

BACKGROUND: Interferon-regulatory factors (IRFs) play a crucial role in immunity, not only influencing interferon expression but also T cell differentiation. IRF-4 was only recently recognized as a further major player in T cell differentiation. OBJECTIVE: As IRF-1 polymorphisms were shown to be associated with atopy and allergy, we comprehensively investigated effects of IRF-4 variants on allergy, asthma and related phenotypes in German children. METHODS: Fifteen tagging single nucleotide polymorphisms (SNPs) in the IRF-4 gene were genotyped by MALDI-TOF MS in the cross-sectional ISAAC phase II study population from Munich and Dresden (age 9-11; N = 3099). Replication was performed in our previously established genome-wide association study (GWAS) data set (N = 1303) consisting of asthma cases from the Multicenter Asthma Genetic in Childhood (MAGIC) study and reference children from the ISAAC II study. RESULTS: SNPs were not significantly associated with asthma but with bronchial hyperresponsiveness, atopy and, most interestingly, with recurrent bronchitis in the first data set. The IRF-4 variant rs9378805 was associated with recurrent bronchitis in the ISAAC population and replicated in the GWAS data set where further SNPs showed associations with recurrent bronchitis and asthma. CONCLUSIONS: We found genetic associations in IRF-4 to be associated with recurrent bronchitis in our two study populations. Associated polymorphisms are localized in a putative regulatory element in the 3'UTR region of IRF-4. These findings suggest a putative role of IRF-4 in the development of bronchitis.

Perspectives on allergen-specific immunotherapy in childhood: an EAACI position statement.

This article is the result of consensus reached by a working group of clinical experts in paediatric allergology as well as representatives from an ethical committee and the European Medicine Agency (EMA). The manuscript covers clinical, scientific, regulatory and ethical perspectives on allergen-specific immunotherapy in childhood. Unmet needs are identified. To fill the gaps and to bridge the different points of view, recommendations are made to researchers, to scientific and patient organizations and to regulators and ethical committees. Working together for the benefit of the community is essential. The European Academy of Allergy and Clinical Immunology (EAACI) serves as the platform of such cooperation.

Specific immunotherapy in children.

Subcutaneous immunotherapy (SCIT) with allergen extracts in children with allergic rhinitis, with or without co-seasonal asthma, has developed into a routine treatment although the scientific evidence for its efficacy is not as strong as for adults. In the hands of experienced allergists, this treatment has been proven to be safe. The development of allergen tablets for sublingual immunotherapy (SLIT) may open a new age of more convenient, safer SIT. In children, in particular, the evidence for the long-term efficacy of SLIT, its ability to prevent the development of asthma and polysensitization and its comparability to SCIT will be required before it will replace the traditional subcutaneous route. Issues of compliance represent an important drawback of SLIT. We need ways of improving this. Treatment of asthma by immunotherapy is still restricted to clearly defined patients with mild to moderate asthma with symptoms that are related to the specific allergen sensitization. In these patients, symptoms and use of anti-inflammatory therapy can be reduced by SIT.

Cardiovascular autonomic neuropathy contributes to left ventricular diastolic dysfunction in subjects with Type 2 diabetes and impaired glucose tolerance undergoing coronary angiography.

AIMS: Left ventricular diastolic dysfunction is considered a precursor of diabetic cardiomyopathy, while diabetic cardiovascular autonomic neuropathy is associated with an increased risk of mortality. This study aimed to evaluate the association between left ventricular diastolic dysfunction and cardiovascular autonomic neuropathy, both diagnosed according to the current guidelines. METHODS: We evaluated 145 patients referred for an elective coronary angiography, 52 of whom had Type 2 diabetes and 48 had impaired glucose tolerance, while 45 subjects had normal glucose tolerance. Cardiovascular autonomic neuropathy was diagnosed using autonomic function tests, while left ventricular diastolic dysfunction was verified by tissue Doppler imaging echocardiography. RESULTS: Cardiovascular autonomic neuropathy was diagnosed in 15 (28.8%) patients with Type 2 diabetes and in six (12.5%) individuals with impaired glucose tolerance. The rates of left ventricular diastolic dysfunction were 81 and 33% in patients with and without cardiovascular autonomic neuropathy, respectively (P < 0.001). In the cardiovascular autonomic neuropathy group (n = 21), early diastolic relaxation velocity (Em) was significantly reduced (5.4 ± 0.9 vs. 7.3 ± 2.1 cm/s; P < 0.001) and the E/Em ratio was significantly higher (13.6 ± 4.6 vs. 10.3 ± 3.4 cm/s, P < 0.001) as compared with the group without cardiovascular autonomic neuropathy (n = 79). These findings remained significant after adjustment for age, sex, coronary artery disease, hypertension and HbA(1c) . A severe form of left ventricular diastolic dysfunction was observed in 33 and 15% of patients with and without cardiovascular autonomic neuropathy, respectively (P = 0.001). CONCLUSION: Cardiovascular autonomic neuropathy is associated with a higher prevalence and a more severe form of left ventricular diastolic dysfunction in patients with diabetes or impaired glucose tolerance undergoing coronary angiography. Because both cardiovascular autonomic neuropathy and left ventricular diastolic dysfunction are associated with increased cardiovascular morbidity and mortality, screening for patients with left ventricular diastolic dysfunction and cardiovascular autonomic neuropathy with diabetes or impaired glucose tolerance may identify those at high risk.

[Indications and evidence base for specific immunotherapy in childhood]. / Indikationen und Studienlage zur spezifischen Immuntherapie im Kindes- und Jugendalter.

Allergic rhinitis (AR) is a common disease affecting 7-17% of children and adolescents who may suffer from a significantly impaired quality of life. For 100 years subcutaneous immunotherapy (SCIT) has been used to treat AR. If patients are chosen carefully, a clinically relevant reduction of seasonal symptoms and medication use can be achieved by SCIT. Serious adverse events are rarely encountered during adequately performed and monitored SCIT. Sublingual immunotherapy (SLIT) may be used as a less painful therapeutic alternative in children if the administration of SCIT is impossible. Recent controlled trials have shown that SLIT performed over one pollen season also ameliorates seasonal symptoms and reduces the need for concomitant medication. Local reactions are frequently encountered during SLIT whereas systemic and life-threatening adverse events under correctly performed SLIT have not been reported so far.

Diagnostic value of outcome measures following allergen exposure in an environmental challenge chamber compared with natural conditions.

BACKGROUND: Efficacy testing of drugs in seasonal allergic rhinitis (SAR) is often disturbed by seasonal variations of environmental allergens, and assessment of onset and duration of action is hardly possible under natural conditions. Allergen provocation in an environmental challenge chamber (ECC) can be of added value in this respect. However, the specificity, sensitivity, and reproducibility of outcome measures under both settings are unclear. OBJECTIVE: The aim of this study was to investigate and compare the diagnostic value (specificity, sensitivity, and reproducibility) of clinical end-points and biomarkers both following allergen provocation in an ECC and under natural conditions. METHODS: Sixty adult patients with SAR to grass and 60 healthy subjects were exposed twice to grass pollen in an ECC and observed twice during the pollen season. Symptoms, nasal flow, as well as exhaled and nasal nitric oxide (NO) were investigated. RESULTS: The total nasal symptom score (TNSS) in the ECC had the best reproducibility (intraclass correlation coefficient ICC=0.86) and sensitivity/specificity [area under receiver operating characteristic curve (AUC)=0.99] of all measures. Symptoms in season also had good sensitivity/specificity but were far less reproducible. Nasal flow in the ECC had good sensitivity/specificity but reproducibility was limited. NO measurements showed good reproducibility but sensitivity/specificity were limited, except for exhaled NO in season (AUC=0.75). CONCLUSION: The high reproducibility and sensitivity/specificity in the ECC suggests that TNSS is a valuable outcome measure. While exhaled NO can be considered to monitor airway inflammation, nasal NO appears to be unspecific.

Relapse and survival in early-stage ovarian cancer.

OBJECTIVE: To analyze the prognostic influence of patient characteristics, diagnostic markers or therapeutic procedures in women diagnosed with early ovarian cancer based on relapse and survival in long term follow-up. MATERIALS AND METHODS: All women diagnosed and treated for early ovarian cancer at our institution between 1992 and 2006 were included in this retrospective study. Patient characteristics, clinical data including operative procedure, serum markers, stage and histology at first diagnosis as well as follow-up data were analyzed with regard to survival times and relapse rates. RESULTS: Altogether, 116 patients were included. Mean follow-up time was 7.0 +/- 3.3 years (range 2-14 years). Histology revealed a serous tumor in 64.7% (75/116), mucinous in 19.0% (22/116) and endometiroid tumors in 7.8% (9/116) of all cases. TNM classification was pT1a in 49.1% (57/116), pT1b in 6% (7/116), pT1c in 32.8% (38/116) and pT2a in 12.1% (14/116). Lymph node involvement (N1) was found in 3.4% of all patients. 17 deaths and 17 relapses (each 14.7%) were documented during follow-up time with a mean time to recurrence of 3.3 +/- 2.1 years (range 1-7 years). The general 1-, 2-, 5- and 10-year survival rates were 99, 95.7 and 88.9 and 81.0%, respectively. Patients with tumor stage pT1a and pT1b had a significantly better survival (P = 0.0003) and significantly lower risk of recurrence (P = 0.0138) compared to higher tumor stages. Moreover, patients who experienced recurrent disease or presented with ascites at primary diagnosis had a significantly worse overall survival (recurrence: hazard ratio 0.17, 95% confidence interval 0.0155-0.2182, P = 0.0001; ascites: HR 2.84, CI 1.1919-10.1131, P = 0.0225). The risk for recurrent disease was significantly elevated for patients with low grade (G3) tumors (P = 0.0330). Interestingly, there was neither a worse survival rate nor a higher relapse rate for patients with primary laparoscopic surgical access. CONCLUSION: Patients with early ovarian cancer stage pT1c and pT2a or low grade tumor have to be monitored closely in oncologic follow-up as they bare a significant risk for disease recurrence. Ascites at primary diagnosis, pT1c or pT2a tumor stage or recurrent disease are associated with a poor survival even in early ovarian cancer.

Validity of the J-CTO Score and the CL-Score for predicting successful CTO recanalization.

BACKGROUND: Percutaneous coronary intervention (PCI) of total chronic coronary occlusion (CTO) still remains a major challenge in interventional cardiology. To predict the probability of a successful intervention different scoring systems are available. We analyzed in this study the validity of two scoring systems, the Japanese CTO score (J-CTO score) and the newly developed Clinical and Lesion-related score (CL Score). METHODS: Between 2012 and 2015 we included 379 consecutive patients. They underwent PCI for at least one CTO. Antegrade and retrograde CTO techniques were applied. The retrograde approach was used only after failed antegrade intervention. RESULTS: Patients undergoing CTO PCI were mainly men (84%). The overall procedural success rate was 84% (±0.4). The mean J-CTO score was 2.9 (±1.3) and the mean CL score was 4.3 (±1.7). The CL score predicted more precisely the interventional results than the J-CTO score. CONCLUSIONS: Our study suggests that the previously presented CL score is superior to the J-CTO score in identifying CTO lesions with a likelihood for successful recanalization. Generally it appears to be a helpful tool for selecting patients and identifying the appropriate operator.

Infant formula containing galacto-and fructo-oligosaccharides and Bifidobacterium breve M-16V supports adequate growth and tolerance in healthy infants in a randomised, controlled, double-blind, prospective, multicentre study.

The objective of the present study was to evaluate the growth and tolerance in healthy, term infants consuming a synbiotic formula with daily weight gain as the primary outcome. In a randomised, controlled, double-blind, multicentre, intervention study infants were assigned to an extensively hydrolysed formula containing a specific combination of Bifidobacterium breve M-16V and a prebiotic mixture (short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides in a 9:1 ratio; scGOS/lcFOS; synbiotic group), or the same formula without this synbiotic concept for 13 weeks (control group). Anthropometry, formula intake, tolerance, stool characteristics, blood parameters, faecal microbiota and metabolic faecal profile were assessed. Medically confirmed adverse events were recorded throughout the study. Equivalence in daily weight gain was demonstrated for the intention-to-treat (ITT) population (n 211). In the per-protocol (PP) population (n 102), the 90 % CI of the difference in daily weight gain slightly crossed the lower equivalence margin. During the intervention period, the mean weight-for-age and length-for-age values were close to the median of the WHO growth standards in both groups, indicating adequate growth. The number of adverse events was not different between both groups. No relevant differences were observed in blood parameters indicative for liver and renal function. At 13 weeks, an increased percentage of faecal bifidobacteria (60 v. 48 %) and a reduced percentage of Clostridium lituseburense/C. histolyticum (0·2 v. 2·6 %) were observed in the synbiotic group (n 19) compared with the control group (n 27). In conclusion, this study demonstrates that an extensively hydrolysed formula with B. breve M-16V and the prebiotic mixture scGOS/lcFOS (9:1) supports an adequate infant growth.

Relevance of human metapneumovirus in exacerbations of COPD.

BACKGROUND AND METHODS: Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR. RESULTS: We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 x 10(5) viral copies/ml in nasal lavage and 1.88 x 10(5) viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD. CONCLUSION: HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely.

Major allergen Phl p Vb in timothy grass is a novel pollen RNase.

A cDNA coding for the major group V allergen Phl p Vb was isolated from a timothy grass pollen cDNA library by immunoscreening with a specific monoclonal antibody. It was discovered for the first time that the recombinant Phl p Vb pollen allergen after expression and purification has ribonuclease activity. High homology of Phl p Vb to other group V allergens in grass pollen indicates similar function. By RNase activity gel of natural pollen extract of timothy grass and consecutive Western blot analysis of the excised proteins, the RNase active bands were shown to be group V allergens. Additionally it was demonstrated that an homologous protein to Phl p Vb in the mother plant could be induced by salicylic acid. This indicates that group Vb allergens may be involved in host-pathogen interactions because in pollen they are quickly exported RNases and in the mother plant they depend on a hormone which is related to expression of plant resistance genes.

Differential expression of IFN-alpha subtypes in human PBMC: evaluation of novel real-time PCR assays.

Studies of the human IFN-alpha subtype system have been hampered by the lack of efficient procedures to quantify and differentiate the expression of the highly homologous IFN-alpha subtypes. Here we evaluate four novel real-time PCR assays for the specific detection and quantification of IFN-alpha mRNA for the subtypes alpha(2), alpha(6), alpha(8) and alpha(1/13) in a combined assay in human peripheral blood mononuclear cells (PBMC). This included (a) the selection of beta-glucuronidase (GUS) as a suitable housekeeping gene for relative quantification; (b) verification of the specificity by using human DNA of different IFN-alpha subtypes; and (c) comparison of the amplification efficiencies among the different assays. This highly sensitive method allows the detection of low-level, constitutive IFN-alpha mRNA and shows differences in the composition of constitutive IFN-alpha subtypes compared to other cell types (HeLa and HEp-2). The in vitro stimulation of PBMC with Newcastle disease virus (NDV), Respiratory syncytial virus (RSV) or an inactivated Herpes simplex (HSV) preparation leads to the transcriptional induction of all IFN-alpha subtypes investigated but to different expression levels. Among the subtypes detected, IFN-alpha(13/1) and alpha(2) are the major transcripts followed by alpha(8), and finally alpha(6) as a minor transcribed subtype. Time-kinetics of IFN-alpha transcriptional activation also revealed variations in the course of IFN-alpha transcription between NDV, RSV or HSV. The data obtained from the real-time PCR assays correlated well with IFN-alpha(2) protein release. In conclusion, we have demonstrated the suitability and reliability of new real-time PCR assays for the rapid and efficient analysis of IFN-alpha subtype expression.