SRAP markers as an alternative tool for Alternaria classification.

Alternaria is one of the main fungal contaminants of cereal grains worldwide with the potential to produce mycotoxins hazardous to human and animal health. Many studies have been carried out to characterize Alternaria sp.-grp. using traditional morphology or polyphasic approach, but a good correlation between morphological sp.-grp., molecular, and chemotaxonomic groups has not always been achieved. For this reason, this study aimed to investigate the usefulness of a cheaper alternative tool, SRAP markers, in identifying Alternaria sp.-grps. obtained from Argentinean barley grains and to compare it with preliminary characterization using morphological traits, phylogeny, and metabolite profiles. Fifty-three Alternaria isolates from barley grains of the main producing regions of Argentina were analyzed with four combinations of SRAP markers. The UPGMA dendrogram, based on the Simple Matching similarity coefficient, revealed three distinct groups. SRAP markers allowed the separation of Alternaria from Infectoriae sections in agreement with the results of a polyphasic approach previously made. Besides, isolates of A. arborescens sp.-grp. were clustered in a separate group from isolates of A. tenuissima and A. alternata sp.-grp., which were grouped in the same cluster. SRAP markers are a recommended tool for classifying Alternaria isolates because of its simplicity, reliability, and cost-effectiveness compared to other molecular markers.

Interaction of methyl-jasmonate and Fusarium poae in bread wheat.

Fusarium Head Blight (FHB) is a devastating disease that affects the grain yield and quality of essential crops such as wheat. In the last years, some Fusarium species have acquired particular importance as Fusarium poae. However, studies to evaluate F. poae-wheat interaction are still scarce. The interaction between F. poae and two bread wheat cultivars with different resistance levels against FHB was evaluated. Moreover, the application of methyl-jasmonate (MeJA) was evaluated as a possible tool to reduce the fungal presence. Our results showed that the MeJA treatment is isolate-dependent, reducing F. poae fungal growth. A decrease in fungal biomass was observed in the susceptible cultivar after MeJA application; however, no differences between inoculated and inoculated-MeJA treatments were observed in the resistant cultivar. Finally, the F. poae inoculation induces the expression of PR1-1 and PDF 1.2, being early in the resistant cultivar compared to the susceptible ones. The application of MeJA combined with the F. poae inoculation increased PR1-1 and PDF1.2 expressions in resistant cultivars. To our knowledge, this is the first study that evaluates the interaction between F. poae and wheat and the MeJA treatment as a possible management strategy against this important pathogen.

Mixed cropping regimes promote the soil fungal community under zero tillage.

Fungi of yield soils represent a significant portion of the microbial biomass and reflect sensitivity to changes in the ecosystem. Our hypothesis was that crops included in cropping regimes under the zero tillage system modify the structure of the soil fungi community. Conventional and molecular techniques provide complementary information for the analysis of diversity of fungal species and successful information to accept our hypothesis. The composition of the fungal community varied according to different crops included in the cropping regimes. However, we detected other factors as sources of variation among them, season and sampling depth. The mixed cropping regimes including perennial pastures and one crop per year promote fungal diversity and species with potential benefit to soil and crop. The winter season and 0-5 cm depth gave the largest evenness and fungal diversity. Trichoderma aureoviride and Rhizopus stolonifer could be used for monitoring changes in soil under zero tillage.

Fusarium pseudograminearum Associated with Barley Kernels in Argentina.

Barley (Hordeum vulgare L.), one of the most widely grown winter cereal crops in Argentina, is primarily grown for use as malted barley for the beer industry. In December 2010, a survey of fungi was conducted in a barley (cv. Shakira) seed lot in a field located in Tres Arroyos, Buenos Aires, Argentina. A sample of 400 seeds was surface sterilized (70% EtOH for 2 min and 5% NaClO for 2 min), rinsed twice in sterilized distilled water, plated on potato dextrose agar (PDA), and incubated at 24 ± 2°C in a 12-h dark/light cycle. One isolate that was morphologically similar to Fusarium graminearum was observed after 6 days of incubation. The isolate was transferred onto PDA and carnation leaf agar (CLA) substrates and grown with the same conditions as described above. On PDA, the isolate produced abundant, white-to-yellow-to-red, aerial mycelium and formed red pigments in the medium. On CLA, macroconidia were abundant, relatively slender and almost straight to moderately curved, and commonly five to six septate. Microconidia were not observed. Chlamydospores were observed after 3 weeks. The fungus was initially identified as F. graminearum on the basis of morphology of the asexual stage (1). Pathogenicity was conducted using a hand sprayer to inoculate five barley (cv. Shakira) heads in potted plants with a 5-ml asexual spore suspension (1 × 104 conidia per ml). Two heads were sprayed with sterile distilled water as a control. Plants were covered with polyethylene bags and incubated for 3 days in a growth chamber under a 12-h day/dark cycle at 22 ± 2°C. Plants were unbagged and moved into a greenhouse. Noninoculated spikelets were asymptomatic and inoculated spikelets showed discoloration or a tan-to-dark brown necrosis. The fungus was reisolated from symptomatic kernels. DNA of the isolate was extracted (3) and the isolate was identified to species by sequencing the reductase (RED), trichothecene 3-O-acetyltransferase (tri101), and translation elongation factor (TEF) regions (4). The sequences were compared with those in GenBank. The RED sequence (Accession No. JQ350697) showed 100% similarity, the tri101 (Accession No. JQ350698) showed 99% similarity, and the TEF (Accession No. JQ350699) showed 100% similarity with several F. pseudograminearum sequences. Additionally, the isolate was tested for the potential to produce deoxinyvalenol (DON) using a PCR approach that allows identification of two acetylated forms of DON: 15-acetyl-DON (15-ADON) and 3-ADON (2). A PCR product indicative of a 3-ADON genotype was obtained. To our knowledge, this is the first report of F. pseudograminerum associated with barley kernels in Argentina. Considering its potential to cause head blight and product mycotoxins, a large-scale survey of F. pseudograminearum on barley crops in Argentina is underway. A voucher culture (No. 1154) has been deposited in the Culture Collection of the La Plata Spegazzini Institute. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK. 2006. (2) A. Quarta et al. Food Addit. Contam. 22:309, 2005. (3) S. A. Stenglein and P. A. Balatti. Physiol. Mol. Plant Pathol. 68:158, 2006. (4) T. J. Ward et al. Fungal Genet. Biol. 45:473, 2008.

First record of Fusarium verticillioides as an entomopathogenic fungus of grasshoppers.

Fusarium verticillioides (Saccardo) Nirenberg (Ascomycota: Hypocreales) is the most common fungus reported on infected corn kernels and vegetative tissues, but has not yet been documented as being entomopathogenic for grasshoppers. Grasshoppers and locusts represent a large group of insects that cause economic damage to forage and crops. Tropidacris collaris (Stoll) (Orthoptera: Acridoidea: Romaleidae) is a large and voracious grasshopper that in recent years has become an increasingly recurrent and widespread pest in progressively more greatly extended areas of some of in Argentina's northern provinces, with chemical insecticides being currently the only means of control. During February and March of 2008-09, nymphs and adults of T. collaris were collected with sweep nets in dense woodland vegetation at a site near Tres Estacas in western Chaco Province, Argentina, and kept in screened cages. F. verticillioides was isolated from insects that died within 10 days and was cultured in PGA medium. Pathogenicity tests were conducted and positive results recorded. Using traditional and molecular-biological methods, an isolate of F. verticillioides was obtained from T. collaris, and its pathogenecity in the laboratory was shown against another harmful grasshopper, Ronderosia bergi (Stål) (Acridoidea: Acrididae: Melanoplinae). The mortality caused by F. verticillioides on R. bergi reached 58 ± 6.53% by 10 days after inoculation. This is the first record of natural infection caused by F. verticillioides in grasshoppers.

Fusarium tricinctum Associated with Head Blight on Wheat in Argentina.

Wheat (Triticum aestivum L.), the most widely grown winter cereal crop in Argentina, is grown on 5 million ha. Fusarium species affect yield and grain quality because of mycotoxins. In December 2009, a screen of fungal species in wheat seeds from a field in Azul, Buenos Aires, Argentina was conducted. Four hundred seeds were surface sterilized by dipping successively into 70% ethanol for 2 min, 5% sodium hypochlorite for 2 min, and finally rinsing twice in fresh sterilized distilled water. The seeds were plated on potato dextrose agar (PDA), pH 6, and incubated at 24 ± 2°C with exposure to 12-h alternate cycles of darkness and light. Eight isolates morphologically similar to Fusarium species were observed after 6 days of incubation. For identification, monosporic isolates were transferred onto PDA and carnation leaf agar (CLA) to grow at the conditions described above (1). One isolate, when grown on PDA, rapidly produced abundant, dense, white, aerial mycelium that became pink with age and formed red pigments in the medium. On CLA, macroconidia were abundant, relatively slender, curved to lunate, and three to five septate. Microconidia were abundant, napiform, oval or pyriform, zero to one septate, and commonly clustered in false heads. Chlamydospores were absent. The fungus was identified as Fusarium tricinctum (Corda) Saccardo on the basis of fungal morphology (1). To complete Koch's postulates, the pathogenicity of the fungus was tested by spraying five healthy inflorescences (on average 16 spikelets per spike) of wheat with a 5-ml suspension (2 × 105 conidia per ml). Another two healthy inflorescences were sprayed with sterile distilled water. Plants were placed in a growth chamber with a 12-h photoperiod at 22 ± 2°C, covered with polyethylene bags that were removed after 3 days, and then moved to a glasshouse. The same procedure was repeated. While control inflorescences were asymptomatic, inoculated inflorescences showed a mean of five bleached spikelets per spike. By using the methodology described above, the fungus was reisolated from all infected grains of inoculated plants but not from the controls. To confirm the morphological diagnosis, the genomic DNA of the isolate was extracted (3) and the internal transcribed spacer (ITS) and the translation elongation factor (TEF) regions were PCR-amplified using primer pairs ITS3/ITS4 (4) and EF-1/EF-2 (2), respectively. The sequences were compared with those in GenBank. The ITS sequence (Accession No. HM635739) showed 100% similarity with several F. tricinctum sequences (e.g., Accession Nos. HM068317, FN598932, and EF589873) but also with other Fusarium species such as F. acuminatum. The TEF sequence (Accession No. HQ214681) showed 99 to 100% similarity with Accession Nos. HM068307, EU744838, and EU744837 of F. tricinctum. To our knowledge, this is the first report of F. tricinctum on wheat in Argentina. This species is known to produce fusarin C, enniatins, and moniliformin toxins. Since F. tricinctum can infect different cereal grains, a large-scale survey of cereals from fields throughout Argentina is in progress. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK. 2006. (2) K. O'Donell et al. Proc. Nat. Acad. Sci. USA 95:2044, 1998. (3) S. A. Stenglein and P. A. Balatti. Physiol. Mol. Plant Pathol. 68:158, 2006. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Phylogenetic relationships of Fusarium poae based on EF-1 alpha and mtSSU sequences.

A molecular phylogenetic analysis of Fusarium poae isolates from South America (Argentina) and Europe (mainly England, Germany, Italy) was performed using 98 F. poae, four Fusarium culmorum, two Fusarium sporotrichioides and one Fusarium langsethiae isolates. Phylogenetic analyses were performed using nuclear (translation elongation factor 1-alpha, EF-1 alpha) and mitochondrial (mitochondrial small subunit rDNA, mtSSU) sequences. Partitioned (each dataset separately) and combined (EF-1 alpha+mtSSU) analyses did not reveal any clear correlations from the inferred branching topology, between the distribution of observed haplotypes and the geographic origin and/or host species. Results from the present study confirmed that isolates from F. poae form a monophyletic group, and the low variability within isolates from a broad geographic range suggests a common lineage history. Among F. poae isolates from Argentina, however, some were found to possess an insert within mtSSU with structural similarities to group IC2 introns. F. poae isolates differing by the presence/absence of a mtSSU insertion were characterized further by analysis of a portion of the Tri5 gene, but this sequence was unable to reveal variability. The presence of this insert only within isolates from Argentina suggests that evolutionary events (insertions/deletions) are probably taking place within the Argentinian F. poae isolates, and that the acquisition of this insert occurred after geographic isolation of the Argentinian and European populations.

Fusarium proliferatum, a New Pathogen Causing Head Blight on Oat in Argentina.

Oat (Avena sativa L.) is widely grown (~200,000 ha) for livestock feed in Argentina. Fusarium spp. affect yield and commercial quality and can cause indirect losses because some Fusarium spp. produce mycotoxins. In December 2008, a study of oat seeds (cv. Graciela INTA) from Trenque Lauquen, Buenos Aires, Argentina was conducted. Seeds (400) were surface sterilized by dipping successively into 70% ethanol for 2 min, 5% sodium hypochlorite for 2 min, rinsed twice in fresh sterilized distilled water, plated on 2% potato dextrose agar (PDA) pH 6, and incubated at 24 ± 2°C with 12-h photoperiods. Six isolates morphologically similar to Fusarium spp. were observed after 6 days of incubation. For identification, monosporic isolates were transferred onto 2% PDA and carnation leaf agar (CLA) to grow with the conditions described above. Two isolates produced abundant, white, aerial mycelium and violet-to-dark (with age) pigments in the PDA. On CLA, macroconidia were abundant, slender, almost straight, thin walled, and usually three to five septate. Microconidia were abundant, usually single celled, oval or club-shaped in chains (less commonly in false heads) on monophialides and polyphialides. Chlamydospores were absent. The fungus was identified as Fusarium proliferatum (Matsushima) Nirenberg on the basis of fungal morphology (1). To complete Koch's postulates, the pathogenicity of the fungus was tested by spraying five healthy inflorescences of oat (cv. Graciela INTA) with a 5-ml suspension (2 × 105 conidia/ml). Another two healthy inflorescences were sprayed with sterile distilled water. Plants were placed in a growth chamber with a 12-h photoperiod at 22 ± 2°C and covered with polyethylene bags that were removed after 3 days and plants were moved to a glasshouse. This procedure was repeated. While control inflorescences were asymptomatic, inoculated inflorescences showed bleaching glumes that sometimes became necrotic with some grains that presented pale brown discoloration and necrotic areas. The fungus was reisolated from glumes and grains of inoculated plants and not from controls using the methodology described above. To confirm the morphological diagnosis, the genomic DNA of the isolates was extracted (3) and a PCR reaction with specific primers 5'-CTTTCCGCCAAGTTTCTTC-3'-forward and 5'-TGTCAGTAACTCGACGTTGTTG-3'-reverse was chosen (2) using the following cycling protocol: initial denaturation step at 95°C for 2 min; 30 cycles at 95°C for 30 s, 55°C for 30 s, 72°C for 45 s; final extension at 72°C for 2 min. Successful amplifications were confirmed by gel electrophoresis. Size of the DNA fragment was estimated using a 100-bp DNA ladder. The reaction was repeated three times. The expected size product (585 bp) was obtained, confirming the identification (2). To our knowledge, this is the first report of F. proliferatum on oat in Argentina. This species is known to produce fumonisins, beauvericin, fusaric acid, fusarins, and moniliformin toxins, among others. Since F. proliferatum can infect different cereal grains, a large-scale survey in the same and different fields is in progress. A voucher culture has been deposited in the LPSC (Culture Collection of the La Plata Spegazzini Institute) No. 1058. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK. 2006. (2) G. Mule et al. Eur. J. Plant Pathol. 110:495, 2004. (3) S. A. Stenglein and P. A. Balatti, Physiol. Mol. Plant Pathol. 68:158, 2006.

Combination of omalizumab and specific immunotherapy is superior to immunotherapy in patients with seasonal allergic rhinoconjunctivitis and co-morbid seasonal allergic asthma.

BACKGROUND: The treatment of allergic asthma by specific immunotherapy (SIT) is hampered by potential side-effects. OBJECTIVE: The aim of this study was to study the effect of omalizumab, a monoclonal anti-IgE antibody, in combination with SIT in patients with seasonal allergic rhinoconjunctivitis (SAR) and co-morbid seasonal allergic asthma (SAA) incompletely controlled by conventional pharmacotherapy. METHODS: A randomized, double-blind, placebo-controlled, multi-centre trial was performed to assess the efficacy and safety of omalizumab (Xolair) vs. placebo in combination with depigmented SIT (Depigoid) during the grass pollen season. Omalizumab or placebo was started 2 weeks before SIT; the whole treatment lasted 18 weeks. Primary endpoint was daily 'symptom load', the sum of daily scores for symptom severity and rescue medication use. RESULTS: A total of 140 patients (age 11-46 years) were randomized; and a total of 130 finished the study. Combination therapy reduced the symptom load by 39% (P=0.0464, Wilcoxon test) over SIT monotherapy. This difference was mainly due to reduced symptom severity (P=0.0044), while rescue medication use did not change significantly. Combination therapy also improved asthma control (Asthma Control Questionnaire, P=0.0295) and quality of life in the case of asthma (Asthma Quality of Life Questionnaire, P=0.0293) and rhinoconjunctivitis (Rhinoconjunctivitis Quality of Life Questionnaire, P=0.0537). Numbers of patients with 'excellent or good' treatment efficacy according to ratings of investigators (75.0% vs. 36.9%) or patients (78.5% vs. 46.1%) were markedly higher in the combination group than under SIT alone. CONCLUSION: Combination of omalizumab with SIT for treatment of patients with SAR and co-morbid SAA was safe and reduced the symptom load in a statistically significant and clinically meaningful manner.

First Report of Angular Leaf Spot Caused by Phaeoisariopsis griseola on Phaseolus coccineus in Argentina.

Angular leaf spot (ALS), caused by Phaeoisariopsis griseola (Sacc.) Ferraris, is one of the most destructive and widespread problems of common bean (Phaseolus vulgaris L.) in Tucumán and other northwestern provinces of Argentina (4). Symptoms similar to those of ALS were observed during April 2005 on most plants of runner bean (P. coccineus L.) in an 80-ha field in Tafí del Valle, Tucumán (2,000 m above sea level). Leaf lesions were brown to gray, irregular to angular to circular, and 0.5 to 1 cm in diameter. Lesions on pods were oval to circular with reddish brown centers surrounded by darker brown borders. Conidia in vivo were curved cylindrical to obclavate with one to five septa and measured 25 to 60 × 3.5 to 7 µm. The conidiophores were 100 to 250 µm high and clustered together to form synnemata measuring 20 to 50 µm in diameter. The pathogen was isolated by placing conidia from diseased leaves onto potato dextrose agar (PDA) at pH 6. Colonies measuring 2 to 3 mm in diameter composed of dense, dark olive mycelium developed after incubation in the dark at 24 ± 2°C for 3 to 4 days. Pathogenicity of the isolate was tested with conidia obtained from the second subculture of 14-day-old colonies on PDA. Conidial suspensions of 2 × 104 conidia per ml were sprayed onto the upper and lower surfaces of the first trifoliolate leaves of six runner bean plants, 18 days after planting. Inoculated and control plants (sprayed with distilled water) were placed in a growth chamber with a 12-h photoperiod at 24 ± 2°C and 95 to 100% relative humidity and 48 h later moved to the greenhouse. Disease symptoms were evaluated 18 days after inoculation. While control plants were healthy, all inoculated plants showed symptoms similar to those observed in the field. The fungus that was consistently reisolated from lesions in the inoculated plants was identified as Phaeoisariopsis griseola on the basis of fungal morphology (1), symptoms produced on leaves (3), and random amplified polymorphic DNA data with primer 5'-CAATCGCCGT-3' (2). Runner bean is a new crop in Tafí del Valle, which is a geographically isolated area. In a period of only 2 years, the area cultivated with beans increased approximately five-fold. Because of this, the presence of a pathogen like Phaeoisariopsis griseola, which causes considerable reduction in yield in most common bean-producing areas of Argentina, is of concern. To our knowledge, this is the first report of ALS occurring on P. coccineus in Argentina. This report may prompt the inclusion of regular testing of seeds for ALS in P. coccineus-production areas. A voucher culture has been deposited in the LPSC (Culture collection of the La Plata Spegazzini Institute) No. 844. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, UK, 1971. (2) P. Guzmán et al. Plant Dis. 83:37, 1999. (3) A. W. Saettler. Pages 15-16 in: Compendium of Bean Diseases. R. Hall, ed, The American Phytopathological Society, St. Paul, 1991. (4) S. A. Stenglein et al. Pages 209-243 in: Advances in Applied Microbiology, Vol. 52. A. I. Laskin et al., eds, Academic Press, San Diego, 2003.

The co-seasonal application of anti-IgE after preseasonal specific immunotherapy decreases ocular and nasal symptom scores and rescue medication use in grass pollen allergic children.

BACKGROUND: Specific immunotherapy (SIT) and treatment with anti-immunoglobulin (Ig)E antibody are complementary approaches to treat allergic rhinoconjunctivitis, which may be used for single or combined treatment. OBJECTIVE: A randomized, double-blind, placebo-controlled trial was conducted to compare the efficacy of single and combined treatment with SIT and anti-IgE (Omalizumab) in reducing symptom severity and rescue medication use. METHODS: A total of 221 subjects with birch and grass pollen allergic rhinoconjunctivitis aged 6-17 years were analysed during the grass pollen season. Group A (SITbirch + placebo) served as a reference group obtaining no effective treatment for grass pollen allergy. Group B received anti-IgE monotherapy during grass pollen season, group C SIT grass pollen monotherapy, and group D the combined treatment of SIT and Omalizumab. RESULTS: Preseasonal treatment with grass pollen SIT alone compared with SIT with the nonrelated allergen did not reduce symptoms or rescue medication use. Anti-IgE monotherapy significantly diminished rescue medication use and number of symptomatic days. The combined treatment with SIT and anti-IgE showed superior efficacy on symptom severity compared with anti-IgE alone. CONCLUSIONS: Co-seasonal Omalizumab therapy showed considerable effects in children with seasonal allergic rhinitis. The combination of SIT plus Omalizumab was clinically superior to each treatment alone during the first year of observation.

[Miflonide/Foradil via Aerolizer compared with other anti-inflammatory and anti-obstructive therapeutic regimens]. / Miflonide/Foradil via Aerolizer im Vergleich zu anderen antiinflammatorischen und antiobstruktiven Therapieregimes.

Standard therapy of asthma consists of combined, antiinflammatory (steroids) and anti-obstructive (long acting/short acting beta-agonist) treatment via inhalation using two separate or a single inhalation device. Here, we report the results of using Miflonide (Budesonide 400 - 800 microg per day) and Foradil (Formoterol 24 - 48 microg per day) in cases of moderate and severe asthma previously treated insufficiently with another combination therapy. 80 patients with asthma previously treated insufficiently with any other combination therapy of steroid and long acting beta-agonist were included. Instead of their previous therapy all were switched to therapy with Miflonide and Foradil for eight weeks (two office visits at 4 and 8 weeks). Lung function (peak flow [l/min], FEV1 [I], FVC [I], R(tot) [kPa*s/l]) was performed at every visit. Doctors' and patients' estimation of disease severity, physical examination, drug related side-effects and the use of short acting beta-agonist aerosols were registered. Lung function parameters improved significantly compared to the run in phase prior to the change in medication (peak flow: + 18.4 %, FEV 1 : + 10.7 %, FVC: + 6.8 %, R(tot) : -18.0 %). Use of additional short acting inhalative beta-agonists was reduced. Subjectively, patients judged their general condition as improved, effectiveness as greater compared to previous medication and side effects as tolerable. The use of a combination of Miflonide/Foradil lead to an improvement in subjective and objective parameters in asthma patients that had previously been treated with a variety of other antiinflammatory and anti-obstructive therapy regimens. Reasons for this observation are beside change in medication, patients training in asthma therapy, change of application system, and increase of patients compliance.

[Onset of action of formoterol in patients with moderate to severe, partially reversible airflow obstruction assessed by bodyplethysmography]. / Bodyplethysmographischer Wirkeintritt von Formoterol bei Patienten mit mittel- bis schwergradiger partiell reversibler Atemwegsobstruktion.

BACKGROUND: Formoterol is a long acting beta2-agonist used for the treatment of obstructive airway diseases. Compared with Salmeterol, Formoterol has a rapid onset of bronchodilation. There are only scant data regarding the comparative onset of action using bodyplethysmography in moderate to severely obstructive patients. METHODS: In a mono-center, single-blinded parallel group study 60 patients (age: 61.9 +/- 12.8 years, 65 % male) with moderate to severe (mean FEV(1) 40.6 +/- 15.3 % of predicted), partially reversible (FEV(1) post bronchodilator > 15 % from baseline) airway obstruction were treated with either formoterol 12 microg bid or salmeterol 50 microg bid over a period of two weeks. Onset of action was measured by airway resistance (sRaw) before and after two weeks of treatment. RESULTS: Compared with Salmeterol, Formoterol had a significantly faster onset of action (10 % decrease of Raw) at baseline (F: 1.4 +/- 0.9 vs. S: 15.1 +/- 34.5 min., p < 0.0001) and after two weeks of treatment (F: 6.2 +/- 21.6 vs. S: 51 +/- 135 min., p < 0.0001). Morning FEV(1) improved similarily during treatment in both study groups, when compared with baseline lung function (F: 1.38 +/- 0.64 vs. 1 +/- 0.41 l; S: 1.43 +/- 0.67 vs. 1 +/- 0.4 l, p < 0.05, both comparisons). Both treatments were well tolerated. CONCLUSION: Formoterol produces a rapid improvement of airway resistance in patients with moderate to severe, partially reversible airway obstruction. The onset of bronchodilation was significantly faster for Formoterol compared with Salmeterol. Both drugs improved lung function similarily after two weeks of treatment. It is important to distinguish beta2-agonists not only into short- and long-acting but also into fast- and slow-acting.

Efficient lytic infection of human arterial endothelial cells by human cytomegalovirus strains.

Endothelial cells (EC) are common targets of permissive infection by human cytomegalovirus (HCMV) in vivo during acute disease. However, studies of HCMV-EC interactions in vitro have generated discordant results. While lytic infection of cultured venous EC has been well established, Fish et al. (K. N. Fish, C. Soderberg Naucler, L. K. Mills, S. Stenglein, and J. A. Nelson, J. Virol. 72:5661-5668) have reported noncytopathic persistence of the virus in cultured aortic EC. We propose that interstrain differences in viral host cell tropism rather than the vascular bed of origin of infected EC might account for these discrepancies. In the present investigation we compared the responses of EC derived from human adult iliac artery, placental microvasculature, and umbilical vein to infection with various HCMV strains. Regardless of the vascular bed of origin, infection with EC-propagated HCMV strains induced 100% efficient cytopathic change progressing to complete lysis of inoculated monolayers. While fibroblast-propagated strains persisted at low titer in infected arterial EC cultures, they were also cytolytic for individual infected cells. The finding of cytopathic lytic infection of arterial EC by HCMV implicates a mechanism of vascular injury in the pathogenesis of HCMV infection.

Human cytomegalovirus infection of immature dendritic cells and macrophages.

A central aspect of human cytomegalovirus (HCMV) pathogenesis is the interaction of the virus with different antigen-presenting cell (APC) types of the host. In principle, a number of various cell types have the potential of antigen presentation when MHC II expression is induced by appropriate stimuli. The most potent antigen presenters are monocytes/macrophages and dendritic cells (DCs), therefore called professional APCs. Interestingly, these cells seem to be targets of productive HCMV infection. The susceptibility of the monocyte/macrophage system has been analyzed intensively during the past decade. Investigation of the role of DCs during HCMV infection, however, has begun only recently.

Human cytomegalovirus persistently infects aortic endothelial cells.

Endothelial cells (EC) have been implicated as constituting an important cell type in the pathogenesis of human cytomegalovirus (HCMV). Microvascular and macrovascular EC exhibit different biochemical and functional properties depending on the organ of origin. Phenotypic differences between microvascular and macrovascular EC may alter the ability of these cells to support HCMV replication. In this study, we compared the replication of HCMV in primary macrovascular aortic EC (AEC) with that in brain microvascular EC (BMVEC). An examination of IE72, pp65, and gB viral antigen expression in BMVEC and AEC by immunoflourescence revealed similar frequencies of infected cells. Intracellular production of virus was 3 log units greater in BMVEC than in AEC, while equal quantities of extracellular virus were produced in both cell types. HCMV infection of BMVEC resulted in rapid cellular lysis, while the virus was nonlytic and continuously released from HCMV-infected AEC for the life span of the culture. An examination of infected cells by electron microscopy revealed the formation of abundant nucleocapsids in both AEC and BMVEC. However, significant amounts of mature viral particles were only detected in the cytoplasm of BMVEC. These observations indicate that levels of HCMV replication in EC obtained from different organs are distinct and suggest that persistently infected AEC may serve as a reservoir of virus.

Morphological and functional characterization of an in vitro blood-brain barrier model.

Cell culture models have been extensively used for studies of blood-brain barrier (BBB) function. However, several in vitro models fail to reproduce some, if not most, of the physiological and morphological properties of in situ brain microvascular endothelial cells. We have recently developed a dynamic, tridimensional BBB model where endothelial cells exposed to intraluminal flow form a barrier to ions and proteins following prolonged co-culturing with glia. We have further characterized this cell culture model to determine whether these barrier properties were due to expression of a BBB phenotype. Endothelial cells of human, bovine or rodent origin were used. When co-cultured with glia, intraluminally grown endothelial cells developed features similar to in vivo endothelial cells, including tight junctional contacts at interdigitating processes and a high transendothelial resistance. This in vitro BBB was characterized by the expression of an abluminal, ouabain-sensitive Na/K pump, and thus favored passage of potassium ions towards the lumen while preventing K+ extravasation. Similarly, the in vitro BBB prevented the passage of blood-brain barrier-impermeant drugs (such as morphine, sucrose and mannitol) while allowing extraluminal accumulation of lipophylic substances such as theophylline. Finally, expression of stereo-selective transporters for Aspartate was revealed by tracer studies. We conclude that the in vitro dynamic BBB model may become an useful tool for the studies of BBB-function and for the testing of drug passage across the brain endothelial monolayer.