We quantitatively determined whether the selective phosphodiesterase (PDE) inhibitor, rolipram, inhibits changes in the adhesion molecules CD11b and L-selectin on platelet-activating factor (PAF)-stimulated human neutrophils and eosinophils in vitro.Incubations were performed in human whole blood obtained from healthy volunteers, to restrict activation by purification procedures and to simulate in vivo conditions, in which different cell types may interact, more closely. Receptor expression was measured after fixation of cells, using monoclonal antibodies and flow cytometry.Concentration-dependent inhibition of the PAF-induced CD11b expression and L-selectin shedding for neutrophils and eosinophils was observed with rolipram, dibutyryl cyclic adenosine monophosphate (cAMP), prostaglandin E 2 (PGE 2 ), and isoproterenol. However, these inhibitions did not exceed 50%. Preincubation with rolipram (10 -8 M) and subsequent incubation with isoproterenol (0.5×10 -8 M) or PGE 2 (10 -8 M) induced a cumulative, but not synergistic, effect. Using the combination of rolipram with isoproterenol or PGE 2 , inhibition of PAF-induced Lselectin shedding from eosinophils was as high as 71±28 and 67±21%, respectively. Other inhibitions were below 50%.In conclusion, rolipram inhibits CD11b expression and L-selectin shedding of platelet-activating factor-stimulated neutrophils and eosinophils in whole blood in a concentration-dependent fashion. Inhibitions did not exceed 50%, even at high concentrations. The inhibition of platelet-activating factor induced shedding of Lselectin from eosinophils with a combination of rolipram and prostaglandin E 2 or isoproterenol, however, was found to be approximately 70%. Inhibition of rolling adhesion of eosinophils may, therefore, be a mode of action of type IV phosphodiesterase inhibitors.

Rapid and reliable identification of bacterial pathogens directly from patient samples is required for optimizing antimicrobial therapy. Although Sanger sequencing of the 16S ribosomal RNA (rRNA) gene is used as a molecular method, species identification and discrimination is not always achievable for bacteria as their 16S rRNA genes have sometimes high sequence homology. Recently, next generation sequencing (NGS) of the 16S–23S rRNA encoding region has been proposed for reliable identification of pathogens directly from patient samples. However, data analysis is laborious and time-consuming and a database for the complete 16S–23S rRNA encoding region is not available. Therefore, a better, faster, and stronger approach is needed for NGS data analysis of the 16S–23S rRNA encoding region. We compared speed and diagnostic accuracy of different data analysis approaches: de novo assembly followed by Basic Local Alignment Search Tool (BLAST), operational taxonomic unit (OTU) clustering, or mapping using an in-house developed 16S–23S rRNA encoding region database for the identification of bacterial species. De novo assembly followed by BLAST using the in-house database was superior to the other methods, resulting in the shortest turnaround time (2 h and 5 min), approximately 2 h less than OTU clustering and 4.5 h less than mapping, and a sensitivity of 80%. Mapping was the slowest and most laborious data analysis approach with a sensitivity of 60%, whereas OTU clustering was the least laborious approach with 70% sensitivity. Although the in-house database requires more sequence entries to improve the sensitivity, the combination of de novo assembly and BLAST currently appears to be the optimal approach for data analysis.

Complement receptors on neutrophils and eosinophils play a role in activation and adhesion. During asthmatic reactions these receptors have been found elevated on circulating granulocytes. In the present study we compared the expression of CD35 (complement receptor type 1) and CD11b (complement receptor type 3) on neutrophils and eosinophils from asthmatic and non-asthmatic children. This was done in whole blood samples using depolarized light scattering for the discrimination of neutrophils and eosinophils. The non-stimulated expression as well as the upregulated expression of receptors by the chemotactic peptide N-formylmethionyl-leucyl-phenyl-alanine (fMLP) were studied. The results showed that without prior stimulation only the expression of CD35 on neutrophils was significantly elevated in children with asthma (P<0.05). After up-regulation with fMLP, the CD11b expression on neutrophils (P<0.005, fMLP: 0.002 microM) and eosinophils (P<0.05, fMLP: 0.02 microM) was significantly higher in asthmatic children than in the controls. These results indicate that the inducible expression of CD11b on neutrophils and eosinophils from allergic asthmatic children is primed in vivo.

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