Neutrophil phagocytosis is down-regulated by nucleotides until encounter with pathogens.

Extracellular nucleotides such as ATP, ADP, UTP, UDP and UDPG can trigger intracellular signal transduction via purinergic (P2Y) receptors, and their interaction induces a wide range of biological effects in various cells. In this study, we investigated P2Y expression and the effects of nucleotides on chemotaxis and phagocytosis in human neutrophils. RT-PCR detected broad expression of P2Y subfamilies in neutrophils, as well as monocytes. Moreover, intracellular Ca(2+) increased in response to ATP, ADP, UTP and UDP in these cells, suggesting that P2Y receptors were functionally expressed. In neutrophils, chemotactic activity was increased significantly in response to ATP and ADP, and moderately in response to UTP and UDP; actin polymerization by ATP, ADP, UTP and UDP was also evident in the cells. Interestingly, we found that ATP and ADP, which enhanced chemotaxis activity significantly, had inhibitory effects on phagocytosis by neutrophils. These findings provide new evidence for the regulation of neutrophil phagocytosis by nucleotides. Furthermore, this inhibitory effect was completely lost upon co-culture with fMLP or LPS, known constituents of bacteria, resulting in recovery of normal phagocytic activity. Taken together, these findings suggest that ATP and ADP constantly stimulate the chemotactic activity of neutrophils in peripheral blood, but may inhibit their phagocytic activity until they encounter pathogens, in order to prevent them acting against self-tissues or cells, as fMLP and LPS commonly present in pathogens would again trigger normal phagocytic activity.

Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt.

Highly pathogenic avian influenza A virus subtype H5N1 is currently widespread in Asia, Europe, and Africa, with 60% mortality in humans. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1 virus infection, with more than 50% of the cases worldwide, but the basis for this high incidence has not been elucidated. A change in receptor binding affinity of the viral hemagglutinin (HA) from α2,3- to α2,6-linked sialic acid (SA) is thought to be necessary for H5N1 virus to become pandemic. In this study, we conducted a phylogenetic analysis of H5N1 viruses isolated between 2006 and 2009 in Egypt. The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity. Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity. Recombinant H5N1 viruses with a single mutation at HA residue 192 or a double mutation at HA residues 129 and 151 had increased attachment to and infectivity in the human lower respiratory tract but not in the larynx. These findings correlated with enhanced virulence of the mutant viruses in mice. Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans. Our findings suggested that emergence of new H5 sublineages with α2,6 SA specificity caused a subsequent increase in human H5N1 influenza virus infections in Egypt, and provided data for understanding the virus's pandemic potential.

Development of two types of rapid diagnostic test kits to detect the hemagglutinin or nucleoprotein of the swine-origin pandemic influenza A virus H1N1.

Since its emergence in April 2009, pandemic influenza A virus H1N1 (H1N1 pdm), a new type of influenza A virus with a triple-reassortant genome, has spread throughout the world. Initial attempts to diagnose the infection in patients using immunochromatography (IC) relied on test kits developed for seasonal influenza A and B viruses, many of which proved significantly less sensitive to H1N1 pdm. Here, we prepared monoclonal antibodies that react with H1N1 pdm but not seasonal influenza A (H1N1 and H3N2) or B viruses. Using two of these antibodies, one recognizing viral hemagglutinin (HA) and the other recognizing nucleoprotein (NP), we developed kits for the specific detection of H1N1 pdm and tested them using clinical specimens of nasal wash fluid or nasopharyngeal fluid from patients with influenza-like illnesses. The specificities of both IC test kits were very high (93% for the HA kit, 100% for the NP kit). The test sensitivities for detection of H1N1 pdm were 85.5% with the anti-NP antibody, 49.4% with the anti-HA antibody, and 79.5% with a commercially available influenza A virus detection assay. Use of the anti-NP antibody could allow the rapid and accurate diagnosis of H1N1 pdm infections.