Key role of regulated upon activation normal T-cell expressed and secreted, nonstructural protein1 and myeloperoxidase in cytokine storm induced by influenza virus PR-8 (A/H1N1) infection in A549 bronchial epithelial cells

Influenza virus infection causes severe respiratory disease such as that due to avian influenza (H5N1). Influenza A viruses proliferate in human epithelial cells, which produce inflammatory cytokines/chemokines as a "cytokine storm" attenuated with the viral nonstructural protein 1 (NS1). Cytokine/chemokine production in A549 epithelial cells infected with influenza A/H1N1 virus (PR-8) or nonstructural protein 1 (NS1) plasmid was examined in vitro. Because tumor necrosis factor-α (TNF-α) and regulated upon activation normal T-cell expressed and secreted (RANTES) are predominantly produced from cells infected with PR-8 virus, the effects of mRNA knockdown of these cytokines were investigated. Small interfering (si)TNF-α down-regulated RANTES expression and secretion of RANTES, interleukin (IL)-8, and monocyte chemotactic protein-1 (MCP-1). In addition, siRANTES suppressed interferon (IFN)-γ expression and secretion of RANTES, IL-8, and MCP-1, suggesting that TNF-α stimulates production of RANTES, IL-8, MCP-1, and IFN-γ, and RANTES also increased IL-8, MCP-1, and IFN-γ. Furthermore, administration of TNF-α promoted increased secretion of RANTES, IL-8, and MCP-1. Administration of RANTES enhanced IL-6, IL-8, and MCP-1 production without PR-8 infection. These results strongly suggest that, as an initial step, TNF-α regulates RANTES production, followed by increase of IL-6, IL-8, and MCP-1 and IFNs concentrations. At a later stage, cells transfected with viral NS1 plasmid showed production of a large amount of IL-8 and MCP-1 in the presence of the H(2)O(2)-myeloperoxidse (MPO) system, suggesting that NS1 of PR-8 may induce a "cytokine storm" from epithelial cells in the presence of an H(2)O(2)-MPO system.     

The role of mannose-binding lectin-associated serine protease-3 in activation of the alternative complement pathway

Mannose-binding lectin (MBL)-associated serine proteases (MASPs) are responsible for activation of the lectin complement pathway. Three types of MASPs (MASP-1, MASP-2, and MASP-3) are complexed with MBL and ficolins in serum. Although MASP-1 and MASP-2 are known to contribute to complement activation, the function of MASP-3 remains unclear. In this study, we investigated the mechanism of MASP-3 activation and its substrate using the recombinant mouse MASP-3 (rMASP-3) and several different types of MASP-deficient mice. A proenzyme rMASP-3 was obtained that was not autoactivated during preparation. The recombinant enzyme was activated by incubation with Staphylococcus aureus in the presence of MBL-A, but not MBL-C. In vivo studies revealed the phagocytic activities of MASP-1/3-deficient mice and all MASPs (MASP-1/2/3)-deficient mice against S. aureus and bacterial clearance in these mice were lower than those in wild-type and MASP-2-deficient mice. Sera from all MASPs-deficient mice showed significantly lower C3 deposition activity on the bacteria compared with that of wild-type serum, and addition of rMASP-3 to the deficient serum restored C3 deposition. The low C3 deposition in sera from all MASPs-deficient mice was probably caused by the low level factor B activation that was ameliorated by the addition of rMASP-3. Furthermore, rMASP-3 directly activated factors B and D in vitro. These results suggested that MASP-3 complexed with MBL is converted to an active form by incubation with bacterial targets, and that activated MASP-3 triggered the initial activation step of the alternative complement pathway.     

Identification of pathogenic dematiaceous fungi and related taxa based on large subunit ribosomal DNA D1/D2 domain sequence analysis

The nucleotide sequences of the D1/D2 domains of large subunit (26S) ribosomal DNA for 76 strains of 46 species of pathogenic dematiaceous fungi and related taxa were determined. Intra-species sequence diversity of medically important dematiaceous fungi including Phialophora verrucosa, Fonsecaea pedrosoi, Fonsecaea compacta, Cladophialophora carrionii, Cladophialophora bantiana, Exophiala dermatitidis, Exophiala jeanselmei, Exophiala spinifera, Exophiala moniliae, and Hortaea werneckii were extremely small; as few as 0 changes were detected in C. bantiana, Fonsecaea and Exophiala species, 1 bp in C. carrionii and H. werneckii, and 2 bp in P. verrucosa. Inter-species nucleotide diversity between most species was higher. These data suggested that the D1/D2 domain is sufficiently variable for identification of pathogenic dematiaceous fungi and relevant species. The phylogenetic trees constructed from the sequence data revealed that most human pathogenic species formed a single cluster and that Cladosporium and Phialophora species were distributed polyphyletically into several clusters.     

Tbx Genes Specify Posterior Digit Identity through Shh and BMP Signaling

Despite extensive studies on the anterior-posterior (AP) axis formation of limb buds, mechanisms that specify digit identities along the AP axis remain obscure. Using the four-digit chick leg as a model, we report here that Tbx2 and Tbx3 specify the digit identities of digits IV and III, respectively. Misexpression of Tbx2 and Tbx3 induced posterior homeotic transformation of digit III to digit IV and digit II to digit III, respectively. Conversely, misexpression of their mutants VP16 Delta Tbx2 and VP16 Delta Tbx3 induced anterior transformation. In both cases, alterations in the expression of several markers (e.g., BMP2, Shh, and HoxD genes) were observed. In addition, Tbx2 and Tbx3 rescued Noggin-mediated inhibition of interdigital BMP signaling, signaling which is pivotal in establishing digit identities. Hence, we conclude that Tbx3 specifies digit III, and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade.     

The Tomato spotted wilt virus cell‐to‐cell movement protein (NSM) triggers a hypersensitive response in Sw‐5‐containing resistant tomato lines and in Nicotiana benthamiana transformed with the functional Sw‐5b resistance gene copy

Although the Sw‐5 gene cluster has been cloned, and Sw‐5b has been identified as the functional gene copy that confers resistance to Tomato spotted wilt virus (TSWV), its avirulence (Avr) determinant has not been identified to date. Nicotiana tabacum ‘SR1‘ plants transformed with a copy of the Sw‐5b gene are immune without producing a clear visual response on challenge with TSWV, whereas it is shown here that N. benthamiana transformed with Sw‐5b gives a rapid and conspicuous hypersensitive response (HR). Using these plants, from all structural and non‐structural TSWV proteins tested, the TSWV cell‐to‐cell movement protein (NS_M) was confirmed as the Avr determinant using a Potato virus X (PVX) replicon or a non‐replicative pEAQ‐HT expression vector system. HR was induced in Sw‐5b‐transgenic N. benthamiana as well as in resistant near‐isogenic tomato lines after agroinfiltration with a functional cell‐to‐cell movement protein (NS_M) from a resistance‐inducing (RI) TSWV strain (BR‐01), but not with NS_M from a Sw‐5 resistance‐breaking (RB) strain (GRAU). This is the first biological demonstration that Sw‐5‐mediated resistance is triggered by the TSWV NS_M cell‐to‐cell movement protein.     

Approaches for enhancement of N2 fixation efficiency of chickpea (Cicer arietinum L.) under limiting nitrogen conditions

Chickpea (Cicer arietinum) is an important pulse crop in many countries in the world. The symbioses between chickpea and Mesorhizobia, which fix N₂ inside the root nodules, are of particular importance for chickpea's productivity. With the aim of enhancing symbiotic efficiency in chickpea, we compared the symbiotic efficiency of C‐15, Ch‐191 and CP‐36 strains of Mesorhizobium ciceri in association with the local elite chickpea cultivar ‘Bivanij’ as well as studied the mechanism underlying the improvement of N₂ fixation efficiency. Our data revealed that C‐15 strain manifested the most efficient N₂ fixation in comparison with Ch‐191 or CP‐36. This finding was supported by higher plant productivity and expression levels of the nifHDK genes in C‐15 nodules. Nodule specific activity was significantly higher in C‐15 combination, partially as a result of higher electron allocation to N₂ versus H⁺. Interestingly, a striking difference in nodule carbon and nitrogen composition was observed. Sucrose cleavage enzymes displayed comparatively lower activity in nodules established by either Ch‐191 or CP‐36. Organic acid formation, particularly that of malate, was remarkably higher in nodules induced by C‐15 strain. As a result, the best symbiotic efficiency observed with C‐15‐induced nodules was reflected in a higher concentration of the total and several major amino metabolites, namely asparagine, glutamine, glutamate and aspartate. Collectively, our findings demonstrated that the improved efficiency in chickpea symbiotic system, established with C‐15, was associated with the enhanced capacity of organic acid formation and the activities of the key enzymes connected to the nodule carbon and nitrogen metabolism.     

Stereoscopic Analysis of Optic Nerve Head Parameters in Primary Open Angle Glaucoma: The Glaucoma Stereo Analysis Study

PurposeThe Glaucoma Stereo Analysis Study (GSAS), a cross sectional multicenter collaborative study, used a stereo fundus camera to assess various morphological parameters of the optic nerve head (ONH) in glaucoma patients and investigated the relationships between these parameters and patient characteristics.ResultsPatient characteristics included refractive error of −3.38±3.75 diopters, intraocular pressure (IOP) of 13.6±2.6 mmHg, and visual field mean deviation (MD) of −4.71±3.26 dB. Representative ONH parameters included a horizontal disc width of 1.66±0.28 mm, vertical disc width of 1.86±0.23 mm, disc area of 2.42±0.63 mm², cup area of 1.45±0.57 mm², and cup volume of 0.31±0.22 mm³. Correlation analysis revealed significant negative associations between vertical cup-to-disc ratio (0.82±0.08) and MD (r = −0.40, P<0.01) and between disc tilt angle (10.5±12.5 degrees) and refractive error (r = −0.36, P<0.01). Seventy-five percent of the eyes had a positive value for rim decentering (0.30±0.42), indicating that rim thinning manifested more often as an inferior lesion than a superior lesion.ConclusionWe used stereoscopic analysis to establish a database of ONH parameters, which may facilitate future studies of glaucomatous changes in ONH morphology.     

Monocytes Infiltrate the Pancreas via the MCP-1/CCR2 Pathway and Differentiate into Stellate Cells

Recent studies have shown that monocytes possess pluripotent plasticity. We previously reported that monocytes could differentiate into hepatic stellate cells. Although stellate cells are also present in the pancreas, their origin remains unclear. An accumulation of enhanced green fluorescent protein (EGFP)⁺CD45^– cells was observed in the pancreases and livers of chimeric mice, which were transplanted with a single hematopoietic stem cell isolated from EGFP-transgenic mice and treated with carbon tetrachloride (CCl₄). Because the vast majority of EGFP⁺CD45^– cells in the pancreas expressed stellate cell-associated antigens such as vimentin, desmin, glial fibrillary acidic protein, procollagen-I, and α-smooth muscle actin, they were characterized as pancreatic stellate cells (PaSCs). EGFP⁺ PaSCs were also observed in CCl₄-treated mice adoptively transferred with monocytes but not with other cell lineages isolated from EGFP-transgenic mice. The expression of monocyte chemoattractant protein-1 (MCP-1) and angiotensin II (Ang II) increased in the pancreas of CCl₄-treated mice and their respective receptors, C-C chemokine receptor 2 (CCR2) and Ang II type 1 receptor (AT1R), were expressed on Ly6C^high monocytes isolated from EGFP-transgenic mice. We examined the effect of an AT1R antagonist, irbesartan, which is also a CCR2 antagonist, on the migration of monocytes into the pancreas. Monocytes migrated toward MCP-1 but not Ang II in vitro. Irbesartan inhibited not only their in vitro chemotaxis but also in vivo migration of adoptively transferred monocytes from peripheral blood into the pancreas. Irbesartan treatment significantly reduced the numbers of EGFP⁺F4/80⁺CCR2⁺ monocytic cells and EGFP⁺ PaSCs in the pancreas of CCl₄-treated chimeric mice receiving EGFP⁺ bone marrow cells. A specific CCR2 antagonist RS504393 inhibited the occurrence of EGFP⁺ PaSCs in injured mice. We propose that CCR2⁺ monocytes migrate into the pancreas possibly via the MCP-1/CCR2 pathway and give rise to PaSCs.