Characterization of granulocytes and mast cells in cultures of mouse bone marrow stimulated with interleukin-3

Bone marrow cells in liquid culture with interleukin 3 produce a population of non-adherent granulocytes and mast cells. Flow cytometry was used to identify granulocytes and mast cells on the basis of the physical properties of perpendicular light scatter (PLS) and coulter volume (CV) as well as the expression lgE and CR3 receptors. Multicolor analysis indicated there were subpopulations of Thy 1.2 positive cells which transiently appeared in these cultures and also expressed lgE receptors, CR3 receptors or neither of these receptors. The data suggested a differentiation scheme in which Thy 1.2 positive precursor cells give rise to granulocytes and mast cells. Further evidence for this differentiation scheme was provided from CV vs. PLS distributions which showed increases in CV and PLS as Thy 1.2 positive cells differentiated into mast cells and decreases in CV and PLS as Thy 1.2 positive cells differentiated into granulocytes.     

Lipid Peroxidation of Lung Surfactant due to Reactive Oxygen Species Released from Phagocytes Stimulated by Bacteria from Children with Cystic Fibrosis

We used Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia, live or heat-killed, isolated from the airways of children with Cystic Fibrosis, to stimulate human neutrophils (PMN) and rat alveolar macrophages (AM) to produce reactive oxygen metabolites in the presence or absence of Curosurf, a natural porcine lung surfactant. We determined: (1) the amount of lipid peroxidation (LPO) as assessed by the amounts of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNE) using the LPO 586 test kit; (2) the production by AM of superoxide with the nitroblue tetrazolium test and (3) of nitric oxide (NO) with the Griess reaction. Stimulation of PMN or AM increases LPO of Curosurf and cell wall lipids. In both types of phagocytes, B. cepacia induced the highest LPO levels followed by P. aeruginosa and S. maltophilia. PMN, stimulated by live bacteria, induced higher LPO than those stimulated by heat-killed bacteria. B. cepacia stimulated AM to produce more superoxide and NO than did P. aeruginosa and S. maltophilia. The high phagocyte-stimulating ability of B. cepacia and its higher surfactant LPO than those of the other bacteria used in this in vitro study may play a role in vivo in the serious clinical condition known as the "Cepacia syndrome".     

Restoration action and species response: oviposition habits of Plebejus icarioides fenderi (Lepidoptera: Lycaenidae) across a restoration chronosequence in the Willamette Valley, Oregon, USA

Habitat restoration is critical to the conservation of rare species. However, restoration efforts often proceed without knowledge of their effects on these species. We investigated the reproductive response of federally endangered Fender’s blue butterfly (Plebejus icarioides fenderi) to prairie restoration in Willamette Valley, Oregon, USA. In 2009 and 2010, we quantified availability of larval host plant, Kincaid’s lupine (Lupinus oreganus), and butterfly oviposition in three restored areas (1–10 years old) and adjacent intact habitat. Oviposition measures in restored areas reached or exceeded intact habitat (0.1 eggs/leaf of host plant and 12 % of time ovipositing) within five years post-restoration. However, none of the restorations provided an equivalent host plant density to intact areas (55 leaves/m²). The different response time of host plants and butterflies to restoration highlights the importance of monitoring both vegetation and oviposition over an ecologically relevant timescale (at least 10 years). For imperiled species, quantifying reproductive response to restoration is critical for adaptive management and successful conservation of the species which restoration efforts are intended to benefit.     

RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis

Background

To elucidate further the pathogenesis of sporadic, idiopathic pulmonary arterial hypertension (IPAH) and identify potential therapeutic avenues, differential gene expression in IPAH was examined by suppression subtractive hybridisation (SSH).

Methods

Peripheral lung samples were obtained immediately after removal from patients undergoing lung transplant for IPAH without familial disease, and control tissues consisted of similarly sampled pieces of donor lungs not utilised during transplantation. Pools of lung mRNA from IPAH cases containing plexiform lesions and normal donor lungs were used to generate the tester and driver cDNA libraries, respectively. A subtracted IPAH cDNA library was made by SSH. Clones isolated from this subtracted library were examined for up regulated expression in IPAH using dot blot arrays of positive colony PCR products using both pooled cDNA libraries as probes. Clones verified as being upregulated were sequenced. For two genes the increase in expression was verified by northern blotting and data analysed using Student's unpaired two-tailed t-test.

Results

We present preliminary findings concerning candidate genes upregulated in IPAH. Twenty-seven upregulated genes were identified out of 192 clones examined. Upregulation in individual cases of IPAH was shown by northern blot for tissue inhibitor of metalloproteinase-3 and decorin (P < 0.01) compared with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase.

Conclusion

Four of the up regulated genes, magic roundabout, hevin, thrombomodulin and sucrose non-fermenting protein-related kinase-1 are expressed specifically by endothelial cells and one, muscleblind-1, by muscle cells, suggesting that they may be associated with plexiform lesions and hypertrophic arterial wall remodelling, respectively.     

Autoantibodies against the replication protein A complex in systemic lupus erythematosus and other autoimmune diseases

Replication protein A (RPA), a heterotrimer with subunits of molecular masses 70, 32, and 14 kDa, is a single-stranded-DNA-binding factor involved in DNA replication, repair, and recombination. There have been only three reported cases of anti-RPA in systemic lupus erythematosus (SLE) and Sjögren syndrome (SjS). This study sought to clarify the clinical significance of autoantibodies against RPA. Sera from 1,119 patients enrolled during the period 2000 to 2005 were screened by immunoprecipitation (IP) of ³⁵S-labeled K562 cell extract. Antigen-capture ELISA with anti-RPA32 mAb, immunofluorescent antinuclear antibodies (ANA) and western blot analysis with purified RPA were also performed. Our results show that nine sera immunoprecipitated the RPA70–RPA32–RPA14 complex and all were strongly positive by ELISA (titers 1:62,500 to 1:312,500). No additional sera were positive by ELISA and subsequently confirmed by IP or western blotting. All sera showed fine speckled/homogeneous nuclear staining. Anti-RPA was found in 1.4% (4/276) of SLE and 2.5% (1/40) of SjS sera, but not in rheumatoid arthritis (0/35), systemic sclerosis (0/47), or polymyositis/dermatomyositis (0/43). Eight of nine patients were female and there was no racial predilection. Other positive patients had interstitial lung disease, autoimmune thyroiditis/hepatitis C virus/pernicious anemia, or an unknown diagnosis. Autoantibody specificities found in up to 40% of SLE and other diseases, such as anti-nRNP, anti-Sm, anti-Ro, and anti-La, were unusual in anti-RPA-positive sera. Only one of nine had anti-Ro, and zero of nine had anti-nRNP, anti-Sm, anti-La, or anti-ribosomal P antibodies. In summary, high titers of anti-RPA antibodies were found in nine patients (1.4% of SLE and other diseases). Other autoantibodies found in SLE were rare in this subset, suggesting that patients with anti-RPA may form a unique clinical and immunological subset.     

Why UV vision and red vision are important for damselfish (Pomacentridae): structural and expression variation in opsin genes

Coral reefs belong to the most diverse ecosystems on our planet. The diversity in coloration and lifestyles of coral reef fishes makes them a particularly promising system to study the role of visual communication and adaptation. Here, we investigated the evolution of visual pigment genes (opsins) in damselfish (Pomacentridae) and examined whether structural and expression variation of opsins can be linked to ecology. Using DNA sequence data of a phylogenetically representative set of 31 damselfish species, we show that all but one visual opsin are evolving under positive selection. In addition, selection on opsin tuning sites, including cases of divergent, parallel, convergent and reversed evolution, has been strong throughout the radiation of damselfish, emphasizing the importance of visual tuning for this group. The highest functional variation in opsin protein sequences was observed in the short‐ followed by the long‐wavelength end of the visual spectrum. Comparative gene expression analyses of a subset of the same species revealed that with SWS1, RH2B and RH2A always being expressed, damselfish use an overall short‐wavelength shifted expression profile. Interestingly, not only did all species express SWS1 – a UV‐sensitive opsin – and possess UV‐transmitting lenses, most species also feature UV‐reflective body parts. This suggests that damsels might benefit from a close‐range UV‐based ‘private’ communication channel, which is likely to be hidden from ‘UV‐blind’ predators. Finally, we found that LWS expression is highly correlated to feeding strategy in damsels with herbivorous feeders having an increased LWS expression, possibly enhancing the detection of benthic algae.