In vitro generation of influenza-specific polyfunctional CD4+ T cells suitable for adoptive immunotherapy

Background aimsInfluenza viruses cause potentially fatal respiratory infections in stem cell transplant patients. Specific T cells provide long-lived host adaptive immunity to influenza viruses, and the potential for generating such cells for clinical use was investigatedMethodsThe inactivated influenza vaccine (Fluvax) approved for human use was used as the antigen source. Monocyte-derived dendritic cells pulsed with Fluvax were used to stimulate autologous peripheral blood mononuclear cells (PBMC) on days 0 and 7. Cells were expanded with interleukin (IL)-2 from day 7 onwards. Cell numbers and phenotype were assessed on day 21. The presence of influenza virus-specific cells was assessed by cytokine production and proliferative responses following restimulation with influenza antigensResultsOver 21 days of culture, a mean fold increase of 26.3 in cell number was observed (n = 7). Cultures were predominantly effector and central memory CD4⁺ cells, and expressed a phenotype characteristic of activated antigen-specific cells capable of B-cell helper function. Cytotoxic CD4⁺ and CD8⁺ cells specific for influenza and a high percentage of CD4⁺ cells specific for each of three influenza viruses targeted by Fluvax (H1N1, H3N2 and Brisbane viruses) were generated. In addition, T cells expanded when restimulated with antigens derived from influenza viruses.ConclusionsWe have demonstrated a clinically usable method for producing influenza virus-specific T cells that yield high numbers of highly reactive CD4⁺ cells suitable for adoptive immunotherapy. We propose that reconstructing host immunity through adoptive transfer of influenza virus-specific T cells will reduce the frequency of influenza-related deaths in the period of severe immune suppression that follows stem cell transplantation.     

H5N1亚型禽流感病毒NS1基因截短体的克隆与原核表达

禽流感病毒H5N1 NS1蛋白是一种非结构蛋白,在病毒感染过程中发挥着重要的作用.构建基因截短的重组蛋白,可为进一步研究NS1不同结构域与宿主蛋白间的相互作用奠定基础.在成功克隆禽流感病毒H5N1全长NS1基因并测序的基础上,将部分截短基因序列克隆到表达栽体pET28a(+)上,构建基因截短的重组表达质粒pET28a-NS1-RBD和pET28a-NS1-ED,转化大肠埃希菌BL21(DE3),阳性重组质粒经IPTG诱导表达后进行SDS-PAGE检测,获得预期蛋白的表达,然后利用Ni-NTA树脂蛋白纯化系统对重组蛋白进行纯化,并通过Western Blotting进一步确认NS1及截短体蛋白的表达.结果表明,实验成功构建禽流感病毒H5N1亚型的NS1蛋白截短体,并在大肠埃希菌中高效表达,这为进一步研究NS1蛋白不同结构域与宿主蛋白的相互作用提供了实验材料,为深入研究NS1蛋白的生物学功能奠定了坚实基础.     

Products of Chlorophyll a Transformation by Selected Benthic Organisms in the Odra Estuary (Southern Baltic Sea)

The transformation of chlorophyll a by two benthic bivalves – Dreissena polymorpha and Mytilus edulis and, for comparison, by the chironomid Chironomus plumosus – was examined. D. polymorpha is a suspension-feeding, freshwater but very salinity-tolerant species, M. edulis a suspension-feeding marine species, and C.␣plumosus a typical detritivorous freshwater species. All three are common in the Odra Estuary. Specimens of the three species were collected from this area in the period 1999–2003. The bivalves were transferred to a compartment on land and kept in tanks filled with water (filtered and unfiltered) collected simultaneously with the organisms, under light and temperature conditions similar to those obtaining in their natural environment. Pigments in the water samples, faeces and pseudofaeces from the tanks, and in the gut content from C. plumosus, were determined with HPLC. The digestion products of the three species were mainly phaeophorbides a, generally thought to originate mainly from zooplankton grazing, and pyrophaeophytin a in minor quantities. The results indicate that the main chlorophyll a derivatives in faeces and pseudofaeces and gut content of the three species were the same as those in the sediments of the Baltic Sea, including the Odra Estuary. Our previous studies have shown that the sediments in this area are richer in phaeophorbides a than sediments from other parts of the southern Baltic Sea. All this suggests that the benthos may play an important role in the transformation of chlorophyll a in the Odra Estuary area.     

The activity profile of the NhaD-type Na+(Li+)/H+ antiporter from the soda Lake Haloalkaliphile Alkalimonas amylolytica is adaptive for the extreme environment

In extreme alkaliphiles, Na(+)/H(+) antiporters play a central role in the Na(+) cycle that supports pH homeostasis, Na(+) resistance, solute uptake, and motility. Properties of individual antiporters have only been examined in extremely alkaliphilic soil Bacillus spp., whereas the most alkaline natural habitats usually couple high pH with high salinity. Here, studies were conducted on a Na(+)(Li(+))/H(+) antiporter, NhaD, from the soda lake haloalkaliphile Alkalimonas amylolytica. The activity profile of A. amylolytica NhaD at different pH values and Na(+) concentrations reflects its unique natural habitat. In membrane vesicles from antiporter-deficient Escherichia coli EP432 (DeltanhaA DeltanhaB), the pH optimum for NhaD-dependent Na(+)(Li(+))/H(+) antiport was at least 9.5, the highest pH that could be tested; no activity was observed at pH < or =8.5. NhaD supported low Na(+)/H(+) antiport activity at pH 9.5 that was detectable over a range of Na(+) concentrations from 10 mM to at least 800 mM, with a 600 mM optimum. Although A. amylolytica nhaD was isolated by complementing the Li(+) sensitivity of the triple mutant E. coli strain KNabc (DeltanhaA DeltanhaB DeltachaA), sustained propagation of nhaD-bearing plasmids in this strain resulted in a glycine (Gly(327))-->serine mutation in a putative cytoplasmic loop of the mutant transporter. The altered activity profile of NhaD-G327S appears to be adaptive to the E. coli setting: a much higher activity than wild-type NhaD at Na(+) concentrations up to 200 mM but lower activity at 400 to 600 mM Na(+), with a pH optimum and minimal pH for activity lower than those of wild-type NhaD.     

Determination of baseline susceptibility to Cry1Ab protein for Asian corn borer (Lep., Crambidae)

Abstract: Although transgenic Bacillus thuringiensis (Bt) corn can provide a new tool for control of the Asian corn borer (ACB), Ostrinia furnacalis (Guenée), concern has been raised regarding the possibility of the target insect evolving resistance to the Bt protein under intensive selection pressure from Bt corn. Therefore, it is necessary to establish baseline data to enable detection of changes in susceptibility in field populations after prolonged exposure to Bt corn. Susceptibility to purified Cry1Ab protein from Bt was determined for 10 populations of ACB from the major corn‐growing regions of China, ranging geographically from Heilongjiang Province in the northeast to Shaanxi Province in the east‐central part. Neonate ACB were exposed to semi‐artificial diet incorporated with increasing Cry1Ab protein concentrations, and mortality and growth inhibition were evaluated after 7 days. The range of LC₅₀ (50% lethal concentration) among the populations was 0.10 to 0.81 μg/g (Cry1Ab protein/diet). Differences (P < 0.05) in susceptibility among the populations were significant. LC₅₀s generated from the Huanghuaihai Summer Corn Region were higher than those from the Spring Corn Regions. Bt was one of the significant natural biomortality factors of overwintering generation ACB. There was a significant correlation between percentage of the larvae infected with Bt and their LC₅₀ values to Cry1Ab protein in geographic distinct populations (r = 0.7350*, d.f. = 8, _r0.05 = 0.632). Based on the background of Bt formulations used for corn insect pests control in these areas, these differences were not caused by prior exposure to Bt insecticides. Instead, the small differences likely reflect natural Bt selection pressure. Because the variation in susceptibility to Cry1Ab was small (<10‐fold), the ACB apparently is susceptible to Cry1Ab across its range within China.     

Effect of insulin on exocrine pancreatic secretion in healthy and diabetic anaesthetised rats

This investigation characterised the effects of exogenous insulin on exocrine pancreatic secretion in anaesthetised healthy and diabetic rats. Animals were rendered diabetic by a single injection of streptozotocin (STZ, 60 mg kg(-1) I.P.). Age-matched controls were injected citrate buffer. Rats were tested for hyperglycaemia 4 days after STZ injection and 7-8 weeks later when they were used for the experiments. Following anaesthesia (1 g kg(-1) urethane I.P.), laparotomy was performed and the pancreatic duct cannulated for collection of pure pancreatic juice. Basal pancreatic juice flow rate in diabetic rats was significantly (p < 0.001) increased whereas protein and amylase outputs were significantly (p < 0.001) decreased compared to control rats. Insulin (1 IU, I.P.) produced in healthy rats significant increases in pancreatic flow rate, amylase secretion and protein output compared to basal (p < 0.05). Insulin action also included a reduction in blood glucose (152.7 +/- 16.9 mg dl(-1), n = 6, prior to insulin and 42.0 +/- 8.4 mg dl(-1), n = 4, 100 min later). In fact, flow rate and glycaemia showed a strong negative correlation (p < 0.01, Pearson). Pretreatment with atropine (0.2 mg kg(-1), I.V.) abolished the effects of insulin on secretory parameters despite a similar reduction in glycaemia; in this series of experiments the correlation between flow rate and blood glucose was lost. In diabetic rats, insulin (4 IU, I.P.) did not modify exocrine pancreatic secretion. There was a fall in blood glucose (467.6 +/- 14.0 mg dl(-1), n = 10, prior to insulin and 386.6 +/- 43.6 mg dl(-1), n = 7, 120 min later). Rats, however, did not become hypoglycaemic. Similar results were observed in diabetic atropinized rats. The results of this study indicate that the effects of insulin on exocrine pancreatic secretion in anaesthetised healthy rats are mediated by hypoglycaemia-evoked vagal cholinergic activation.     

白氏枝厉螨雄螨及成螨前期描述(蜱螨亚纲:革螨股:胭螨科)

描述白氏枝厉螨Dendrolaelaps baixuelii Ma,1977雄螨及成螨前期。     

Studies on Pollen Morphology and Taxonomy of Lespedeza and Its Allied Genera from NE China

The pollen morphology of 11 species and 1 variety in the genus Lespedeza and its allied genera (Campylotropis, Kummerowia) from NE China was examined under light and scanning electron microscopes. 1. Lespedeza Michx. (plate 1:1-6; 2:1-6; 3:1-6; 4:1-2) Pollen grains prolate, rarely subprolate or spheroidal, elliptic or rarely suborbicular in equatorial view, 3-lobed-rounded in polar view, tricolporate, colpus margins smooth or jagged. Polar axis 20.7-33.1μm long, equatorial axis 15.4-20.9 μm long. Exine reticulate or foveolate, lumina verrucose or smooth under SEM. 2. Campylotropis Bge. (plate 4:3-4) One species in NE China, C. macrocarpa (Bge.) Rehd. Pollen grains prolate, elliptic in equatorial view, 3-lobed-rounded in polar view, 3-colporate, colpus linear, 25.1μm long, 1.79μm broad, colpus margins jagged, with a series of verrucae equal in size along one side visible under SEM. Polar axis 19.7μm long, equatorial axis 14.6μm long. Exine reticulate, lumina nearly rounded, verrucose at periphery under SEM. 3. Kummerowia Schindl. (plate 4:5-6) Pollen grains spheroidal, oblate or prolate, elliptic in equatorial view, obtuse-triangular in polar view, tricolporate, colups linear, 25.1μm long, 2.01μm broad, colpus margins sinuate. Polar axis 24.7-27.9μm long, equatorial axis 19.7-26.6μm long. Exine reticulate or subreticulate, lumina nearly rounded, with verrocae visible under SEM. According to the pollen morphology of Lespedeza and its allied genera, the division of Lespedeza (s. lat.) into Lespedeza (S. str.), Campylotropis and Kummarowia by Schindler (1912) is reasonable. The subdivision of Lespedeza (s. str.) into Sect. Macrolespedeza and Sect. Lespedeza by many botanists, and the treatment of Lespedeze juncea (L. f.) Pers. var. inschanica Maxim. as an independent species (i.e. Lespedeza inschanics (Maxim). Schindl.) are also supported by the pollen morphology shown in the present work.