Phosphoenolpyruvate in the rejuvenation of stored red cells in SAGM medium: optimal conditions and the indirect effect of methemoglobin formation

Red cells stored in saline-adenine-glucose-mannitol (SAGM) medium were rejuvenated by incubation with phosphoenolpyruvate (PEP) under conditions that can be achieved easily in ordinary blood banking. Regeneration of 2,3 diphosphoglycerate (2,3 DPG) and adenine nucleotides of stored red cells was dependent on the pH of the incubation medium and the incubation time. In red cells stored for 3 and 5 weeks, the optimal pH and incubation time for regeneration of 2,3 DPG and adenine nucleotides were 5.8 and 90 minutes and 6.1 and 60 minutes, respectively. During the incubation of red cells with PEP, methemoglobin was formed; it increased when the medium pH was below 6.0 and the incubation time exceeded 60 minutes. We conclude that incubation at a medium pH of 6.1 for 60 minutes is optimal for the rejuvenation of stored red cells with PEP. Under such incubation conditions, the concentrations of 2,3 DPG and adenine nucleotides in red cells stored for 5 weeks were restored to normal without methemoglobin formation.     

Identification and characterization of an endogenous chemotactic ligand specific for FPRL2

Chemotaxis of dendritic cells (DCs) and monocytes is a key step in the initiation of an adequate immune response. Formyl peptide receptor (FPR) and FPR-like receptor (FPRL)1, two G protein-coupled receptors belonging to the FPR family, play an essential role in host defense mechanisms against bacterial infection and in the regulation of inflammatory reactions. FPRL2, the third member of this structural family of chemoattractant receptors, is characterized by its specific expression on monocytes and DCs. Here, we present the isolation from a spleen extract and the functional characterization of F2L, a novel chemoattractant peptide acting specifically through FPRL2. F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein, an intracellular tetrapyrolle-binding protein. The peptide binds and activates FPRL2 in the low nanomolar range, which triggers intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases through the G(i) class of heterotrimeric G proteins. When tested on monocytes and monocyte-derived DCs, F2L promotes calcium mobilization and chemotaxis. Therefore, F2L appears as a new natural chemoattractant peptide for DCs and monocytes, and the first potent and specific agonist of FPRL2.     

Susceptibility of Encephalitozoon cuniculi to several drugs in vitro.

In the light of the increased incidence of human Encephalitozoon infections and the absence of an established treatment protocol, a simple in vitro testing method to compare activities of drugs against Encephalitozoon cuniculi was developed. With this in vitro method, the 50% inhibitory concentrations of fumagillin, thiabendazole, albendazole, oxibendazole, and propamidine isethionate for E. cuniculi in rabbit kidney cells were determined. Itraconazole, toltrazuril, metronidazole, ronidazole, and ganciclovir were ineffective in this testing system.     

Antimalarial and toxic effects of the acyclic nucleoside phosphonate (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine in Plasmodium berghei-infected mice.

Plasmodium berghei-infected mice died with low levels of parasitemia after repeated intraperitoneal administration (five times at 15 mg kg of body weight-1 every other day) of the in vitro active antimalarial acyclic nucleoside phosphonate (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA]. Toxicological studies showed that the main cause of death resulted from (S)-HPMPA-induced nephrotoxicity. Although concomitant intraperitoneal administration of the tubular epithelium transport blocker probenecid prevented (S)-HPMPA-induced toxicity, mice eventually died with a high level of parasitemia, despite repeated administration of high doses of (S)-HPMPA. The short half-life of (S)-HPMPA in plasma combined with the insusceptibility of the nonreplicative stages of the parasite to (S)-HPMPA could explain this failure to eradicate all parasites. Indeed, a low but sustained (calculated) level of 200 nM (S)-HPMPA in plasma completely cured P. berghei-infected mice. However, these mice, which received a total dose of only 28 mg kg-1 administered via osmotic pumps for 7 days, died because of the toxicity of the drug. These findings indicate that nephrotoxicity hinders the use of (S)-HPMPA as a drug against blood stage parasites. An alternative application of (S)-HPMPA as a potent prophylactic drug is discussed.     

Simple, fast, and accurate fluorometric method to determine drug susceptibility of Plasmodium falciparum in 24-well suspension cultures.

An in vitro test which quantifies drug inhibition of Plasmodium falciparum replication by measuring the fluorescence intensity of Hoechst 33258 dye bound to DNA is described. The procedure does not require expensive reagents or equipment and can be completed in less than 10 min. The assay was highly accurate and sensitive: cultures with as few as 0.4% schizont-infected erythrocytes could reliably be analyzed. The method was not biased by the actual parasite stage used; i.e., the amount of fluorescence detected in a sample of a culture of mature schizonts equaled the amount detected with the ring form culture derived from these schizonts. Even the presence of large proportions of free merozoites, which are easily neglected in microscopic estimates, did not bias the results. Furthermore, measurement of the chloroquine susceptibility of the multidrug-resistant K1 strain and the chloroquine-susceptible NF54 strain showed that the method is most suitable for quantifying the drug resistance of P. falciparum.     

有氧运动延缓衰老线粒体DNA突变的机制

目的:线粒体DNA突变在衰老过程中起核心作用。总结有氧运动在延缓机体衰老过程中对线粒体DNA突变的影响,探讨有氧运动延缓衰老的机制。资料来源:应用计算机检索Medline数据库1996-01/2006-01期间关于线粒体DNA突变与有氧运动延缓衰老的文章,检索词为"aerobics exercise,aging,mtDNA mutation",限定文章语言种类为English。同时计算机检索中国期刊全文数据库、万方数据库1994-01/2006-12期间的有氧运动、衰老和线粒体DNA突变相关的文章,检索词"衰老,线粒体DNA突变,有氧运动",并限定文章语言种类为中文。资料选择:对资料进行初审,纳入标准:①有氧运动延缓衰老关系的理论研究。②线粒体DNA突变与衰老关系的基础与临床研究。③有氧运动对线粒体DNA突变的影响研究。资料提炼:共收集到36篇线粒体DNA突变与有氧运动延缓衰老相关的文献,均为全文,32篇符合纳入标准,排除4篇重复性研究。资料综合:①有氧运动可以通过减少线粒体DNA突变,从而达到延缓机体的衰老。有氧运动可能通过影响自由基及抗氧化系统、细胞凋亡、线粒体的结构和功能,对衰老进程产生重要影响。②线粒体DNA突变随增龄而积累,达到一定阈值后,可导致细胞能量供应的严重障碍,从而造成组织器官生理功能的减退。③长期有氧运动可通过刺激心肌、骨骼肌线粒体的生成及蛋白质的合成而延缓线粒体形态结构的改变,有利于维持线粒体功能以满足机体对其能源的需求。结论:中、低强度有氧运动可以提高机体有氧工作能力,增进线粒体氧化磷酸化的功能,对延缓衰老具有一定的积极作用。有氧运动可以减少心肌、骨骼肌等线粒体DNA突变,提示有氧运动在延缓衰老机制中,减少线粒体DNA突变是可能机制之一。     

大鼠眼外肌成肌细胞的增殖及分化特征

目的:完善眼外肌成肌细胞体外培养、鉴定的方法及观察其生物学特性。方法:实验于2005-02/08在青岛大学医学院附院中心实验室(省级实验室)完成。取出生后3～7d的大鼠,通过大鼠眼外肌细胞的取材、分离、消化、培养等技术,观察细胞的形态、生长曲线、细胞融合率,观察大鼠眼外肌卫星细胞的增殖与分化能力,利用成肌细胞标记物α-横纹肌肌动蛋白、中间丝结蛋白免疫细胞化学染色对所获得的细胞进行鉴定。结果:①成肌细胞的生长情况:在生长培养基作用下,细胞增殖旺盛;在分化培养基条件下,细胞分化良好,可融合成肌管。②成肌细胞融合率:在24h和48h融合率提高明显,72h达高峰,之后不再变化。③细胞免疫化学检测结果:α-横纹肌肌动蛋白和中间丝结蛋白免疫细胞化学染色阳性。结论:体外培养的大鼠眼外肌卫星细胞具有良好的增殖与分化能力,其生物学特征同骨骼肌卫星细胞。     

Augmentation of spontaneous macrophage-mediated cytolysis by eosinophil peroxidase

Eosinophil peroxidase (EPO), a cationic protein purified from horse blood, adhered to four different types of tumor cells, markedly potentiating their lysis by preformed or enzymatically generated H(2)0(2) (up to 76-fold, as assayed in serum-containing tissue culture medium without supplemental halide). Similarly, compared with uncoated tumor cells, EPO-coated tumor cells were up to 32 times more sensitive to lysis when incubated with macrophages or granulocytes whose respiratory burst was triggered by PMA. However, EPO-coated tumor cells were also readily lysed by bacillus Calmette- Guerin-activated macrophages in the absence of exogenous triggering agents. This spontaneous cytolysis was rapid (50 percent at 2 h) and potent (50 percent lysis at macrophage/tumor cell ratios of 1.5 to 4.6), and was observed with both a peroxide-sensitive tumor (TLX9) and a peroxide-resistant tumor (NK lymphoma). Under the conditions used, neither EPO alone nor macrophages alone were spontaneously cytolytic. Neither EPO nor EPO-coated tumor cells triggered a detectable increment in H(2)0(2) release from macrophages. Nonetheless, spontaneous macrophage-mediated cytolysis of EPO- coated tumor cells was completely inhibitable by catalase (50 percent inhibition, 23 U/ml), although not by heated catalase, indicating a requirement for H(2)0(2). Cytolysis was also completely inhibitable by azide (50 percent inhibition, 2.6 X 10 -5 M), indicating a requirement for enzymatic activity of EPO. Thus, a cytophilic peroxidase from eosinophils and H(2)0(2) spontaneously released from activated macrophages interacted synergistically in a physiologic medium to destroy tumor cells.     

Septic arthritis caused by Legionella dumoffii in a patient with systemic lupus erythematosus-like disease

We describe a patient with systemic lupus erythematosus (SLE)-like disease on immunosuppressive treatment who developed septic arthritis of the knee involving Legionella dumoffii. Cultures initially remained negative. A broad-range 16S PCR using synovial fluid revealed L. dumoffii rRNA genes, a finding that was subsequently confirmed by positive Legionella culture results.     

Ticks and associated pathogens collected from domestic animals in the Netherlands

Following an outbreak of autochthonous canine babesiosis in the Netherlands, a request made to veterinarians and the public to collect ticks from companion animals resulted in 4298 ticks submitted between July 2005 and October 2006 to our center. Ticks were identified as Ixodes ricinus adults (2907/4298, 67.6%), Ixodes sp. nymphs (529/4298, 12.3%) and Ixodes sp. larvae (385/4298, 9.0%), I. hexagonus adults (328/4298, 7.6%), Dermacentor reticulatus (72/4298, 1.7%), and several other exotic tick species such as Amblyomma flavomaculatum (formerly Aponomma flavomaculatum), Hyalomma marginatum rufipes, Rhipicephalus sanguineus, and R. turanicus (55/4298, 1.3%). Eight localities were surveyed for the presence of local D. reticulatus, a tick not indigenous to the Netherlands, based on multiple submissions of D. reticulatus ticks from these areas. D. reticulatus was collected from the vegetation in six of these localities, confirming the presence of populations of this tick in the Netherlands. Adult I. ricinus (n=251), I. hexagonus (n=237), and D. reticulatus (n=344) ticks were selected at random and subsequently screened by polymerase chain reaction (PCR) and reverse line blot (RLB) hybridization for the presence of Borrelia, Babesia, Theileria, Anaplasma, Ehrlichia, and Rickettsia species. I. ricinus ticks were infected with Rickettsia helvetica (24.7%), spirochetes belonging to the Borrelia burgdorferi sensu lato group (7.2%), the Ehrlichia-like "Schotii" variant (2.4%), Anaplasma phagocytophilum (1.6%), Babesia sp. (EU1) (1.2%), Babesia divergens (0.4%), and Babesia microti (0.4%). A. phagocytophilum (5.9%) and R. helvetica (0.8%) were also detected in adult I. hexagonus ticks. Spotted fever group Rickettsiae, previously reported as Rickettsia sp. DnS14/RpA4 (14.0%), and Borrelia burgdorferi sensu lato (0.3%) were detected in the D. reticulatus ticks, which appeared to be free from B. canis infection. We concluded that a much broader spectrum of ticks and tick-borne pathogens is present in the Netherlands than previously thought, including several potential zoonotic pathogens.