Efficacy of ozonated water against various food-related microorganisms.

The antimicrobial effects of ozonated water in a recirculating concurrent reactor were evaluated against four gram-positive and four gram-negative bacteria, two yeasts, and spores of Aspergillus niger. More than 5 log units each of Salmonella typhimurium and Escherichia coli cells were killed instantaneously in ozonated water with or without addition of 20 ppm of soluble starch (SS). In ozonated water, death rates among the gram-negative bacteria--S. typhimurium, E. coli, Pseudomonas aeruginosa, and Yersinia enterocolitica--were not significantly different (P > 0.05). Among gram-positive bacteria, Listeria monocytogenes was significantly P < 0.05) more sensitive than either Staphylococcus aureus or Enterococcus faecalis. In the presence of organic material, death rates of S. aureus compared with L. monocytogenes and E. coli compared with S. typhimurium in ozonated water were not significantly (P > 0.05) affected by SS addition but were significantly reduced (P < 0.05) by addition of 20 ppm of bovine serum albumin (BSA). More than 4.5 log units each of Candida albicans and Zygosaccharomyces bailii cells were killed instantaneously in ozonated water, whereas less than 1 log unit of Aspergillus niger spores was killed after a 5-min exposure. The average ozone output levels in the deionized water (0.188 mg/ml) or water with SS (0.198 mg/ml) did not differ significantly (P < 0.05) but were significantly lower in water containing BSA (0.149 mg/ml).     

Incorporation of the Tat cell‐penetrating peptide into nanofibers improves the respective antitumor immune response

A major challenge for the development of anticancer vaccines is the induction of a safe and effective immune response, particularly mediated by CD8+ T lymphocytes, in an adjuvant‐free manner. In this respect, we present a simple strategy to improve the specific CD8+ T cell responses using KFE8 nanofibers bearing a Class I (Kb)‐restricted peptide epitope (called E. nanofibers) without the use of adjuvant. We demonstrate that incorporation of Tat, a cell‐penetrating peptide (CPP) of the HIV transactivator protein, into E. nanofibers remarkably enhanced tumor‐specific CD8+ T cell responses. E. nanofibers containing 12.5% Tat peptide (E.Tat_12.5 nanofiber) increased antigen cross‐presentation by bone marrow‐derived dendritic cells as compared with E. nanofibers, or E. nanofibers containing 25 or 50% the Tat peptide. Uptake of KFE8.Tat_12.5 nanofibers by dendritic cells (DCs) was significantly increased compared with KFE8 nanofiber lacking Tat. Peritoneal and lymph node DCs of mice immunized with E.Tat_12.5 nanofibers exhibited increased presentation of the H2kb‐epitope (reminiscent for cross‐presentation) compared with DCs obtained from E. nanofiber vaccinated mice. Tetrameric and intracellular cytokine staining revealed that vaccination with E.Tat_12.5 triggered a robust and specific CD8+ T lymphocyte response, which was more pronounced than in mice vaccinated with E. nanofibers alone. Furthermore, E.Tat_12.5 nanofibers were more potent than E. nanofiber to induce antitumor immune response and tumor‐infiltrating IFN‐γ CD8 T lymphocyte. In terms of cancer vaccine development, we propose that harnessing the nanofiber‐based vaccine platform with incorporated Tat peptide could present a simple and promising strategy to induce highly effective antitumor immune response.     

Screening of the Open Source Malaria Box Reveals an Early Lead Compound for the Treatment of Alveolar Echinococcosis

The metacestode (larval) stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), a very severe and in many cases incurable disease. To date, benzimidazoles such as albendazole and mebendazole are the only approved chemotherapeutical treatment options. Benzimidazoles inhibit metacestode proliferation, but do not act parasiticidal. Thus, benzimidazoles have to be taken a lifelong, can cause adverse side effects such as hepatotoxicity, and are ineffective in some patients. We here describe a newly developed screening cascade for the evaluation of the in vitro efficacy of new compounds that includes assessment of parasiticidal activity. The Malaria Box from Medicines for Malaria Venture (MMV), comprised of 400 commercially available chemicals that show in vitro activity against Plasmodium falciparum, was repurposed. Primary screening was carried out at 10 μM by employing the previously described PGI assay, and resulted in the identification of 24 compounds that caused physical damage in metacestodes. Seven out of these 24 drugs were also active at 1 μM. Dose-response assays revealed that only 2 compounds, namely MMV665807 and MMV665794, exhibited an EC₅₀ value below 5 μM. Assessments using human foreskin fibroblasts and Reuber rat hepatoma cells showed that the salicylanilide MMV665807 was less toxic for these two mammalian cell lines than for metacestodes. The parasiticidal activity of MMV665807 was then confirmed using isolated germinal layer cell cultures as well as metacestode vesicles by employing viability assays, and its effect on metacestodes was morphologically evaluated by electron microscopy. However, both oral and intraperitoneal application of MMV665807 to mice experimentally infected with E. multilocularis metacestodes did not result in any reduction of the parasite load.     

Culture of Echinococcus multilocularis metacestodes: an alternative to animal use

This article reviews the use of an in vitro culture model for the maintenance and proliferation of Echinococcus multilocularis metacestodes and the formation of protoscoleces. This model has been used to identify and characterize parasite molecules involved in host-parasite interactions, and is a suitable tool to perform in vitro drug-screening assays. The development of a simple and easy-to-handle assay to determine the effects of drugs on parasite viability, without the need for time-consuming animal experimentation, has opened the way for larger-scale in vitro drug screening.     

A European perspective on Neospora caninum

Since the identification of Neospora caninum in 1984 as a parasite separate from Toxoplasma gondii by Bjerkas et al., and its subsequent characterization and classification in 1988 by Dubey and co-workers, this parasite has attracted increasing attention, primarily as an important causative agent of abortion in cattle and neuromuscular disease in dogs, but also as a complementary model system to T. gondii for investigating the basic biology of intracellular parasitism. During November 11-14, 1999, the COST 820 Annual meeting (Vaccines against coccidioses) took place in Interlaken, Switzerland. Almost half of the papers presented at that meeting were on N. caninum and neosporosis, reflecting the increasing awareness of the importance of this parasite on part of the scientific community in Europe. On the occasion of the meeting, participants in this COST Action involved in Neospora research in Europe were asked to participate in this invited review in order to document the growing interest in N. caninum and the disease it causes. Thus, this paper is a unique collection of contributions provided by several European experts in the field. It is comprised of 10 reviews or original papers on different aspects of Neospora research including epidemiology, immunology, application and development of serological tools, and molecular characterisation of the parasite currently carried out throughout Europe. In addition, two distinguished invited speakers from overseas (Milton McAllister and John Ellis) provided valuable contributions. This invited review demonstrates that the COST 820 Action has brought together scientists from all over Europe and other parts of the world, and has laid the basis for many fruitful collaborations. The studies described here will contribute in assessing the relevance of neosporosis as a potential risk factor not only for animals, but also for human health.     

556例多发伤患者的急诊救治临床分析

目的探讨多发伤患者的救治策略。方法回顾分析我科2000年1月至2008年5月急诊抢救的556例多发伤患者的临床资料。结果 16例患者经抢救无效死亡,死亡率2.88%;其余患者均经紧急抢救及行必要实验室检查,病情稳定,好转率达97.12%。平均抢救时间为(1.37±1.05)h。结论强化多发伤的急诊科早期救治,树立创伤急救"黄金1 h"观念,是提高多发伤患者生存率及降低死亡率的关键。     

Vaccination with microneme protein NcMIC4 increases mortality in mice inoculated with Neospora caninum

NcMIC4 is a Neospora caninum microneme protein that has been isolated and purified on the basis of its unique lactose-binding properties. We have shown that this protein binds to galactosyl residues of lactose; antibodies directed against NcMIC4 inhibit host cell interactions in vitro, thus making it a vaccine candidate. Because of this feature, NcMIC4 was first purified on a larger scale in its native, functionally active form using lactose-agarose affinity chromatography. Second, NcMIC4 was expressed in Escherichia coli as a histidine-tagged recombinant protein (recNcMIC4) and purified through Ni-affinity chromatography. Third, NcMIC4 cDNA was cloned into the mammalian pcDNA3.1 DNA vector and expression was confirmed upon transfection of Vero cells in vitro. For vaccination studies, we employed the murine cerebral infection model based on C57Bl/6 mice, employing experimental groups of 10 mice each. Two groups were injected intraperitoneally with purified native NcMIC4 and recNcMIC4, respectively, employing RIBI adjuvant. The third group was vaccinated intramuscularly with pcDNA-NcMIC4. Control groups included an infection control, an adjuvant control, and a pcDNA3.1 control group. Following 3 injections at 4-wk intervals, mice were challenged by i.p. inoculation of 2 x 10(6) N. caninum tachyzoites (Nc-1 isolate). During the course of parasite challenge (3 wk), mice from the 3 different test groups showed varying degrees of symptoms bearing a semblance to neosporosis, i.e., walking disorder, rounded back, apathy, and paralysis of the hind limbs. Control groups showed no symptoms at all. Most notably, vaccination with pcDNA-MIC4 proved antiprotective, with 60% of mice succumbing to infection within 3 wk, and all mice lacking a measurable anti-NcMIC4 IgG response. NcMIC4 in its native form elicited a substantial humoral IgG1 immune response and a reduction in cerebral parasite load compared to the controls, but 20% of mice succumbed to infection. Vaccination with recNcMIC4 also resulted in 20% of mice dying; however, in this group, cerebral parasite load was similar to the controls, and recNcMIC4 vaccination elicited a mixed IgG1/IgG2 response. In conclusion, vaccines based on NcMIC4, especially pcDNA-NcMIC4, render mice more susceptible to cerebral disease upon challenge with N. caninum tachyzoites.