The mouse neutralization test in comparison with the rapid fluorescent focus inhibition test: differences in the results in rabies antibody determinations

Sixteen lots of rabies immune globulin (RIG) and six lots of rabies immune horse serum (RIS) from different producers were examined for rabies antibody by the standard mouse neutralization test (MNT) and the rapid fluorescent focus inhibition test (RFFIT). An equine rabies standard serum was assayed in parallel. In comparisons of RIS with this standard the MNT and RFFIT gave comparable results. In comparisons of RIG the antibody values in the MNT was two to ten times higher than that in the RFFIT in 15 out of 16 lots. The MNT and RFFIT are thus not fully comparable when measuring rabies antibodies in RIG. The choice of the titration method is obviously important in the measurement of the antibody concentration in RIG or RIS in IU against an equine rabies reference preparation. The described differences could have consequences for the use of RIG.     

Long-term effects of a multidisciplinary treatment of uncomplicated obesity on carotid intima-media thickness

Obesity is associated with well-known cardiovascular risk factors and a lower life expectancy. This study investigated whether nonoperative nutritional treatment of obesity without comorbidities influenced the carotid intima-media thickness (c-IMT) in the long run. Fifty-four subjects of an original cohort of 251 subjects were re-evaluated 10 years after a medical nutritional treatment (MNT) with cognitive-behavioral approach for uncomplicated obesity. Forty subjects were classified as failure (10-year body weight change > 0.5 kg) and 14 (body weight change ≤ 0.5 kg) as a success of the MNT. Ten years after MNT, c-IMT significantly increased (0.06 ± 0.02 mm; P = 0.004) in the failure group and significantly decreased (-0.07 ± 0.03 mm; P = 0.027) in the success group. Ten-year change in c-IMT correlated significantly with 10-year change in body weight (r = 0.28; P = 0.040). Multiple stepwise linear regression analysis demonstrated that age, final BMI, and group (success or failure) influenced independently the 10-year c-IMT. In conclusion, this study is in agreement with the possibility that the successful MNT of obesity may be an effective choice in the long run and seems to indicate that it may be able to reduce the cardiovascular risk as reflected by the change in c-IMT.     

Modular nanotransporters of anticancer drugs conferring cell specificity and higher efficiency

This review deals with artificial modular nanotransporters (MNT) of polypeptide nature for drug delivery into target cells and then into a specified cell compartment like the nucleus. The developed approach is based on the use of intra-cellular transport processes characteristic of practically all cells, including cancer cells. The first MNT module ligand carries out a double function: specific recognition of a cancer target cell and penetration into the cell via receptor-mediated endocytosis. The movement of the MNT within the cell along this path specifies the need to supply the MNT with an endosomolytic module making it possible to leave the endocytotic pathway before getting into lysosomes in order to have time for interaction with importins. For this purpose, a polypeptide fragment able to make defects in membranes only at the pH of endosomes is used as the second module. Delivery into the cell nucleus is provided by the third module containing an amino acid sequence of nuclear localization, “recognized” by importins located in the hyaloplasm. And finally, the fourth module, a carrier for joining the transported drug, is incorporated into the MNT. Depending on the type of ligand module, MNT for different target cell types have been produced. Each module retains its activity within the MNT, ligand modules bind target receptors with high affinity, while the module with the nuclear localization sequence binds importins. The endosomolytic module forms pores in lipid membranes through which MNT are able to leave acidifying cell compartments (endosomes). Modules within MNT can be replaced or transposed, which makes it possible to use them for delivery of different drugs into different target cells and their compartments. It was shown that photosensitizers and radionuclides used for cancer therapy acquire pronounced cell specificity as well as the 10-1000-fold higher efficiency resulting from their delivery into the most vulnerable compartment — the cell nucleus.     

Absence of histone H2B in nucleosomes containing histone TH2B and interaction of immunoglobulin with nucleosomes

Nucleosomes containing histone TH2B were isolated from chromatin subunits of rat testis nuclei (MNT) by incubating with anti-TH2B immunoglobulin (IgTH2B) which was covalently attached to agarose gels. Electrophoretic separation of histones of these isolated nucleosomes revealed that histone H2B was completely absent, suggesting that histone TH2B, the variant of H2B, existed in nucleosomes only as TH2B X TH2B and that TH2B X H2B was not likely to exist in chromatin. Sucrose gradient ultracentrifugation of mixtures of MNT and IgTH2B revealed that when excess amounts of immunologically active IgTH2B were present, complexes of higher sedimentation coefficients than MNT X IgTH2B were formed, but with limited amounts of active IgTH2B, only MNT X IgTH2B was formed. When purified IgTH2B was coated on polystyrene tubes and incubated with MNT, those MNT immobilized by the tube-coated IgTH2B adsorbed IgTH2B from diluted antiserum during subsequent incubation. Those results suggested the absence of steric hindrance in the binding of IgTH2B to MNT X IgTH2B. When MNT was coated on polystyrene tubes and incubated with DNase and then with dilute anti-TH2B antiserum, it was found that DNase digestion increased the binding of immunoglobulin to the tubes approximately 76%. Interaction of chromatin subunits of rat liver nuclei (MNL) with anti-TH2B antiserum was negligible, but DNase digestion of MNL coated on tubes was followed by considerable interaction with anti-TH2B antiserum. Those results indicated DNase unmasked at least part of the determinants encased by DNA. Anti-H2B immunoglobulin (IgH2B) interacted with histone H2B and TH2B to the same extent, and interacted significantly to a lesser extent with either MNT or MNL. DNase digestion of MNT and MNL increased binding of IgH2B approximately 170 and 117%, respectively.     

微核试验在中国的应用、发展与展望

微核试验是反映染色体损伤的重要试验方法,已广泛应用于遗传、食品、药物、环境等多领域的遗传毒性评估,以及作为有害职业和生活环境暴露人群遗传损害的生物标志物。我国开展微核试验已有二十余年的历史,本文通过检索《中国生物医学文献数据库》(1980~2000),共检索到有关微核的专题研究文献共1316篇,研究内容涉及微核的文献共2083篇。本文对微核的相关信息进行了分析,并结合作者自己的研究工作,回顾了微核试验在中国的应用和发展,总结了我国学者开展该项工作的优势与不足,对微核试验在我国的进一步发展进行了展望。     

Potency assay of antibodies against rabies. A report on a collaborative study

The relative potencies of a number of rabies immunoglobulin preparations were estimated in an international collaborative study comprising eight laboratories in five countries. Two assay methods were used: a virus neutralization test in mice (MNT) and a virus neutralization test in cell culture (RFFIT). Differences between the results obtained by the two methods, which have been reported, could not be generally corroborated. The results indicate that in some laboratories the MNT cause difficulties and give results different from those obtained by RFFIT. In other laboratories such difficulties are not encountered. The results seem to indicate that the RFFIT is a more reliable method than the MNT.     

Antitumor efficacy of Auger electron emitter <Superscript>111</Superscript>In delivered by modular nanotransporter into the nuclei of cells with folate receptor overexpression

A new modular nanotransporter (MNT) for the delivery of anticancer agents into the nuclei of cells with folate receptor overexpression was created. An effective method for acceding labeling of this MNT with Auger electron emitter ¹¹¹In has been developed. A significant therapeutic effect was observed after a single intratumoral injection of the new ¹¹¹In-labeled MNT to mice grafted with human cervical carcinoma characterized by folate receptor overexpression.     

Rabies neutralizing antibody detection by indirect immunperoxidase serum neutralization assay performed on chicken embryo related cell line

The aim of this study was to evaluate the indirect immunoperoxidase virus neutralization (IPVN) and mouse neutralization test (MNT) to detect antibodies against rabies virus from vaccinated dogs and cattle. The IPVN was set up for the ability to measure 0.5 International Units/ml (IU) of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. IPVN was developed and standardized in chicken embryo related (CER) cell line when 141 dog and 110 cattle sera were applied by serial five-fold dilutions (1:5, 1:25, 1:125) as well as the positive and negative reference controls, all added in four adjacent wells, of 96-well microplates. A 50 microl amount of CVS32 strain dilution containing 50-200 TCID50/ml was mixed to each serum dilution, and after 90 min 50 microl of 3 x 10(5) cells/mlcell suspension added to each well. After five days of incubation, the monolayers were fixed and the IPVN test performed. The correlation coefficient between the MNT and IPVN performed in CER cells was r = 0.9949 for dog sera (n = 100) and r = 0.9307 for cattle sera (n = 99), as well as good specificity (94.7%), sensitivity (87.5%), and agreement (96.6%) were also obtained. IPVN technique can adequately identify vaccinated and unvaccinated animals, even from low-responding vaccinated animals, with the advantage of low cost and faster then MNT standard test.     

Studies on Morphological and Physio-Ecological Variations of the Reniform Nematode,Rotylenchulus reniformis Linford and Oliveira, 1940 with an Emphasis on Differential Geographical Distribution of Amphimictic and Parthenogenetic Populations in Japan

The geographical distribution and polymorphism in morphological and biological characters of the reniform nematode, Rotylenchulus reniformis, in Japan were studied. The northern limit of habitat of this nematode was found on the 14 °C isothermal line of annual average-air temperature. Three morphologically different groups were ascertained which corresponded to three biological types based on male frequency. Incidence of males was consistent within populations and was not affected by environmental factors. Sexual attraction of males by females indicated reproductive isolation between the male-numerous type (MNT) and male-rare type (MRT) or male-absent type (MAT). Reproduction was amphimictic in the MNT and parthenogenetic in the MRT and MAT. Larval development in both MRT and MAT, but not that of MNT popula-tions, was inhibited at 34 °C. Differences in host preference were also observed among populations.     

Mnt1p and Mnt2p of Candida albicans are partially redundant alpha-1,2-mannosyltransferases that participate in O-linked mannosylation and are required for adhesion and virulence

The MNT1 gene of the human fungal pathogen Candida albicans is involved in O-glycosylation of cell wall and secreted proteins and is important for adherence of C. albicans to host surfaces and for virulence. Here we describe the molecular analysis of CaMNT2, a second member of the MNT1-like gene family in C. albicans. Mnt2p also functions in O-glycosylation. Mnt1p and Mnt2p encode partially redundant alpha-1,2-mannosyltransferases that catalyze the addition of the second and third mannose residues in an O-linked mannose pentamer. Deletion of both copies of MNT1 and MNT2 resulted in reduction in the level of in vitro mannosyltransferase activity and truncation of O-mannan. Both the mnt2Delta and mnt1Delta single mutants were significantly reduced in adherence to human buccal epithelial cells and Matrigel-coated surfaces, indicating a role for O-glycosylated cell wall proteins or O-mannan itself in adhesion to host surfaces. The double mnt1Deltamnt2Delta mutant formed aggregates of cells that appeared to be the result of abnormal cell separation. The double mutant was attenuated in virulence, underlining the importance of O-glycosylation in pathogenesis of C. albicans infections.     

Efficient isolation of human parainfluenza viruses 1 and 3 using MNT‐1, a human malignant melanoma cell line system that exhibits an apparent cytopathic effect

Isolation of human parainfluenza virus (HPIV) serotypes 1 and 3 from clinical specimens is not very efficient because of the lack of a cell culture system capable of inducing CPE. In this study, the utility of a melanoma cell line, MNT‐1, that allows HPIV growth and displays CPE was demonstrated. In particularly, the efficiency of isolating HPIV1 and HPIV3 using MNT‐1 was greater than for cell lines conventionally used for HPIV isolation. Our demonstrated efficacy of HPIV1 and HPIV3 isolation with apparent CPE using the MNT‐1 cell culture system has the potential to improve virus isolation from clinical specimens.     

Chemically induced micronucleus formation in V79 cells--comparison of three different test approaches

The in vitro micronucleus test (MNT) is a useful assay for the detection of mutagenic events on both the chromosomal and the genomic level. The main disadvantage for introducing the in vitro MNT into official test guidelines seems to be the disparity of existing protocols. To contribute to the aim of standardisation, three different methodological approaches of the in vitro MNT with V79 cells were compared: the standard assay using an asynchronically growing mixed cell population, the cytokinesis block (CB) assay and a modified MNT, the so-called mitotic shake-off (MSO) method. V79 cells were thus treated with two known aneugens (colcemide and griseofulvin) and two clastogens (mitomycin C and cyclophosphamide) over various time periods. The cultures of the CB assay were additionally exposed to cytochalasin B (Cyt-B), an inhibitor of cell, but not of nucleus division. After treatment, the cells were harvested and analysed for the appearance of micronuclei (MN). All three assays yielded positive results for all test substances. These results support the suitability of the MNT with V79 cells with regard to the ability to detect the genotoxic potential of both clastogens and aneugens independent of the test protocol applied. Thus, all three methods are appropriate for MN detection, but due to the fact that the application of Cyt-B has no advantages for a cell line like V79 in which nearly all cells undergo a normal cell cycle, its use is not recommended.     

Modular Nanotransporter with P21 Fragment Inhibits DNA Repair after Bleomycin Treatment

Modular nanotransporter (MNT) with C-terminal fragment of the p21 protein was synthesized and characterized, and its effect on DNA lesions was studied. This p21 fragment in MNT can significantly inhibit DNA repair in A431 human carcinoma cells after bleomycin treatment.     

破伤风类毒素抗体酶联双抗原夹心法定量检测试剂的研制及评价

研制破伤风类毒素抗体酶联双抗原夹心法定量检测试剂,用于破伤风免疫血浆抗体效价检测。以精制破伤风类毒素经Sephacryl S-300柱层析纯化后作为包被抗原,用辣根过氧化物酶以改良过碘酸钠法标记精制破伤风类毒素作为酶标记抗原,以破伤风人免疫球蛋白国家标准品采用小鼠中和试验法标定试剂盒定量标准品,制备双抗原夹心法定量检测试剂;进行试剂盒检测范围、特异性、重复性、精密度及稳定性考核,并与小鼠中和试验法、琼脂双扩散法及国外破伤风类毒素抗体酶联试剂盒进行比较。结果显示,试剂盒的检测范围为10~150mIU/ml,灵敏度为10mIU/ml,线性好(r>0.996),板内孔间变异度小(CV<8%),特异性强(100%),重复性好(CV<13%),于37℃放置6天测定结果无明显差异,与小鼠中和试验法、英国Biding Site酶联试剂有良好的一致性。试验证明所研制的试剂盒适用于破伤风免疫血浆中的破伤风抗体效价定量检测。     

Comparison between the counter immunoelectrophoresis test and mouse neutralization test for the detection of antibodies against rabies virus in dog sera

The detection of rabies antibodies is extremely valuable for epidemiological studies, determination of immune status in man, animals, and for the diagnosis of the disease. Several serological procedures have been described for this purpose. The present study reports a comparison between counterimmunoelectrophoresis test (CIET) and mouse neutralization test (MNT) in the detection of antibodies against rabies virus from 212 serum samples of vaccinated dogs. The agreement between both techniques was 79.7% and a significative association was demonstrated. The correlation coefficients between MNT and the CIET titers was determined considering 88 samples showing positive results in both techniques [CIET = 2 and MNT = 5 (0.13 IU/ml)] and resulted r2 = 0.7926 (p < 0.001). The performance of CIET system was technically simple, cheap and rapid, and thereby it could be useful for serological monitoring of dog vaccination campaigns as well as for individual analysis.     

Monoclonal antibodies against nucleoside triphosphate hydrolase-II can reduce the replication of Toxoplasma gondii

Nucleoside triphosphate hydrolase (NTPase) is an abundant protein secreted by the obligate protozoan parasite Toxoplasma gondii, which has a wide specificity toward NTP. In the present study, two monoclonal antibodies (mAbs, MNT1 and MNT2) against recombinant T. gondii NTPase-II (rTgNTPase-II) were developed. Western blot analysis displayed that these two mAbs can recognize specifically rTgNTPase-II as well as a 63 kDa molecule in tachyzoites soluble antigens that corresponded to native NTPase-II. T. gondii tachyzoites pretreated with two mAbs were observed under Confocal Laser Microscope and a specific reaction was displayed on tachyzoites after indirect fluorescence antibody test (IFAT). When COS-7 cells were co-cultured with tachyzoites pretreated with two mAbs, the number of intracellular parasites per infected cell was significantly decreased compared with the control. Furthermore, incubation of T. gondii tachyzoites with two mAbs can inhibit NTPase activity in the presence of dithiothreitol, which hinted that the reduction of tachyzoite replication might be owing to the inhibition of NTPase-II by the mAbs. The passive immunization test indicated that the transferred mAbs can significantly prolong the survival time of challenge infected mice. Taken together, we concluded that the mAbs against NTPase-II can reduce the replication of T. gondii and have a crucial effect on the protection of host from T. gondii infection.     

Nitric Oxide Implicates c-Fos Expression in the Cuneate Nucleus Following Electrical Stimulation of the Transected Median Nerve

In this study, we investigated whether nitric oxide (NO) modulated injury-induced neuropeptide Y (NPY) releasing and c-Fos expression in the cuneate nucleus (CN) after median nerve transection (MNT). We first examined the temporal changes of neuronal nitric oxide synthase (nNOS) expression in the dorsal root ganglion (DRG) and CN after MNT. Following MNT, the amounts of nNOS-like immunoreactive (nNOS-LI) neurons in the DRG and CN significantly increased as compared with those of the sham-operated rats. Furthermore, 4 weeks after MNT, the increases of nNOS-LI neurons in the DRG and CN were attenuated by pre-emptive lidocaine treatment in a dose-dependent manner. Finally, 4 weeks after MNT, pre-stimulation administration of L-NAME (N _ω-Nitro-l-arginine methyl ester) or 7-NI (7-nitroindazole) suppressed the amount of NPY release from the stimulated terminals and thus attenuated c-Fos expression in the CN. Our data implied that NO would modulate neuronal activity in the DRG and CN both after MNT.     

Knockdown of miR-210 decreases hypoxic glioma stem cells stemness and radioresistance

Glioma contains abundant hypoxic regions which provide niches to promote the maintenance and expansion of glioma stem cells (GSCs), which are resistant to conventional therapies and responsible for recurrence. Given the fact that miR-210 plays a vital role in cellular adaption to hypoxia and in stem cell survival and stemness maintenance, strategies correcting the aberrantly expressed miR-210 might open up a new therapeutic avenue to hypoxia GSCs. In the present study, to explore the possibility of miR-210 as an effective therapeutic target to hypoxic GSCs, we employed a lentiviral-mediated anti-sense miR-210 gene transfer technique to knockdown miR-210 expression and analyze phenotypic changes in hypoxic U87s and SHG44s cells. We found that hypoxia led to an increased HIF-2α mRNA expression and miR-210 expression in GSCs. Knockdown of miR-210 decreased neurosphere formation capacity, stem cell marker expression and cell viability, and induced differentiation and G0/G1 arrest in hypoxic GSCs by partially rescued Myc antagonist (MNT) protein expression. Knockdown of MNT could reverse the gene expression changes and the growth inhibition resulting from knockdown of miR-210 in hypoxic GSCs. Moreover, knockdown of miR-210 led to increased apoptotic rate and Caspase-3/7 activity and decreased invasive capacity, reactive oxygen species (ROS) and lactate production and radioresistance in hypoxic GSCs. These findings suggest that miR-210 might be a potential therapeutic target to eliminate GSCs located in hypoxic niches.     

Efficient isolation of human metapneumovirus using MNT‐1, a human malignant melanoma cell line with early and distinct cytopathic effects

Isolation of human metapneumovirus (HMPV) from clinical specimens is currently inefficient because of the lack of a cell culture system in which a distinct cytopathic effect (CPE) occurs. The cell lines LLC‐MK2, Vero and Vero E6 are used for isolation of HMPV; however, the CPE in these cell lines is subtle and usually requires a long observation period and sometimes blind passages. Thus, a cell line in which an early and distinct CPE occurs following HMPV inoculation is highly desired by clinical virology laboratories. In this study, it was demonstrated that, in the human malignant melanoma cell line MNT‐1, obvious syncytium formation occurs shortly after inoculation with HMPV‐positive clinical specimens. In addition, the growth and efficiency of isolation of HMPV were greater using MNT‐1 than using any other conventional cell line. Addition of this cell line to our routine viral isolation system for clinical specimens markedly enhanced isolation frequency, allowing isolation‐based surveillance. MNT‐1 has the potential to facilitate clinical and epidemiological studies of HMPV.