The AMeX method: a multipurpose tissue-processing and paraffin-embedding method. III. Extraction and purification of RNA and application to slot-blot hybridization analysis.

RNA was extracted from tissues processed by a new fixation and paraffin-embedding method (the AMeX method) and examined by Northern blot analysis and slot-blot analysis. The RNA extraction method for AMeX-processed tissue sections after the deparaffinization step was the same as that for fresh materials. The total amount of cellular RNA extracted from AMeX-processed mouse liver tissue was slightly less than that extracted from fresh tissue. In tissues of malignant lymphoma, the total amount of cellular RNA extracted from 25 sections each 20 microns thick was about 1.6-1.8 micrograms/mm2, regardless of the histological subtype and period of storage. The extracted RNA was moderately degraded, and usually could not be used for Northern blot hybridization analysis. The intensity of ethidium bromide staining and the hybridization signals of RNA extracted from AMeX-processed tissues were usually reduced in comparison with RNA from fresh material, but specific signals could be detected by slot-blot hybridization analysis. We have demonstrated previously that the AMeX method preserves high-molecular-weight DNA and various antigens. Since the present study showed that information on mRNA can be obtained from AMeX-processed tissue, the versatility and usefulness of this method were further proven.     

Topoanatomical measurements of protein-bound radioactivity in the rat brain after intraventricular application of [14C]leucine

The pattern of distribution of protein-bound radioactivity was studied in rats 4 h after stereotaxic injection of [¹⁴C]leucine into the lateral ventricle. For quantitative estimation of the distribution of radioactivity, the semiconductographic method was used. The patterns revealed in this way were in general agreement with patterns obtained by the autoradiographic technique. The semiconductographic measurements indicated a high labelling, especially of some thalamic and hypothalamic nuclei. With the exception of the base of the telencephalon, the cortical structures were very poorly labelled. A steep gradient from the ventricular system to the brain tissue was observed. Very high labelling of blood vessels and leptomeningeal cells, especially in the vicinity of the optic chiasma, was observed.This quantitative analysis of the distribution pattern indicated a marked difference in uptake of the radiolabelled amino acid from the cerebrospinal fluid and calls for caution in the interpretation of experiments in which the relation between brain function and macromolecular synthesis is studied by the intraventricular injection of a radiolabelled precursor.A method is also described which makes it possible to determine the density distribution within a non-radioactive tissue slice in a simple way.     

Nucleic acid vaccines

There are no adequate vaccines against some of the new or reemerged infectious scourges such as HIV and TB. They may require strong and enduring cell-mediated immunity to be elicited. This is quite a task, as the only known basis of protection by current commercial vaccines is antibody. As DNA or RNA vaccines may induce both cell-mediated and humoral immunity, great interest has been shown in them. However, doubt remains whether their efficacy will suffice for their clinical realization. We look at the various tactics to increase the potency of nucleic acid vaccines and divided them broadly under those affecting delivery and those affecting immune induction. For delivery, we have considered ways of improving uptake and the use of bacterial, replicon or viral vectors. For immune induction, we considered aspects of immunostimulatory CpG motifs, coinjection of cytokines or costimulators and alterations of the antigen, its cellular localization and its anatomical localization including the use of ligand-targeting to lymphoid tissue. We also thought that mucosal application of DNA deserved a separate section. In this review, we have taken the liberty to discuss these enhancement methods, whenever possible, in the context of the underlying mechanisms that might argue for or against these strategies.     

IL-4–BATF signaling directly modulates IL-9 producing mucosal mast cell (MMC9) function in experimental food allergy

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Evaluation of a novel internally controlled real-time PCR assay targeting the 16S rRNA gene for confirmation of Neisseria gonorrhoeae infections.

A novel HybProbe real-time LightCycler PCR assay was developed for confirmation of Neisseria gonorrhoeae in samples positive according to the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae PCR assay. The new assay amplifies 375 bp of the N. gonorrhoeae 16S rRNA gene and includes an internal amplification control introduced during DNA purification. The assay had 100% specificity because of the high specificity of the HybProbes and primers. Other Neisseria spp. failed to generate positive crossing-point values and melting peaks. The analytical sensitivity for N. gonorrhoeae DNA was 0.5 fg/PCR, corresponding to 0.3 CFU/PCR. Sensitivity was not impaired in the presence of higher DNA concentrations (>or=1000-fold) from Neisseria spp. other than N. gonorrhoeae. The sensitivity was similar to that reported for the COBAS AMPLICOR assay with cervical swab samples. To assess its clinical applicability as a confirmatory test, 38 (2.9%) of 1313 swabs that were positive according to the COBAS AMPLICOR assay were tested using the new in-house assay and the commercially available GenFlow Neisseria test. Twenty-one samples negative according to COBAS AMPLICOR also underwent confirmatory testing. Both confirmatory tests yielded identical results; the 21 negative samples remained negative, and only 11 (28.9%) of the samples positive according to COBAS AMPLICOR were positive after retesting, suggesting a low prevalence (0.84%) of N. gonorrhoeae infection in the study population. These data suggest that the novel real-time PCR assay is an excellent and easy to interpret confirmatory test for the existing COBAS AMPLICOR assay for N. gonorrhoeae.     

Assessment of CMV load in solid organ transplant recipients by pp65 antigenemia and real-time quantitative DNA PCR assay: correlation with pp67 RNA detection

After bone marrow (BM) or solid-organ (SO) transplantation viremic Cytomegalovirus (CMV) infection is observed frequently. Quantitative assay of CMV in blood helps the management of this clinical condition. In the present report, 83 samples from 39 solid organ recipients, three CMV assays were compared simultaneously for the first time: the Nuclisens CMV pp67 assay (nucleic acid sequence-based amplification, NASBA), an "in-house" quantitative real-time PCR assay (TaqMan) for CMV DNA, and pp65 antigenemia. The relation between CMV DNA and pp65 antigenemia, the quantitative assays, was evaluated on a larger group including 251 blood samples from 118 solid organ recipients. Real-time PCR provided the best results; > or =130 CMV DNA copies/2 x 10(5) peripheral blood leukocytes (PBLs) predicted > or =1 pp65 antigen positive (Ag+) cell/2 x 10(5) PBLs. By taking pp65 antigenemia as the "gold standard," the sensitivity of CMV DNA quantitation and of the pp67 RNA assay were 0.95 and 0.20, respectively, while the corresponding specificity values were 0.50 and 0.93. When real-time PCR was considered as the "gold standard," the sensitivity and specificity of the pp65 antigenemia were 0.65 and 0.91, respectively. Among the three tests examined, the sensitivity of the pp67 RNA assay was the lowest. On the other hand, the pp67 RNA assay was highly specific and effective in pinpointing high viremia patients. The present report, by providing predictive values for all three diagnostic profiles, DNA load, antigenemia, and pp67RNA, is a contribution for validation of real-time PCR as a new standard for quantitative assessment of CMV viremia in clinical settings.     

The effect of a series of fixatives on the AgNOR technique

With increasing interest being shown in nucleolar organizer regions (NORs) in pathology, it was considered of great importance to evaluate the effect of some of the more commonly used and more specialized fixatives on the demonstration of these moieties. NORs can be demonstrated in paraffin sections by a silver technique (AgNOR method) which was developed from a method used by cytogeneticists for the demonstration of NORs in chromosome spreads. The degree of staining is dependent on the fixation regime employed and results may vary greatly from one fixative to another. The fixative schedules and post-treatments used in this study were based on standard sequences from the literature. We have shown that, in general, alcohol-based fixatives give optimal results, Carnoy's fluid being especially recommended. Mercurial and dichromate-containing fixatives were found to have highly detrimental effects on NOR staining. 'Routine' 10 per cent formol saline fixation gave adequate results whereas 10 per cent neutral buffered formalin gave optimal staining, similar to alcohol-based fixation.     

3'-Terminal RNA secondary structures are important for accumulation of tomato bushy stunt virus DI RNAs

The plus-strand RNA genome of tomato bushy stunt virus (TBSV) contains a 351-nucleotide (nt)-long 3'-untranslated region. We investigated the role of the 3'-proximal 130 nt of this sequence in viral RNA accumulation within the context of a TBSV defective interfering (DI) RNA. Sequence comparisons between different tombusviruses revealed that the 3' portion of the 130-nt sequence is highly conserved and deletion analysis confirmed that this segment is required for accumulation of DI RNAs in protoplasts. Computer-aided sequence analysis and in vitro solution structure probing indicated that the conserved sequence consists of three stem-loop (SL) structures (5'-SL3-SL2-SL1-3'). The existence of SLs 1 and 3 was also supported by comparative secondary structure analysis of sequenced tombusvirus genomes. Formation of the stem regions in all three SLs was found to be very important, and modification of the terminal loop sequences of SL1 and SL2, but not SL3, decreased DI RNA accumulation in vivo. For SL3, alterations to an internal loop resulted in significantly reduced DI RNA levels. Collectively, these data indicate that all three SLs are functionally relevant and contribute substantially to DI RNA accumulation. In addition, secondary structure analysis of other tombusvirus replicons and related virus genera revealed that a TBSV satellite RNA and members of the closely related genus Aureusvirus (family Tombusviridae) share fundamental elements of this general structural arrangement. Thus, this secondary structure model appears to extend beyond tombusvirus genomes. These conserved 3'-terminal RNA elements likely function in vivo by promoting and/or regulating minus-strand synthesis.     

丙型肝炎病毒感染的血清学检测

目的了解丙型肝炎病毒（ＨＣＶ）感染及病毒血症存在情况。方法用酶联免疫吸附试验（ＥＬＩＳＡ）和聚合酶链反应（ＰＣＲ）对不同人群的血清标本做了抗－ＨＣＶ、抗－ＨＣＶＩｇＭ和ＨＣＶＲＮＡ的检测，并对三项指标间的关系进行了对比分析。结果抗－ＨＣＶ在普通成年人、献血员、急性肝炎和肝硬化患者中的检出率分别是３５７％，８５８％，６２５％和４８３８％；与ＨＣＶＲＮＡ的符合率分别是１１４３％，６１１１％，８００％和７３３３％。相同人群抗－ＨＣＶＩｇＭ与ＨＣＶＲＮＡ的符合率分别是７５％，９０９％，８１８１％和１００％。结论抗－ＨＣＶＩｇＭ比抗－ＨＣＶ能更客观地反映ＨＣＶ病毒血症的情况，个别抗－ＨＣＶ阴性血清检测到了抗－ＨＣＶＩｇＭ和ＨＣＶＲＮＡ。