Nested real-time PCR for hepatitis A detection.

AIMS: The hepatitis A virus (HAV) is one of the most important human foodborne pathogens causing a number of worldwide outbreaks each year. The detection of HAV in food samples remains a complex issue, because commonly used detection tools, such as conventional or even real-time PCR assays, are often unable to detect HAV with sufficient sensitivity. The aims of this study were to develop highly sensitive and specific nested real-time PCR (NRT-PCR)-based method for HAV detection in food and to compare it with currently available methods. METHODS AND RESULTS: By combining conventional PCR, nested PCR and real-time PCR techniques, we have developed a specific NRT-PCR assay for the detection of HAV. The procedure involves two consecutive PCRs, the first of which is performed as a conventional RT-PCR using primers specific for HAV 5' noncoding region. The second reaction involves a real-time PCR using a nested primer pair specific for the first PCR product and a TaqMan probe. CONCLUSIONS: We have developed a novel NRT-PCR method capable of detecting as little as 0.2 PFU of HAV, which is significantly more sensitive than any other PCR technique tested in our system. SIGNIFICANCE AND IMPACT OF THE STUDY: NRT-PCR provides a potentially useful method for detecting HAV at extremely low levels, as frequently found in food samples, and can be potentially adopted as a regulatory method to ensure food safety.

BAC-mediated transgenic expression of fluorescent autophagic protein Beclin 1 reveals a role for Beclin 1 in lymphocyte development.

Beclin 1/Atg6 is an essential component of the evolutionary conserved PtdIns(3)-kinase (Vps34) protein complex that regulates macroautophagy (autophagy) in eukaryotic cells and also interacts with antiapoptotic Bcl-2 family members, Bcl-2, and Bcl-x(L). To elucidate the physiological function of Beclin 1, we generated transgenic mice producing a green fluorescent Beclin 1 protein (Beclin 1-GFP) under Beclin 1 endogenous regulation. The beclin 1-GFP transgene is functional because it completely rescues early embryonic lethality in beclin 1-deficient mice. The transgenic mice appear normal, with undetected change in basal autophagy levels in different tissues, despite the additional expression of functional Beclin 1-GFP. Staining of Beclin 1-GFP shows mostly diffuse cytoplasmic distribution in various tissues. Detailed analysis of the transgene expression by flow cytometry reveals a Bcl-2-like biphasic expression pattern in developing T and B cells, as well as differential regulation of expression in mature versus immature thymocytes following in vitro stimulation. Moreover, thymocytes expressing high Beclin 1-GFP levels appear increasingly sensitive to glucocorticoid-induced apoptosis in vitro. Our results, therefore, support a role for Beclin 1 in lymphocyte development involving cross talk between autophagy and apoptosis.

Modulation of CD8+ T cell response to antigen by the levels of self MHC class I.

The response of H-Y-specific TCR-transgenic CD8(+) T cells to Ag is characterized by poor proliferation, cytolytic activity, and IFN-gamma secretion. IFN-gamma secretion, but not cytotoxic function, can be rescued by the B7.1 molecule, suggesting that costimulation can selectively enhance some, but not all, effector CD8(+) T cell responses. Although the H-Y epitope binds H-2D(b) relatively less well than some other epitopes, it can induce potent CTL responses in nontransgenic mice, suggesting that the observed poor responsiveness of transgenic CD8(+) T cells cannot be ascribed to the epitope itself. Previously reported reactivity of this TCR to H-2A(b) is also not the cause of the poor responsiveness of the H-Y-specific CD8(+) T cells, as H-Y-specific CD8(+) T cells obtained from genetic backgrounds lacking H-2A(b) also responded poorly. Rather, reducing the levels of H-2(b) class I molecules by breeding the mice to (C57BL/6 x B10.D2)F(1) or TAP1(+/-) backgrounds partially restored cytotoxic activity and enhanced proliferative responses. These findings demonstrate that the self MHC class I gene dosage may regulate the extent of CD8(+) T cell responsiveness to Ag.

Dual MHC class I and class II restriction of a single T cell receptor: distinct modes of tolerance induction by two classes of autoantigens.

In the final stages of thymic development, immature T cells undergo three distinct processes (positive selection, negative selection, and lineage commitment) that all depend on interactions of thymocyte TCRs with MHC molecules. It is currently thought that TCRs are preferentially restricted by either MHC class I or class II molecules. In this report, we present direct evidence that the TCR previously described as H-Y/H-2Db specific cross-reacts with H-2IAb if expressed in CD4+ cells. We also demonstrate an increase in thymocyte numbers in H-Y TCR-trangenic mice deficient in MHC class II, suggesting a relatively discrete form of negative selection by MHC class II compared with that induced by H-Y/H-2Db. We propose that inability to generate CD4+ T cells expressing H-Y TCR in different experimental settings may be due to tolerance to self-MHC class II. These results, therefore, support an intriguing possibility that tolerance to self may influence and/or interfere with the outcome of the lineage commitment.

Altered effector responses of H-Y transgenic CD8+ cells.

The primary role of CD8+ T cells is to destroy virus-infected or tumor cells expressing cognate antigens in the form of peptide-MHC class I complexes. This destruction is primarily achieved by the actions of lytic mediators and/or lymphokines. In this report, we show that mature, H-Y/Db-specific CD8+ T cells from H-Y TCR transgenic mice were unable to efficiently release lytic mediators after antigenic stimulation. However, anti-TCR antibody induced granule exocytosis and target cell lysis, arguing against signaling and/or cytolytic machinery defects in CD8+ cells, and demonstrating that male antigen induced differentiation of 'naive' into effector CD8+ cells. Stimulation of H-Y-specific effector CD8+ T cells with male stimulators, although insufficient to induce lytic granule release, was sufficient for H-Y-specific IFN-gamma production. Unexpectedly, this effector-phase IFN-gamma production was dependent on B7-2 engagement. We hypothesize that altered effector functions in H-Y-specific CD8+ cells are due to the low affinity of TCR-antigen-MHC interaction and/or the elevated threshold of CD8+ T cell activation.

Retronasal or internasal olfaction can mediate odor-guided behaviors in newborn mice.

Studies of olfactory deprivation have most frequently used unilateral naris occlusion to effect deprivation. Recent psychophysical evidence suggests that adult rodents with either acute or chronic naris occlusion show little decrement in olfactory ability. In this study the effect of naris occlusion coupled with ipsilateral or contralateral olfactory bulb deafferentation on odor-guided behaviors was tested in neonatal mice. Animals that received bilateral bulbectomy or control manipulation were also included. In Experiment 1, olfactory lesions were produced by bulb aspiration on the second day after birth (P2). Daily weight gain, a reliable measure of suckling success, was recorded until P21. In Experiment 2, olfactory lesions consisted of bulb transection on P2. Daily weights were recorded until subjects were P10. Animals with bilateral bulbectomy had the highest mortality rate and slowest growth rate. Both naris occlusion groups grew more slowly than controls but were not significantly different at P5 or P10. They diverged, thereafter, such that at P20 the group with naris occlusion ipsilateral to bulbectomy was similar to controls, while the contralateral group was similar to the bilateral bulbectomy group. In Experiment 2, the weights of the naris occlusion groups diverged by the day after surgery, with the contralateral group suffering the most arrested growth. Behavioral tests were combined from the two experiments for analysis. Subjects were tested at P5 for their ability to find the nipple, at P8 for their ability to find the nest, and at P10 for unwashed vs. washed nipple preferences. Only the contralateral group had a significantly depressed ability to find the nipple, while all lesions groups had a significant but similar decline in nest-finding ability.(ABSTRACT TRUNCATED AT 250 WORDS)

[Analysis of genetically determined susceptibility to experimental allergic encephalomyelitis in autoimmune diabetes mellitus in rats]. / Analiza naslednih faktora osetljivosti nazazivanje eksperimentalnog alergijskog encefalomijelitisa u autoimunskog dijabetesa melitusa kod pacova.

Back cross animals were obtained by mating DA rats, which are highly susceptible to the induction of experimental low-dose streptozotocin induced diabetes, and AO rats, which are resistant to both diseases. Susceptibility to the induction of EAE in AO and DA rats correlates with IL-2 production. after 7 cycles of mating and selection for DA-susceptibility to EAE, the number of EAE susceptible animals decreased, and animals were uniformly resistant to diabetes induction. IL-2 testing revealed lower IL-2 activities in the supernatants of lymphocytes from these animals in comparison to DA rats. Our results reveal the dissociation of genes involved in the regulation of the susceptibility to EAE and autoimmune diabetes. In addition, IL-2 testing, even though argues against the critical role of IL-2, does not preclude the necessity of this lymphokine in the pathogenesis of EAE.