Development of a molecular-beacon assay to detect the G1896A precore mutation in hepatitis B virus-infected individuals

The 1896 precore (PC) mutation is the most frequent cause of hepatitis B virus e-antigen (HBeAg)-negative chronic hepatitis B virus (HBV) infection. Detection of the 1896 PC mutation has application in studies monitoring antiviral therapy and the natural history of the disease. Identification of this mutation is usually performed by direct sequencing, which is both costly and laborious. The aim of this study was to develop a rapid, high-throughput assay to detect the 1896 PC mutation using real-time PCR and molecular-beacon technology. The assay was initially standardized on oligonucleotide targets and plasmids containing the wild-type (WT) and PC mutation and then tested on plasma samples from children with HBV DNA of >10(6) copies/ml. Nine individuals were HBeAg negative and suspected to harbor HBeAg mutations, while 12 children were HBeAg positive and selected as controls. Ninety percent (19 of 21) of plasma samples tested with molecular beacons were in complete agreement with sequencing results. The remaining 10% (2 of 21) of samples were identified as heterogeneous mixtures of WT and mutant virus by molecular beacons, though sequencing found only a homogeneous mutant in both cases. Overall, the 1896 PC mutation was detected by this assay in 55.5% of the children with HBeAg-negative infection. In summary, this assay is a rapid, sensitive, and specific technique that effectively discriminates WT from 1896 PC mutant HBV and may be useful in clinical and epidemiological studies.     

Isolation and characterization of Mycoplasma sphenisci sp. nov. from the choana of an aquarium-reared jackass penguin (Spheniscus demersus)

Strain UCMJ was isolated from the choana of a jackass penguin (Spheniscus demersus) with recurrent mucocaseous choanal discharge. Isolation of this mycoplasma expands the known range of species hosting mycoplasmas. The name Mycoplasma sphenisci sp. nov. is proposed for this new species, for which strain UCMJ is the type strain.     

Chondromyxoid fibroma resembles in vitro chondrogenesis, but differs in expression of signalling molecules

Chondromyxoid fibroma is a rare benign cartilaginous bone tumour characterized by morphological features that resemble different steps of chondrogenesis in terms of both cellular morphology, ranging from spindled to rounded cells, and the extracellular matrix formed, which ranges from fibrous to cartilaginous. The presence in chondromyxoid fibroma of signalling molecules that regulate the spatial expression of proteins involved in normal cartilage proliferation and differentiation was investigated in samples from 20 patients and compared with articular chondrocytes from 11 normal donors cultivated in 3D pellet culture. Sections were stained with safranin-O and H&E, and immunohistochemistry was performed for p16, cyclin D1, FGFR3, BCL2, p21, PTHLH, PTHR1 and N-cadherin. Expression patterns were analysed using hierarchical clustering. In chondromyxoid fibroma, specific morphological features correlated with a distinct pattern of expression. Comparison with normal chondrocytes in pellet culture showed a striking morphological resemblance, but with an unmistakably different pattern of expression. N-cadherin, PTHLH, and PTHR1 were expressed to a significantly higher level (p < 0.01) in articular chondrocyte pellets but, conversely, there was significantly lower expression of cyclin D1, p16 and BCL2 (p < 0.05) in these cells. Morphological similarities reflect common steps in cartilage differentiation, albeit driven by different molecular mechanisms. The proteins we have found to be differentially expressed seem crucial for neoplastic chondrogenesis.     

Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis

A TaqMan-based real-time PCR qualitative assay for the detection of three species of malaria parasites-Plasmodium falciparum, P. ovale, and P. vivax-was devised and evaluated using 122 whole-blood samples from patients who had traveled to areas where malaria is endemic and who presented with malaria-like symptoms and fever. The assay was compared to conventional microscopy and to an established nested-PCR assay. The specificity of the new assay was confirmed by sequencing the PCR products from all the positive samples and by the lack of cross-reactivity with Toxoplasma gondii and Leishmania infantum DNA. Real-time PCR assay showed a detection limit (analytical sensitivity) of 0.7, 4, and 1.5 parasites/ micro l for P. falciparum, P. vivax, and P. ovale, respectively. Real-time PCR, like nested PCR, brought to light errors in the species identification by microscopic examination and revealed the presence of mixed infections (P. falciparum plus P. ovale). Real-time PCR can yield results within 2 h, does not require post-PCR processing, reduces sample handling, and minimizes the risks of contamination. The assay can therefore be easily implemented in routine diagnostic malaria tests. Future studies are warranted to investigate the clinical value of this technique.     

A2 adenosine receptors inhibit calcium influx through L-type calcium channels in rod photoreceptors of the salamander retina.

Presynaptic inhibition is a major mechanism for regulating synaptic transmission in the CNS and adenosine inhibits Ca(2+) currents (I(Ca)) to reduce transmitter release at several synapses. Rod photoreceptors possess L-type Ca(2+) channels that regulate the release of L-glutamate. In the retina, adenosine is released in the dark when L-glutamate release is maximal. We tested whether adenosine inhibits I(Ca) and intracellular Ca(2+) increases in rod photoreceptors in retinal slice and isolated cell preparations. Adenosine inhibited both I(Ca) and the [Ca(2+)]i increase evoked by depolarization in a dose-dependent manner with approximately 25% inhibition at 50 microM. An A2-selective agonist, (N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine) (DPMA), but not the A1- or A3-selective agonists, (R)-N(6)-(1-methyl-2-phenylethyl)adenosine and N(6)-2-(4-aminophenyl)ethyladenosine, also inhibited I(Ca) and depolarization-induced [Ca(2+)]i increases. An inhibitor of protein kinase A (PKA), Rp-cAMPS, blocked the effects of DPMA on both I(Ca) and the depolarization-evoked [Ca(2+)]i increase in rods. The results suggest that activation of A2 receptors stimulates PKA to inhibit L-type Ca(2+) channels in rods resulting in a decreased Ca(2+) influx that should suppress glutamate release.     

Outbreak of infection with hepatitis A virus (HAV) associated with a foodhandler and confirmed by sequence analysis reveals a new HAV genotype IB variant

An outbreak of infection with hepatitis A virus associated with a foodhandler and involving 26 subjects occurred in Southern Italy. Sequence analysis of the VP3-VP1 and VP1-P2A junctions confirmed that the outbreak was due to a point source and allowed the identification of a new genotype IB variant. This report confirms the usefulness of sequence-based molecular fingerprinting during outbreaks.     

Detection of clarithromycin-resistant Helicobacter pylori in stool samples

The recognition of the role of Helicobacter pylori in gastric diseases has led to the widespread use of antibiotics in the eradication of this pathogen. The most advocated therapy, triple therapy, often includes clarithromycin. It is well known that clarithromycin resistance is one of the major causes of eradication failure. The development of a rapid noninvasive technique that could easily be performed on fecal samples and that could also provide information about the antibiotic resistance of this microorganism is therefore advisable. Previous findings have demonstrated that clarithromycin resistance is due to a single point mutation in the 23S rRNA. All the mutations described have been associated with specific restriction sites, namely BsaI (A2143G), MboII (A2142C/G), and HhaI (T2717C). On this basis we have developed a new method, a seminested PCR, allowing screening for clarithromycin resistance of H. pylori directly on stool samples. This method furnished a 783-bp fragment of the 23S rRNA, which was subsequently digested by MboII, BsaI, and HhaI, in order to identify single point mutations associated with clarithromycin resistance. Of a total of 283 stool samples examined, 125 were H. pylori positive and two of them were shown to contain clarithromycin-resistant strains due to the presence of a mutation at position 2717, whereas no PCR products contained mutations at position 2142 or 2143. In order to evaluate the reliability of the new system, we compared the results of restriction analysis of the PCR products with the MICs shown by the H. pylori isolates by culturing gastric biopsies from the same patients.     

The Vi capsular antigen of Salmonella enterica serotype Typhi reduces Toll-like receptor-dependent interleukin-8 expression in the intestinal mucosa

Human infections with nontyphoidal Salmonella serotypes, such as S. enterica serotype Typhimurium, are characterized by a massive neutrophil influx in the colon and terminal ileum. In contrast, neutrophils are scarce in intestinal infiltrates of typhoid fever patients. Here, we show that in S. enterica serotype Typhi, the causative agent of typhoid fever, expression of the Vi capsular antigen reduced expression of the neutrophil chemoattractant interleukin-8 (IL-8) in host cells. Capsulated bacteria elicited IL-8 expression in polarized human epithelial cells (T84) and human macrophage-like cells (THP-1) in vitro at significantly reduced levels compared to noncapsulated bacteria. Experiments with a human cell line (HEK293) transfected with human Toll-like receptors (TLRs) demonstrated that in the presence of TLR5 or TLR4/MD2/CD14, a noncapsulated serotype Typhi mutant was able to induce the expression of IL-8, while this host response was significantly reduced when cells were infected with the capsulated serotype Typhi wild type. The relevance of these in vitro observations for the interaction of serotype Typhi with its human host was further studied ex vivo using human colonic tissue explants. Expression of IL-8 was detected in human colonic tissue explants infected with serotype Typhimurium or a noncapsulated serotype Typhi mutant. In contrast, infection with the serotype Typhi wild type did not elicit IL-8 expression in colonic tissue explants. Collectively, these data suggest that the scarcity of neutrophils in intestinal infiltrates of typhoid fever patients is due to a capsule-mediated reduction of TLR-dependent IL-8 production in the intestinal mucosa.     

Neural control of fast-regular saccades and antisaccades: an investigation using positron emission tomography

 Regional cerebral blood flow changes related to the performance of two oculomotor tasks and a central fixation task were compared in ten healthy human subjects. The tasks were: (a) performance of fast-regular saccades; (b) performance of voluntary antisaccades away from a peripheral cue; (c) passive maintenance of central visual fixation in the presence of irrelevant peripheral stimulation. The saccadic task was associated with a relative increase in activity in a number of occipitotemporal areas. Compared with both the fixation and the saccadic task, the performance of antisaccades activated a set of areas including: the superior and inferior parietal lobules, the precentral and prefrontal cortex, the cingulate cortex, and the supplementary motor area. The results of the present study suggest that: (a) compared with self-determined saccadic responses the performance of fast regular, reflexive saccades produces a limited activation of the frontal eye fields; (b) in the antisaccadic task the inferior parietal lobes subserve operations of sensory-motor integration dealing with attentional disengagement from the initial peripheral cue (appearing at an invalid spatial location) and with the recomputation of the antisaccadic vector on the basis of the wrong (e.g., spatially opposite) information provided by the same cue. Received: 20 May 1996 / Accepted: 28 January 1997     

Auditory brainstem response in premenopausal women taking oral contraceptives

BACKGROUND: The aim of this prospective study was to evaluate the effects of the new monophasic oral contraceptives on the audiological system in premenopausal women. METHODS: The auditory brainstem response (ABR) was measured in 94 women during the follicular, periovular and luteal phases of one menstrual cycle in which ovulation was confirmed using sonography and serum progesterone concentration. The latencies for waves I, III and V were determined, and the inter-peak intervals were calculated for waves I-III, I-V and III-V. All 94 women began taking oral contraceptives: 23 women used 20 microg ethinyl estradiol (EE) plus 150 microg desogestrel, 24 women used 30 microg EE plus 75 microg gestodene, and 47 women used 15 microg EE plus 60 microg gestodene. During the third month of contraceptive intake, each subject was again tested for ABR, as above. RESULTS: The wave latencies and inter-peak intervals showed shorter values during the periovular phase with respect to the luteal phase (P < 0.05), the follicular phase for wave I and for inter-peak interval I-V (P < 0.05) of the menstrual cycle. All of the ABR results in pill users were statistically different from those of the periovular phase (P < 0.05), though similar to those of both the luteal and follicular phases (P = NS). CONCLUSIONS: ABR seems to depend on the variations of ovarian steroids during the menstrual cycle and during oral contraceptive intake.