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.     

Aeroallergen-induced immediate asthmatic responses and late-phase associated pulmonary eosinophilia in the guinea pig: effect of methylprednisolone and mepyramine

Guinea pigs were sensitized by intraperitoneal injection of ovalbumin, 10 micrograms mixed with 100 mg A1(OH)3 in saline. On days 15-30 sensitized guinea pigs were challenged with ovalbumin aerosol (0.5 mg/ml, 30 s, 15 psi) which produced immediate asthmatic responses characterized by dyspnea, convulsions, and some deaths during the first 14 min. Twenty to 24 h later the animals were sacrificed with an overdose of pentobarbital, and lungs, bronchi, and lower trachea were dissected and fixed in 10% neutral buffered formalin. Histopathological examination of randomly coded tissues of the respiratory tract revealed a pulmonary eosinophilic cellular infiltrate in the epithelium/subepithelium of trachea, bronchi, and bronchioles as well as the peribronchial, peribronchiolar, and perivascular areas of the lungs. Oral administration of mepyramine (10 mg/kg) 2 h before aeroallergen challenge provided complete protection against immediate asthmatic responses and prevented deaths during the first 14 min without influencing the late phase associated lung eosinophilic cellular infiltrate. The immediate asthmatic responses were not influenced by methylprednisolone (30 mg/kg) administered orally 24 and 2 h before aeroallergen challenge. Following an additional dose of methylprednisolone 4 h after challenge, there was a significant inhibition of pulmonary eosinophilia (30 mg/kg; -24 h, -2 h, and +4 h). These observations suggest that histamine is the principal mediator of immediate asthma attacks in guinea pigs. Methylprednisolone may be acting by inhibiting the production of eosinophil chemotactic factor of anaphylaxis (platelet-activating factor or leukotriene B4) from the alveolar macrophages, T lymphocytes, and perhaps other cells, thus preventing pulmonary eosinophilia.     

Pregnancy following gamete intra-fallopian transfer (GIFT) with cryopreserved semen from infertile men following therapy to lymphomas or testicular tumour: report of three cases

Cryopreserved spermatozoa from men having treatment for lymphoma or testicular tumour have been reported to function sub-optimally. We report on three successful pregnancies following gamete intra-Fallopian transfer (GIFT) using such cryopreserved sperm samples. In one case, ovum donation was required since the woman had premature ovarian failure.     

Ceratobium mosaic potyvirus: another virus from orchids

Summary.  A potyvirus, which we call ceratobium mosaic virus, has been detected in about one third of more than 100 plants representing c. 33 orchid genera in two collections in Australia. It was detected using RT-PCR with redundant primers that are Potyviridae-specific and have additional sequences corresponding to either the SP6 or T7 bacteriophage promoters at their 5′-termini. Thus the nucleotide sequence of the resulting PCR fragments, consisting of about 1.7 kb of the 3′ portion of the viral genome, could be determined directly. Viral sequences obtained from five infected orchids indicate that they contained different isolates of a single potyvirus species most closely related to the bean common mosaic group of potyviruses, but clearly distinct from all whose virion protein genes have been reported to the international gene sequence databases. Accepted December 18, 1997 Received September 9, 1997     

Pulmonary cholesterol granulomas in patients with pulmonary artery hypertension: chest radiographic and CT findings

OBJECTIVE: We describe the chest radiographic and CT findings of pulmonary cholesterol granulomas in patients with pulmonary artery hypertension. CONCLUSION: Histopathologic evidence of cholesterol granulomas was found in five (25%) of 20 patients with severe pulmonary hypertension. In three of these five patients, the granulomas manifested on chest radiographs and CT as small centrilobular nodules mimicking the appearance of sarcoidosis, bronchiolitis, hypersensitivity pneumonitis, or aspiration.     

N-methyl D-aspartate induced mechanical allodynia is blocked by nitric oxide synthase and cyclooxygenase-2 inhibitors

The role of spinal NMDA receptors in mechanical nociceptive processing was assessed in sheep. Intrathecal NMDA (2 nmol-1 micromol) produced a significant reduction in mechanical withdrawal thresholds. This effect was attenuated by pretreatment with the NMDA receptor antagonist MK801 (100 nmol), the cyclooxygenase-2 (COX-2) inhibitor 5,5-dimethyl-3-(3-flourophenyl)-4-(4-methylsulphonyl)phenyl-2(5H) furanone DFU; 200 nmol) and the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 2 micromol), but not by the metabotropic glutamate receptor antagonist (S)-alpha-methyl-4-carboxyphenylglycine (MCPG; 200 nmol-2 micromol) or the non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX; 200 nmol-1 micromol). This first report of NMDA-induced mechanical allodynia suggests that spinal NMDA receptors are involved in mediating acute mechanical nociceptive processing through activation of NOS and COX-2 enzymes.