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.     

Inhibition of aeroallergen-induced bronchial eosinophilia by azelastine in guinea pigs.

The ability of azelastine to influence allergic bronchial eosinophil infiltration in guinea pigs was studied. Aeroallergen challenge of actively sensitized guinea pigs produces eosinophil infiltration in bronchoalveolar lavage fluid collected 20-24 h after aeroallergen exposure. Azelastine and methylprednisolone, administered orally 2 h before challenge, inhibited eosinophilic infiltration yielding the ED50s of 1.55 and 4.48 mg/kg, respectively. WEB-2086, a platelet-activating factor antagonist (3 mg/kg), and theophylline, a phosphodiesterase inhibitor (30 mg/kg), also suppressed allergic bronchial infiltration of eosinophils by 44%. The data obtained in this study demonstrate that azelastine exerts direct bronchial anti-inflammatory activity in guinea pigs.     

Enhancement of attention in man with ACTH/MSH 4–10

Normal men were infused for 4 hr with ACTH/MSH 4–10 or a control solution. Behavioral testing after the infusion indicated that subjects who received ACTH/MSH 4–10 were less anxious and had better visual memory than control subjects but the predominant effect of the heptapeptide was to increase visual attention. It was specualted that ACTH/MSH 4–10 may be uniquely coded for attentional functioning.     

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