Partial zona dissection of human oocytes when failure of zona pellucida penetration is anticipated

Partial zona dissection (PZD) of human oocytes facilitates spermpenetration through mechanically made holes in the zona pellucida.Only 1 of 69 eggs was damaged when sucrose was used to shrinkthe ooplasm during micromanipulation. The fertilization rateof micromanipulated oocytes in 18 couples with male factor infertilitywas 68% (34/50), which compared favourably with inseminationof non-micromanipulated controls (21/45, 47%). PZD was advantageousin oligozoospermic patients, but not in cases of asthenozoospermia,combined semen problems or immunological infertility. Threetwin and two singleton pregnancies resulted following replacementof 23 micromanipulated and eight control embryos in 14 patients.No differences in embryo morphology and development rates werefound between the micromanipulated and control groups. The incidenceof polyspermy in couples with abnormal semen analyses was relativelylow (<20%) possibly due to partial activation of the oocytesfollowing exposure to sucrose. Polyspermy was high (57%) innormozoospermic patients with either immunological infertility(n= 3) or failure of fertilization in previous cycles (n= 4).In the three immunological patients, nine of 11 hyaluronidaseand sucrose-exposed control embryos fertilized and six implanted,possibly indicating that cumulus and corona cells are contributingfactors inhibiting fertilization in such cases.     

Development and application of an in vitro model for screening anti-hepatitis B virus therapeutics.

The development of effective anti-hepatitis B virus agents has been hampered by the lack of reliable in vitro systems for the screening of new therapeutics. In an effort to circumvent this problem, we have developed an in vitro system for screening anti-hepatitis B virus drugs using hepatitis B virus DNA-transfected Hep G2 cells. The cell line designated 2.2.15 produces replicative viral DNA intermediates, mature Dane particles and high levels of viral antigens. Subconfluent 2.2.15 cells were treated with a variety of commonly used anti-hepatitis B virus therapeutics, and their efficacy was determined by analyzing changes in the replicative cellular or extracellular hepatitis B virus DNA content by Southern blotting or slot-blot hybridization. The slot-blot method was sensitive, reproducible and rapid and correlated well with Southern blotting. Analysis of the media for hepatitis B virus DNA was indicative of changes in intracellular, replicative hepatitis B virus DNA, permitting sampling of the media. Therefore 2.2.15 cells may provide a valuable method for identifying and monitoring effective anti-hepatitis B virus therapeutics. Using this system to test various agents, we confirm that 2'-deoxyguanosine strongly inhibited viral replication, whereas others tested were less effective. Correlation with in vivo systems is now needed.     

Anti-Abeta: The good, the bad, and the unforeseen

Alzheimer's disease (AD) is characterized in part by the deposition of amyloid beta protein (Abeta) in compact fibrillar plaques. These structures can induce an innate immune response in the brain, which triggers progressive inflammation, neuronal loss, and further acceleration of Abeta plaque formation. Compared with the case in normal individuals, the T and B lymphocytes in AD patients and murine models are hyporesponsive to Abeta. However, depending on the route of delivery, tolerance can be overcome by vaccination, with the induction of an anti-Abeta-mediated immune response. Through mechanisms that are incompletely understood, immunized APP transgenic animals show markedly reduced Abeta deposition, preservation of normal neuronal architecture, and improved performance in memory and spatial learning tasks. In human trials, Abeta vaccination stabilized cognition and slowed the progression of dementia. Neuropathologic examination of a vaccinated subject showed reduced cortical Abeta without changes in other AD-associated pathology. However, in some patients, vaccination induced severe meningoencephalitis, causing the trial to be terminated. Thus, vaccination appears to activate both beneficial and deleterious anti-Abeta immunity, suggesting that the vaccine can have potent clinical utility if an appropriate immunologic response can be generated.     

Extracellular signal-regulated kinase mediates granulocyte-macrophage colony-stimulating factor messenger RNA stabilization in tumor necrosis factor-alpha plus fibronectin-activated peripheral blood eosinophils

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is critical for promoting the long-term survival of lung- or airway-based eosinophils. Previously, we have shown that fibronectin and tumor necrosis factor alpha induced autocrine production of GM-CSF that markedly enhanced eosinophil survival. Cytokine release was preceded by and dependent on messenger RNA (mRNA) stabilization. Here, we show that mitogen-activated protein kinase (MAPK) activation is responsible for GM-CSF mRNA stabilization in peripheral blood eosinophils (pbeos). Activation of extracellular signal-regulated kinase (ERK) but not p38 correlated with GM-CSF mRNA stability. Although ERK inhibition completely prevented GM-CSF mRNA stabilization, p38 inhibition had a partial effect. To establish which MAPK was crucial, we transduced pbeos with dominant-active TatMEK1(E) or TatMKK3b(E) proteins that selectively phosphorylate ERK or p38, respectively. These studies showed that ERK but not p38 was sufficient for GM-CSF mRNA stabilization. These data are in contradistinction to the c-Jun NH(2)-terminal kinase-mediated regulation of interleukin 2 and 3 mRNAs and suggest unique regulatory features for GM-CSF mRNA in eosinophils.