Monoclonal antibody-based competitive ELISA for simultaneous detection of rinderpest virus and peste des petits ruminants virus antibodies

An experimental competitive enzyme-linked immunosorbent assay (morbillivirus cELISA) using a recombinant N antigen (rRPV N) expressed in a baculovirus and a ruminant morbillivirus (RPV and PPRV)-specific monoclonal antibody (P-13A9) was developed for simultaneous detection of rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) antibodies and its diagnostic performance was evaluated. A set of known reference antisera against RPV and PPRV belonging to different lineages, experimental sera from cattle vaccinated for a RPV of Asian lineage, and field sera from cattle and sheep/goat populations known to be positive (West Africa) and negative (Korea) for RPV and PPRV were used for the evaluation. Morbillivirus cELISA results on the panel of experimental RPV and PPRV antisera showed high correlation (r=0.97) between the whole virus and the rRPV N antigens, suggesting that the rRPV N contains a ruminant morbillivirus-specific antigenic determinant recognized by the P-13A9 and it may be suitable as an ELISA antigen in place of the whole virus. Morbillivirus cELISA detected anti-RPV and anti-PPRV antibodies in all reference RPV and PPRV antisera containing VN titers >/=1:8, suggesting that the assay can simultaneously detect antibodies against RPV and PPRV. Anti-RPV antibody was detected by morbillivirus cELISA in vaccinated cattle as early as the VNT and continued to be detectable by both the cELISA and the VNT until termination of the study. When applied to field samples from Africa, morbillivirus cELISA showed good agreement with a RP cELISA kit (kappa value of 0.86) in bovine sera and with a peste des petits ruminant cELISA kit (kappa value of 0.81) in caprine/ovine sera. Usefulness of morbillivirus cELISA using the rRPV N protein was discussed.     

Evidence for glaucoma-induced horizontal cell alterations in the human retina

In this study we investigated changes to horizontal cells in human retinae affected by glaucoma. Glaucoma is characterized by raised intraocular pressure and is responsible for retinal ganglion cell and, possibly, photoreceptor degeneration. It was therefore assumed that horizontal cells might also be affected. The carbocyanine dye DiI was placed at discrete points on fixed, whole-mounted retinae obtained from normal and glaucomatous patients. After allowing 6-24 weeks for intramembranous diffusion within the lipid layers of the nerve cells and, therefore, fluorescent labeling, we measured horizontal cell soma and dendritic field sizes. Selected cells were then embedded in Araldite and cut at 4 microns. Horizontal cells in glaucomatous eyes appeared larger and had a granulated outline as compared with cells from normal retinae. Analysis of the mean cell soma size indicated that cells were 26% larger in the glaucomatous retinae and that this increase was significantly different from that seen in normal retinae (P < 0.05). The dendritic field size was unaffected (P > 0.05). As seen in cross section there was a clear loss of photoreceptor outer segments, and shrunken silhouettes of photoreceptor inner segments with pyknotic nuclei were observed. It is proposed that the increase in some size is indicative of horizontal cell responses that are likely to culminate in degeneration as a result of heightened intraocular pressure. In addition, this paper provides further evidence that photoreceptors are affected by advanced glaucoma.     

Extrahepatic metabolism of 4-methylumbelliferone and lidocaine in the anhepatic rabbit

OBJECTIVE: The initially well-fixed implants of total hip replacement (THR) are in the long-term subject to aseptic loosening. Many cytokines can contribute to osteolysis due to osteoclast recruitment and/or activation. However, in this respect tumor necrosis factor-alpha (TNF-alpha) plays a pivotal role, because it upregulates interleukin-1 and 6 and granulocyte-macrophage colony stimulating factor. The aim of this study was to assess the eventual presence, cellular localization and extent of expression of TNF-alpha in the synovial-like membrane at the implant or at the cement to bone interface compared to control synovial membrane. METHODS: Twenty samples from the synovial-like membrane of the periprosthetic tissues were compared to control samples. TNF-alpha containing cells were visualized using an avidin-biotin-peroxidase complex (ABC) method and analyzed by light microscopy, double labelling and image analysis. RESULTS: TNF-alpha was found in the periprosthetic tissues in fibroblasts and vascular endothelial cells, but mainly in the macrophages was it found to coincide with areas containing implant-derived debris. TNF-alpha containing cells were more numerous in the synovial-like membrane in the interface tissue from the proximal stem area (2816 +/- 318 cells) than in the control synovial membrane (565 +/- 93 cells, p < 0.01). Interestingly, similarly high TNF-alpha expression (3452 +/- 582 cells) was also seen in the synovial-like membrane of the pseudocapsule. CONCLUSION: These findings suggest that the foreign body-type host reaction caused by THR is characterized by the high expression of TNF-alpha. Because such expression occurred in the interface tissue between the implant and surrounding bone, TNF-alpha, due to its pivotal direct and indirect role in the activation and recruitment of osteoclasts, may contribute to periprosthetic osteolysis and to the loosening of THR.     

External surface and lamellarity of lipid vesicles: a practice-oriented set of assay methods

Three methods are presented for the determination of external surface of large lipid vesicles of different lamellarity with 2% absolute accuracy. These methods (referred to as EPR, NBD and TNBS assays) use different marker lipids which provide signals (electron paramagnetic resonance, fluorescence of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) residues, and UV absorption increase of 2,4,6-trinitrobenzenesulfonic acid after reaction with aminolipids, respectively). The signals change upon addition of different membrane-impermeant reagents due to reaction with marker lipids at the external vesicle surface. They were applied to the same vesicle samples, including unilamellar and multilamellar vesicles, both at two different lipid compositions. External surface data matched for the three assays within 2%, but only after appropriate redesign or adaptation of so far published procedures. Main improvements related to slow influx of reagents (TNBS and NBD assays) or to redistribution of marker lipids (EPR assay), obscuring determination of outer vesicle surface from fast reaction between reagent and readily accessible marker lipids. Furthermore, suitable strategies were found to obtain accurate 100% values (reaction of all marker lipids present), required to relate external vesicle surface to total surface. This included corrections for light scattering (NBD assay) and for turbidity (TNBS assay). These three methods appear to close a gap in the methodology to determine external surface of vesicles for typical practical needs. In particular, the reliability range of the NBD assay could be extended to marker lipid densities as low as 1 marker lipid per 3000 lipids.     

Idiopathic hypoparathyroidism--a rare disease?

Five patients with nonfamilial idiopathic hypoparathyroidism were observed in a peripheral hospital. There was no association with other autoimmune disorders such as hypothyroidism, adrenal insufficiency or pernicious anemia. Only in one patient with tetany was the diagnosis clinically obvious; all the others presented with unusual clinical symptoms. These manifestations of chronic hypocalcemia are presented, as well as the diagnostic workup and therapeutic management. We suggest that idiopathic hypoparathyroidism is not a very rare disease, but one which is often missed because of the unusual clinical picture.     

Evidence for McKusick''s hypothesis of deficient collagen cross-linking in patients with homocystinuria

Fractionation of the venom of the spider Phoneutria nigriventer revealed that it was a mixture of several neurotoxic peptides. The peptides so far characterized either inhibited or induced neurotransmitter release. These effects were mediated by Ca2+ channels or increasing Na+ permeability through voltage sensitive Na(+)-channels, respectively. The pooled toxic components (fraction P4) showed stimulatory effects on acetylcholine release from brain cortical slices. In addition, a component of the observed effects resembling that of alpha-latrotoxin was identified, which was characterized by the ability to provoke release of acetylcholine (ACh) at low temperature and in a manner independent of extracellular Ca2+ and of voltage sensitive Na(+)-channels.