Comprehensive screening for human immunodeficiency virus type 1 subtype-specific CD8 cytotoxic T lymphocytes and definition of degenerate epitopes restricted by HLA-A0207 and -C(W)0304 alleles

For this report, the rapid identification and characterization of human immunodeficiency virus type 1 (HIV-1)-derived broadly cross-subtype-reactive CD8 cytotoxic T lymphocyte (CTL) epitopes were performed. Using a gamma interferon (IFN-gamma) Elispot assay-based approach and a panel of recombinant vaccinia viruses expressing gag, env, pol, and nef genes representing the seven most predominant subtypes and one circulating recombinant form of HIV-1, the subtype specificity and cross-subtype reactivity of a CD8 response were directly measured from circulating peripheral blood mononuclear cells (PBMC). Enhanced sensitivity of detection of CD8 responses from cryopreserved PBMC was achieved using autologous vaccinia virus-infected B-lymphoblastoid cell lines as supplemental antigen-presenting cells. Of eleven subjects studied, six exhibited broadly cross-subtype-reactive CD8-mediated IFN-gamma production (at least seven of eight subtypes recognized) to at least one major gene product from HIV-1. Screening of subjects showing broadly cross-subtype-specific responses in the vaccinia virus-based enzyme-linked immunospot (Elispot) assay using a panel of overlapping peptides resulted in the identification of cross-subtype responses down to the 20-mer peptide level in less than 3 days. Three subjects showed broad cross-subtype reactivity in both the IFN-gamma Elispot assay and the standard chromium release cytotoxicity assay. Fine mapping and HLA restriction analysis of the response from three subjects demonstrated that this technique can be used to define epitopes restricted by HLA-A, -B, and -C alleles. In addition, the ability of all three epitopes to be processed from multiple subtypes of their parent proteins and presented in the context of HLA class I molecules following de novo synthesis is shown. While all three minimal epitopes mapped here had previously been defined as HIV-1 epitopes, two are shown to have novel HLA restriction alleles and therefore exhibit degenerate HLA binding capacity. These findings provide biological validation of HLA supertypes in HIV-1 CTL recognition and support earlier studies of cross-subtype CTL responses during HIV-1 infection.     

Glycerol Phosphate Dehydrogenase, Glucose-6-Phosphate Dehydrogenase, and Lactate Dehydrogenase: Activities in Oligodendrocytes, Neurons, Astrocytes, and Myelin Isolated from Developing Rat Brains

Abstract: Glycerol phosphate dehydrogenase (GPDH), glucose-6-phosphate dehydrogenase (G6PDH), and lactate dehydrogenase (LDH) activities were determined in Oligodendrocytes, neurons, and astrocytes isolated from the brains of developing rats. The activity of each enzyme was significantly lower in both neurons and astrocytes than in Oligodendrocytes. The GPDH activity in Oligodendrocytes increased more than 4-fold during development, and at 120 days cells of this type had 1.4-fold the specific activity of forebrain homogenates. The G6PDH activities in Oligodendrocytes from 10-day-old rats were 1.4-fold the activities in the forebrain homogenates. The activities of this enzyme in Oligodendrocytes were progressively lower at later ages, such that at 120 days the cells had 0.8 times the specific activities of homogenates. The Oligodendrocytes had 0.6 times the homogenate activities of LDH at 10 days, and this ratio had decreased to 0.2 by 120 days. These enzymes were also measured in myelin isolated from 20-, 60-, and 120-day-old rats. By 120 days the specific activities of G6PDH and LDH in myelin were <8% of the respective activities in homogenates. The GPDH activity in myelin was, however, at least 20% the specific activity in the homogenates, even in the oldest animals. It is proposed that LDH could be used as a marker for oligodendroglial cytoplasm in subfractions of myelin and in myelin-related membrane vesicles.     

Cryopreservation of organotypic brain spheroid cultures

The cryopreservation of hen and rat brain spheroids was investigated. Brain spheroid cultures were prepared from 7-day-old hen embryos or 16-day-old rat embryos, by using a rotation-mediated culture system. The spheroids were cryopreserved in medium containing 5-15% dimethyl sulphoxide (DMSO) and stored in liquid nitrogen, by using a two-stage cooling procedure. The results show that the viability, as indicated by the total protein content of hen embryo brain spheroids at 24 hours, and at 3, 7 and 28 days after thawing, ranged from 45.5% to 64.2% of control values. It took 3 days for the post-thaw brain spheroids to stabilise, as indicated by their morphology and selected neural markers of functionality. These functions were maintained over a 28-day observation period. Spheroids cultured for 12-15 days in vitro before cryopreservation survived better than those that were cryopreserved after 5-7 days in vitro. The viability and biochemical functionality of spheroids after long-term (up to 6 months) storage were similar to those following short-term storage. The viability of rat brain spheroids cryopreserved in 15% DMSO, as indicated by total protein content, at 24 hours, and at 3 or 7 days after thawing, ranged from 23.1% to 32.1% of control values. This study shows for the first time that brain spheroids prepared from primary tissue can be successfully cryopreserved.     

The response of a three trophic level soil food web to the identity and diversity of plant species and functional groups

Despite considerable recent interest in how biodiversity may influence ecosystem properties, the issue of how plant diversity and composition may affect multiple trophic levels in soil food webs remains essentially unexplored. We conducted a glasshouse experiment in which three plant species of each of three functional groups (grasses, N‐fixing legumes and forbs) were grown in monoculture and in mixtures of three species (with the three species being in the same or different functional groups) and all nine species. Plant species identity had important effects on the biomasses or population densities of belowground primary consumers (microbial biomass, herbivorous nematodes) and two groups of secondary consumers (microbe‐feeding nematodes and enchytraeids); the third consumer trophic level (predatory nematodes) was marginally not significantly affected at P=0.05. Plant species also influenced the relative importance of the bacterial‐based and fungal‐based energy channels for both the primary and secondary consumer trophic levels. Within‐group diversity of only the soil microflora and herbivorous nematodes (both representing the basal consumer trophic level) were affected by plant species identity. However, community composition within all trophic groupings considered (herbivorous nematodes, microbes, microbe‐feeding nematodes, predatory nematodes) was strongly influenced by what plant species were present. Despite the strong responses of the soil biota to plant species identity, there were few effects of plant species or functional group richness on any of the belowground response variables measured. Further, net primary productivity (NPP) was unaffected by plant diversity. Since some belowground response variables were correlated with NPP across treatments, it is suggested that belowground responses to plant diversity might become more apparent in situations when NPP itself responds to plant diversity. Our results point to plant species identity as having important multitrophic effects on soil food webs, both at the whole trophic group and within‐group levels of resolution, and suggest that differences in plant traits across species may be important in driving the decomposer subsystem.     

E-cadherin engagement stimulates proliferation via Rac1

E-cadherin has been linked to the suppression of tumor growth and the inhibition of cell proliferation in culture. We observed that progressively decreasing the seeding density of normal rat kidney-52E (NRK-52E) or MCF-10A epithelial cells from confluence, indeed, released cells from growth arrest. Unexpectedly, a further decrease in seeding density so that cells were isolated from neighboring cells decreased proliferation. Experiments using microengineered substrates showed that E-cadherin engagement stimulated the peak in proliferation at intermediate seeding densities, and that the proliferation arrest at high densities did not involve E-cadherin, but rather resulted from a crowding-dependent decrease in cell spreading against the underlying substrate. Rac1 activity, which was induced by E-cadherin engagement specifically at intermediate seeding densities, was required for the cadherin-stimulated proliferation, and the control of Rac1 activation by E-cadherin was mediated by p120-catenin. Together, these findings demonstrate a stimulatory role for E-cadherin in proliferative regulation, and identify a simple mechanism by which cell-cell contact may trigger or inhibit epithelial cell proliferation in different settings.     

Ebola virus glycoprotein 1: identification of residues important for binding and postbinding events

The filoviruses Ebola virus (EBOV) and Marburg virus (MARV) are responsible for devastating hemorrhagic fever outbreaks. No therapies are available against these viruses. An understanding of filoviral glycoprotein 1 (GP1) residues involved in entry events would facilitate the development of antivirals. Towards this end, we performed alanine scanning mutagenesis on selected residues in the amino terminus of GP1. Mutant GPs were evaluated for their incorporation onto feline immunodeficiency virus (FIV) particles, transduction efficiency, receptor binding, and ability to be cleaved by cathepsins L and B. FIV virions bearing 39 out of 63 mutant glycoproteins transduced cells efficiently, whereas virions bearing the other 24 had reduced levels of transduction. Virions pseudotyped with 23 of the poorly transducing GPs were characterized for their block in entry. Ten mutant GPs were very poorly incorporated onto viral particles. Nine additional mutant GPs (G87A/F88A, K114A/K115A, K140A, G143A, P146A/C147A, F153A/H154A, F159A, F160A, and Y162A) competed poorly with wild-type GP for binding to permissive cells. Four of these nine mutants (P146A/C147A, F153A/H154A, F159A, and F160A) were also inefficiently cleaved by cathepsins. An additional four mutant GPs (K84A, R134A, D150A, and E305/E306A) that were partially defective in transduction were found to compete effectively for receptor binding and were readily cleaved by cathepsins. This finding suggested that this latter group of mutants might be defective at a postbinding, cathepsin cleavage-independent step. In total, our study confirms the role of some GP1 residues in EBOV entry that had previously been recognized and identifies for the first time other residues that are important for productive entry.