Anti-idiotypic antibody response to monoclonal anti-CD4 preparations in nonhuman primate species.

A series of mouse monoclonal anti-CD4 preparations was characterized for the ability to recognize overlapping epitopes on CD4 and to inhibit HIV/simian immunodeficiency virus (SIV) syncytium formation. Based on this characterization, mAb able to recognize CD4 epitopes overlapping the HIV binding site were selected and used to immunize nonhuman primates to elicit the production of specific anti-Id antibodies. Five baboons and five rhesus monkeys were immunized with either individual or a cocktail consisting of several monoclonal anti-CD4 preparations. All the nonhuman primates produced specific anti-Id that recognized either private or cross-reactive Id depending on the monoclonal anti-CD4 used to generate the anti-Id response. Inhibition assays were performed to ascertain the ability of: 1) soluble CD4 to inhibit the Id-anti-Id reaction and 2) the various anti-Id to inhibit the CD4-monoclonal anti-CD4 reaction. These studies demonstrated that some of the anti-Id recognized a cross-reactive Id that was associated with the Ag-combining site. In addition, some of the anti-Id weakly recognized SIV gp120 by Western blot analysis. These studies may be useful in designing experiments that may lead to a better understanding of the CD4-HIV gp120 interaction and to the production of Id and/or anti-Id reagents that might be used to manipulate this virus-receptor interaction.     

Mutant plant viruses with cell binding motifs provide differential adhesion strengths and morphologies

The ability of Tobacco mosaic virus (TMV) to tolerate various amino acid insertions near its carboxy terminus is well-known. Typically these inserts are based on antigenic sequences for vaccine development with plant viruses as carriers. However, we determined that the structural symmetries and the size range of the viruses could also be modeled to mimic the extracellular matrix proteins by inserting cell-binding sequences to the virus coat protein. The extracellular matrix proteins play important roles in guiding cell adhesion, migration, proliferation, and stem cell differentiation. Previous studies with TMV demonstrated that the native and phosphate-modified virus particles enhanced stem cell differentiation toward bone-like tissues. Based on these studies, we sought to design and screen multiple genetically modified TMV mutants with reported cell adhesion sequences to expand the virus-based tools for cell studies. Here, we report the design of these mutants with cell binding amino acid motifs derived from several proteins, the stabilities of the mutants against proteases during purification and storage, and a simple and rapid functional assay to quantitatively determine adhesion strengths by centrifugal adhesion assay. Among the mutants, we found that cells on TMV expressing RGD motifs formed filopodial extensions with weaker attachment profiles, whereas the cells on TMV expressing collagen I mimetic sequence displayed little spreading but higher attachment strengths.     

Dirofilaria repens in Vietnam: detection of 10 eye and subcutaneous tissue infection cases identified by morphology and molecular methods

From 2006 to 2010, hospitals in Hanoi treated 10 human patients for dirofilariasis. The worms were collected from parasitic places, and identification of the species was completed by morphology and molecular methods. Ten parasites were recovered either from the conjunctiva (n=9) or subcutaneous tissue (n=1). The parasites were 4.0-12.5 cm in length and 0.5-0.6 mm in width. Morphological observations suggested all parasites as Dirofilaria repens. Three of the 10 parasites (1 from subcutaneous tissue and 2 from eyes) were used for molecular confirmation of the species identification. A portion of the mitochondrial cox1 (461 bp) was amplified and sequenced. Nucleotide and amino acid homologies were 95% and 99-100%, respectively, when compared with D. repens (Italian origin, GenBank AJ271614; DQ358814). This is the first report of eye dirofilariasis and the second report of subcutaneous tissue dirofilariasis due to D. repens in Vietnam.     

Development of a panel of genome-wide ancestry informative markers to study admixture throughout the Americas

Most individuals throughout the Americas are admixed descendants of Native American, European, and African ancestors. Complex historical factors have resulted in varying proportions of ancestral contributions between individuals within and among ethnic groups. We developed a panel of 446 ancestry informative markers (AIMs) optimized to estimate ancestral proportions in individuals and populations throughout Latin America. We used genome-wide data from 953 individuals from diverse African, European, and Native American populations to select AIMs optimized for each of the three main continental populations that form the basis of modern Latin American populations. We selected markers on the basis of locus-specific branch length to be informative, well distributed throughout the genome, capable of being genotyped on widely available commercial platforms, and applicable throughout the Americas by minimizing within-continent heterogeneity. We then validated the panel in samples from four admixed populations by comparing ancestry estimates based on the AIMs panel to estimates based on genome-wide association study (GWAS) data. The panel provided balanced discriminatory power among the three ancestral populations and accurate estimates of individual ancestry proportions (R2 > 0.9 for ancestral components with significant between-subject variance). Finally, we genotyped samples from 18 populations from Latin America using the AIMs panel and estimated variability in ancestry within and between these populations. This panel and its reference genotype information will be useful resources to explore population history of admixture in Latin America and to correct for the potential effects of population stratification in admixed samples in the region.     

Expansion of human cytomegalovirus (HCMV) immediate-early 1-specific CD8+ T cells and control of HCMV replication after allogeneic stem cell transplantation

Recovery of human cytomegalovirus (HCMV)-specific T immunity is critical for protection against HCMV disease in the early phase after allogeneic stem cell transplantation (SCT). Using an enzyme-linked immunospot assay with overlapping 15-mer peptides spanning pp65 and immediate-early 1 HCMV proteins, we investigated which HCMV-specific CD8⁺ gamma interferon-positive (IFN-γ⁺) T-cell responses against pp65 and IE-1 were associated with control of HCMV replication in 48 recipients of unmanipulated HLA-matched allografts at 3 months (M3) and 6 months (M6) after SCT and in 23 donors. At M3 after SCT, the magnitude of the pp65-specific IFN-γ-producing CD8⁺ T-cell response was greater in recipients than in donors, regardless of HCMV status. In contrast, expansion of IE-1-specific CD8⁺ T cells at M3 was associated with protection against HCMV, and no patient with this expansion had HCMV replication at M3. At M6, the number of HCMV-specific CD8⁺ T cells against both pp65 and IE-1 had expanded in all recipients, regardless of their previous levels of HCMV replication. The recipients' HCMV-specific CD8⁺ T cells already detectable in related donors were predominantly targeting pp65. In contrast, in 40% of the cases, the HCMV-specific CD8⁺ T cells in recipients involved new CD8⁺ T-cell specificities undetectable in their related donors and preferentially targeting IE-1. Taken together, these results showed that the delay in reconstituting IE-1-specific CD8⁺ T cells is correlated with the lack of protection against HCMV in the first 3 months after SCT. They also show that IE-1 is a major antigenic determinant of the early restoration of protective immunity to HCMV after SCT.     

Mutations within the membrane domain of HMG-CoA reductase confer resistance to sterol-accelerated degradation

The pivotal event for sterol-induced degradation of the cholesterol biosynthetic enzyme HMG-CoA reductase is binding of its membrane domain to Insig proteins in the endoplasmic reticulum. Insigs are carriers for gp78, an E3 ubiquitin ligase that marks reductase for proteasomal degradation. We report here the isolation of mutant Chinese hamster ovary cell lines, designated SRD-16, -17, and -18, in which sterol-induced ubiquitination and degradation of reductase are severely impaired. These cells were produced by chemical mutagenesis and selection with SR-12813, a compound that mimics sterols in stimulating ubiquitination and degradation of reductase. Each SRD cell line was found to contain a point mutation in one reductase allele, resulting in substitutions of aspartate for serine-60 (SRD-16), arginine for glycine-87 (SRD-17), and proline for alanine-333 (SRD-18). Sterols failed to promote ubiquitination and degradation of these reductase mutants, owing to their decreased affinity for Insigs. Thus, three different point mutations in reductase, all of which localize to the membrane domain, disrupt Insig binding and abolish sterol-accelerated degradation of the enzyme.     

Species-diagnostic single-nucleotide polymorphism and sequence-tagged site markers for the parasitic wasp genus Nasonia (Hymenoptera: Pteromalidae)

Wasps of the genus Nasonia are important biological control agents of house flies and related filth flies, which are major vectors of human pathogens. Species of Nasonia (Hymenoptera: Pteromalidae) are not easily differentiated from one another by morphological characters, and molecular markers for their reliable identification have been missing so far. Here, we report eight single-nucleotide polymorphism and three sequence-tagged site markers derived from expressed sequenced tag libraries for the two closely related and regionally sympatric species N. giraulti and N. vitripennis. We studied variation of these markers in natural populations of the two species, and we mapped them in the Nasonia genome. The markers are species-diagnostic and evenly spread over all five chromosomes. They are ideal for rapid species identification and hybrid recognition, and they can be used to map economically relevant quantitative trait loci in the Nasonia genome.     

Chromatin compaction and cell death by high molecular weight FGF-2 depend on its nuclear localization, intracrine ERK activation, and engagement of mitochondria

Fibroblast growth factor 2 (FGF-2) is produced as CUG-initiated, 22-34 kDa or AUG-initiated 18 kDa isoforms (hi- and lo-FGF-2, respectively), with potentially distinct functions. We report that expression of hi-FGF-2 in HEK293 cells elicited chromatin compaction preceding cell death with apoptotic features. Nuclear localization of the intact protein was required as expression of a non-nuclear hi-FGF-2 mutant failed to elicit chromatin compaction. Equally ineffective, despite nuclear localization, was the over-expression of the 18 kDa core sequence (lo-FGF-2). Chromatin compaction by hi-FGF-2 was accompanied by increased cytosolic cytochrome C, and was attenuated either by over-expression of Bcl-2 or by a peptide inhibitor of the pro-apoptotic protein Bax. In addition hi-FGF-2 elicited sustained activation of total and nuclear extracellular signal regulated kinase (ERK1/2) by an intracrine route, as it was not prevented by neutralizing anti-FGF-2 antibodies. Inhibition of the ERK1/2 activating pathway by dominant negative upstream activating kinase, or by PD 98059, prevented chromatin compaction by hi-FGF-2. ERK1/2 activation was not affected by the Bax-inhibiting peptide suggesting that it occurred upstream of mitochondrial involvement. We conclude that the hi-FGF-2-induced chromatin compaction and cell death requires its nuclear localization, intracrine ERK1/2 activation and mitochondrial engagement.     

pH-induced conformational change of the influenza M2 protein C-terminal domain

The M2 protein from influenza A is a pH-activated proton channel that plays an essential role in the viral life cycle and serves as a drug target. Using spin labeling EPR spectroscopy, we studied a 38-residue M2 peptide spanning the transmembrane region and its C-terminal extension. We obtained residue-specific environmental parameters under both high- and low-pH conditions for nine consecutive C-terminal sites. The region forms a membrane surface helix at both high and low pH, although the arrangement of the monomers within the tetramer changes with pH. Both electrophysiology and EPR data point to a critical role for residue Lys 49.