Truncation of the cytoplasmic domain induces exposure of conserved regions in the ectodomain of human immunodeficiency virus type 1 envelope protein

We have described a CD4-independent variant of HXBc2, termed 8x, that binds directly to CXCR4 and mediates CD4-independent virus infection. Determinants for CD4 independence map to residues in the V3 and V4-C4 domains together with a single nucleotide deletion in the transmembrane domain which introduces a frameshift (FS) at position 706. This FS results in a truncated cytoplasmic domain of 27 amino acids. We demonstrate here that while introduction of the 8x FS mutation into heterologous R5, X4, or R5X4 Env proteins did not impart CD4 independence, it did affect the conformation of the gp120 surface subunit, exposing highly conserved domains involved in both coreceptor and CD4 binding. In addition, antigenic changes in the gp41 ectodomain were also observed, consistent with the idea that the effects of cytoplasmic domain truncation must in some way be transmitted to the external gp120 subunit. Truncation of gp41 also resulted in the marked neutralization sensitivity of all Env proteins tested to human immunodeficiency virus-positive human sera and monoclonal antibodies directed against the CD4 or coreceptor-binding sites. These results demonstrate a structural interdependence between the cytoplasmic domain of gp41 and the ectodomain of the Env protein. They also may help explain why the length of the gp41 cytoplasmic domain is retained in vivo and may provide a way to genetically trigger the exposure of neutralization determinants in heterologous Env proteins that may prove useful for vaccine development.     

Multipoint linkage detection in the presence of heterogeneity

Linkage heterogeneity is common for complex diseases. It is well known that loss of statistical power for detecting linkage will result if one assumes complete homogeneity in the presence of linkage heterogeneity. To this end, Smith (1963, Annals of Human Genetics 27, 175-182) proposed an admixture model to account for linkage heterogeneity. It is well known that for this model, the conventional chi-squared approximation to the likelihood ratio test for no linkage does not apply even when the sample size is large. By dealing with nuclear families and one marker at a time for genetic diseases with simple modes of inheritance, score-based test statistics (Liang and Rathouz, 1999, Biometrics 55, 65-74) and likelihood-ratio-based test statistics (Lemdani and Pons, 1995, Biometrics 51, 1033-1041) have been proposed which have a simple large-sample distribution under the null hypothesis of linkage. In this paper, we extend their work to more practical situations that include information from multiple markers and multi-generational pedigrees while allowing for a class of general genetic models. Three different approaches are proposed to eliminate the nuisance parameters in these test statistics. We show that all three approaches lead to the same asymptotic distribution under the null hypothesis of no linkage. Simulation results show that the proposed test statistics have adequate power to detect linkage and that the performances of these two classes of test statistics are quite comparable. We have applied the proposed method to a family study of asthma (Barnes et al., 1996), in which the score-based test shows evidence of linkage with p-value <0.0001 in the region of interest on chromosome 12. Additionally, we have implemented this score-based test within the frequently used computer package GENEHUNTER.     

Access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1

Anti-human immunodeficiency virus type 1 (HIV-1) antibodies whose binding to gp120 is enhanced by CD4 binding (CD4i antibodies) are generally considered nonneutralizing for primary HIV-1 isolates. However, a novel CD4i-specific Fab fragment, X5, has recently been found to neutralize a wide range of primary isolates. To investigate the precise nature of the extraordinary neutralizing ability of Fab X5, we evaluated the abilities of different forms (immunoglobulin G [IgG], Fab, and single-chain Fv) of X5 and other CD4i monoclonal antibodies to neutralize a range of primary HIV-1 isolates. Our results show that, for a number of isolates, the size of the neutralizing agent is inversely correlated with its ability to neutralize. Thus, the poor ability of CD4i-specific antibodies to neutralize primary isolates is due, at least in part, to steric factors that limit antibody access to the gp120 epitopes. Studies of temperature-regulated neutralization or fusion-arrested intermediates suggest that the steric effects are important in limiting the binding of IgG to the viral envelope glycoproteins after HIV-1 has engaged CD4 on the target cell membrane. The results identify hurdles in using CD4i epitopes as targets for antibody-mediated neutralization in vaccine design but also indicate that the CD4i regions could be efficiently targeted by small molecule entry inhibitors.     

Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans

Melanin is a virulence factor for many pathogenic fungal species, including Cryptococcus neoformans. Melanin is deposited in the cell wall, and melanin isolated from this fungus retains the shape of the cells, resulting in hollow spheres called "ghosts". In this study, atomic force, scanning electron, and transmission electron microscopy revealed that melanin ghosts are covered with roughly spherical granular particles approximately 40-130 nm in diameter, and that the melanin is arranged in multiple concentric layers. Nuclear magnetic resonance cryoporometry indicated melanin ghosts contain pores with diameters between 1 and 4 nm, in addition to a small number of pores with diameters near 30 nm. Binding of the antibodies to melanin reduced the apparent measured volume of these pores, suggesting a mechanism for their antifungal effect. We propose a model of cryptococcal melanin structure whereby the melanin granules are held together in layers. This structural model has implications for cell division, cell wall remodeling, and antifungal drug discovery.     

Inheritance of total serum IgE in the isolated Tangier Island population from Virginia: complexities associated with genealogical depth of pedigrees in segregation analyses

OBJECTIVES: This study was aimed at performing a segregation analysis of total serum immunoglobulin E (tIgE) in an isolated population using maximal genealogical information permitted by current software and computer capacities, while assessing the reliability of the best-fitting model of inheritance for tIgE through simulations. METHODS: All current Tangier Island, VA, residents (n = 664) belonged to one large extended pedigree (n = 3,501) spanning 13 generations, with an average inbreeding coefficient of 0.009. Phenotype data were obtained on 453 (68.2%) of the residents using a population-based recruitment scheme. Due to computational limitations resulting from the extremely complex pedigree structure, analysis on only two pedigree reconstructions was feasible: a reduced pedigree retaining all phenotyped individuals and their parents as 57 distinct families, and 922 nuclear families. RESULTS: Familial correlations and heritability calculations reveal a significant genetic component to tIgE in these data (heritability = 26%). The most parsimonious model to explain tIgE distribution indicated by the reduced pedigree structure was a two-distribution Mendelian model. However, larger and non-genetic models could not be rejected. Simulations over 200 replicates performed to evaluate the reliability of this model, indicated that using restricted genealogical information had minimal impact on results of segregation analyses performed here.     

Covalent cross-linking of the NC1 domain of collagen type IX to collagen type II in cartilage

From a study to understand the mechanism of covalent interaction between collagen types II and IX, we present experimental evidence for a previously unrecognized molecular site of cross-linking. The location relative to previously defined cross-linking sites predicts a specific manner of interaction and folding of collagen IX on the surface of nascent collagen II fibrils. The initial evidence came from Western blot analysis of type IX collagen extracted by pepsin from fetal human cartilage, which showed a molecular species that had properties indicating an adduct between the alpha1(II) chain and the C-terminal domain (COL1) of type IX collagen. A similar component was isolated from bovine cartilage in sufficient quantity to confirm this identity by N-terminal sequence analysis. Using an antibody that recognized the putative cross-linking sequence at the C terminus of the alpha1(IX) chain, cross-linked peptides were isolated by immunoaffinity chromatography from proteolytic digests of human cartilage collagen. They were characterized by immunochemistry, N-terminal sequence analysis, and mass spectrometry. The results establish a link between a lysine near the C terminus (in the NC1 domain) of alpha1(IX) and the known cross-linking lysine at residue 930 of the alpha1(II) triple helix. This cross-link is speculated to form early in the process of interaction between collagen IX molecules and collagen II polymers. A model of molecular folding and further cross-linking is predicted that can spatially accommodate the formation of all six known cross-linking interactions to the collagen IX molecule on a fibril surface. Of particular biological significance, this model can accommodate potential interfibrillar as well as intrafibrillar links between the collagen IX molecules themselves, so providing a mechanism whereby collagen IX could stabilize a collagen fibril network.     

Lipoprotein receptor binding, cellular uptake, and lysosomal delivery of fusions between the receptor-associated protein (RAP) and alpha-L-iduronidase or acid alpha-glucosidase

Enzyme replacement therapy for lysosomal storage disorders depends on efficient uptake of recombinant enzyme into the tissues of patients. This uptake is mediated by oligosaccharide receptors including the cation-independent mannose 6-phosphate receptor and the mannose receptor. We have sought to exploit alternative receptor systems that are independent of glycosylation but allow for efficient delivery to the lysosome. Fusions of the human lysosomal enzymes alpha-l-iduronidase or acid alpha-glucosidase with the receptor-associated protein were efficiently endocytosed by lysosomal storage disorder patient fibroblasts, rat C6 glioma cells, mouse C2C12 myoblasts, and recombinant Chinese hamster ovary cells expressing individual members of the low-density lipoprotein receptor family. Uptake of the fusions exceeded that of phosphorylated enzyme in all cases, often by an order of magnitude or greater. Uptake was specifically mediated by members of the low-density lipoprotein receptor protein family and was followed by delivery of the fusions to the lysosome. The advantages of the lipoprotein receptor system over oligosaccharide receptor systems include more efficient cellular delivery and the potential for transcytosis of ligands across tight endothelia, including the blood-brain barrier.     

A tumor-associated glycoprotein that blocks MHC class II-dependent antigen presentation by dendritic cells

Tumors evade immune surveillance despite the frequent expression of tumor-associated Ags (TAA). Tumor cells escape recognition by CD8(+) T cells through several mechanisms, including down-regulation of MHC class I molecules and associated Ag-processing machinery. However, although it is well accepted that optimal anti-tumor immune responses require tumor-reactive CD4(+) T cells, few studies have addressed how tumor cells evade CD4(+) T cell recognition. In this study, we show that a common TAA, GA733-2, and its murine orthologue, mouse epithelial glycoprotein (mEGP), function in blocking MHC class II-restricted Ag presentation by dendritic cells. GA733-2 is a common TAA that is expressed normally at low levels by some epithelial tissues and a subset of dendritic cells, but at high levels on colon, breast, lung, and some nonepithelial tumors. We show that ectopic expression of mEGP or GA733-2, respectively, in dendritic cells derived from murine bone marrow or human monocytes results in a dose-dependent inability to stimulate proliferation of Ag-specific or alloreactive CD4(+) T cells. Dendritic cells exposed to cell debris from tumors expressing mEGP are similarly compromised. Furthermore, mice immunized with dendritic cells expressing mEGP from a recombinant adenovirus vector exhibited a muted anti-adenovirus immune response. The inhibitory effect of mEGP was not due to down-regulation of functional MHC class II molecules or active suppression of T cells, and did not extend to T cell responses to superantigen. These results demonstrate a novel mechanism by which tumors may evade CD4(+) T cell-dependent immune responses through expression of a TAA.