Identification of a mutation causing mucopolysaccharidosis type IIIA in New Zealand Huntaway dogs

Mucopolysaccharidosis type IIIA (MPS IIIA) is an autosomal recessive disease that occurs due to a deficiency of heparan sulfate sulfamidase (SGSH). The deficiency of SGSH results in the lysosomal accumulation and urinary excretion of the glycosaminoglycan heparan sulfate. The clinical severity of MPS IIIA is predominantly characterized by severe central nervous system degeneration. Naturally occurring MPS IIIA has recently been described in New Zealand Huntaway dogs, with similar disease progression and biochemical characteristics observed in severely affected MPS IIIA patients. Here, we identify the disease-causing mutation in the MPS IIIA Huntaway dog as 708-709insC. The frequency of the 708-709insC mutation in a sample group of 203 New Zealand Huntaway dogs was determined to be 3.8%. The identification of the 708-709insC mutation will permit the identification of heterozygous carriers as an initial step toward establishing a breeding colony of MPS IIIA dogs for the study of various therapeutic strategies targeted to the central nervous system.     

The Resistance of Retroviral Vectors Produced from Human Cells to Serum Inactivation In Vivo and In Vitro Is Primate Species Dependent

The ability to deliver genes as therapeutics requires an understanding of the vector pharmacokinetics similar to that required for conventional drugs. A first question is the half-life of the vector in the bloodstream. Retroviral vectors produced in certain human cell lines differ from vectors produced in nonhuman cell lines in being substantially resistant to inactivation in vitro by human serum complement (F. L. Cosset, Y. Takeuchi, J. L. Battini, R. A. Weiss, and M. K. Collins, J. Virol. 69:7430-7436, 1995). Thus, use of human packaging cell lines (PCL) may produce vectors with longer half-lives, resulting in more-efficacious in vivo gene therapy. However, survival of human PCL-produced vectors in vivo following systemic administration has not been explored. In this investigation, the half-lives of retroviral vectors packaged by either canine D17 or human HT1080 PCL were measured in the bloodstreams of macaques and chimpanzees. Human PCL-produced vectors exhibited significantly higher concentrations of circulating biologically active vector at the earliest time points measured (>1, 000-fold in chimpanzees), as well as substantially extended half-lives, compared to canine PCL-produced vectors. In addition, the circulation half-life of human PCL-produced vector was longer in chimpanzees than in macaques. This was consistent with in vitro findings which demonstrated that primate serum inactivation of vector produced from human PCL increased with increasing phylogenetic distance from humans. These results establish that in vivo retroviral vector half-life correlates with in vitro resistance to complement. Furthermore, these findings should influence the choice of animal models used to evaluate retroviral-vector-based therapies.     

Human immunodeficiency virus type 1 assembly, budding, and cell-cell spread in T cells take place in tetraspanin-enriched plasma membrane domains

Human immunodeficiency virus type-1 (HIV-1) egress from infected CD4+ T cells is thought to be via assembly and budding at the plasma membrane and may involve components of the T-cell secretory apparatus, including tetraspanins. However, many studies on HIV-1 assembly have examined the trafficking of viral proteins in isolation, and most have used immortalized epithelial, fibroblastic, or hematopoietic cell lines that may not necessarily reflect natural infection of susceptible T cells. Here we have used immunofluorescence and cryoimmunoelectron microscopy (CEM) to examine protein transport during HIV-1 assembly in productively infected Jurkat CD4+ T cells and primary CD4+ T cells. The HIV-1 envelope glycoprotein (Env) and the core protein (Gag) colocalize strongly with CD63 and CD81 and less strongly with CD9, whereas no colocalization was seen between Env or Gag and the late endosome/lysosomal marker Lamp2. CEM revealed incorporation of CD63 and CD81 but not Lamp2 into virions budding at the plasma membrane, and this was supported by immunoprecipitation studies, confirming that HIV-1 egress in T cells is trafficked via tetraspanin-enriched membrane domains (TEMs) that are distinct from lysosomal compartments. CD63, CD81, and, to a lesser extent, CD9 were recruited to the virological synapse (VS), and antibodies against these tetraspanins reduced VS formation. We propose that HIV-1 promotes virus assembly and cell-cell transfer in T cells by targeting plasma membrane TEMs.     

Kinda baboons (Papio kindae) and grayfoot chacma baboons (P. ursinus griseipes) hybridize in the Kafue river valley, Zambia

The ranges of small kinda (Papio kindae) and much larger grayfooted chacma (P. ursinus griseipes) baboons adjoin in the Kafue National Park, Zambia. In a visual survey of baboons at 48 sites in the Kafue River drainage we found that, contrary to previous reports, groups at the species interface near the town of Ngoma are phenotypically diverse and presumably formed by multigenerational hybridization. Mitochondrial and/or Y-chromosome genetic markers from fecal samples (N=164) collected at 29 sites support this conclusion. Groups with phenotypic signs of a history of hybridization also had taxon-specific mitochondria and Y-haplotypes from both parental species. Although the distribution of mitochondrial haplotypes largely mirrored that of external phenotypes, a significant proportion of male specimens from grayfoot as well as hybrid groups carried kinda Y-chromosomes, and kinda Y-chromosomes were involved in all observed cases of mitochondrial/Y-chromosome discordance. These observations are consistent with, though they do not prove, a population history in which the range of chacmas and the hybrid zone have advanced at the expense of the kinda range. They also suggest that, unexpectedly, kinda male×chacma female matings are much more common than the reciprocal cross in the ancestry of hybrids. We suggest that distinctive male kinda behavior and the "juvenile" appearance of kinda baboons of both sexes, perhaps combined with obstetric difficulties of a small kinda female carrying the large offspring of a chacma male, may account for this bias.     

Autoimmune disease risk variant of IFIH1 is associated with increased sensitivity to IFN-α and serologic autoimmunity in lupus patients

Increased IFN-α signaling is a heritable risk factor for systemic lupus erythematosus (SLE). IFN induced with helicase C domain 1 (IFIH1) is a cytoplasmic dsRNA sensor that activates IFN-α pathway signaling. We studied the impact of the autoimmune-disease-associated IFIH1 rs1990760 (A946T) single nucleotide polymorphism upon IFN-α signaling in SLE patients in vivo. We studied 563 SLE patients (278 African-American, 179 European-American, and 106 Hispanic-American). Logistic regression models were used to detect genetic associations with autoantibody traits, and multiple linear regression was used to analyze IFN-α-induced gene expression in PBMCs in the context of serum IFN-α in the same blood sample. We found that the rs1990760 T allele was associated with anti-dsDNA Abs across all of the studied ancestral backgrounds (meta-analysis odds ratio = 1.34, p = 0.026). This allele also was associated with lower serum IFN-α levels in subjects who had anti-dsDNA Abs (p = 0.0026). When we studied simultaneous serum and PBMC samples from SLE patients, we found that the IFIH1 rs1990760 T allele was associated with increased IFN-induced gene expression in PBMCs in response to a given amount of serum IFN-α in anti-dsDNA-positive patients. This effect was independent of the STAT4 genotype, which modulates sensitivity to IFN-α in a similar way. Thus, the IFIH1 rs1990760 T allele was associated with dsDNA Abs, and in patients with anti-dsDNA Abs this risk allele increased sensitivity to IFN-α signaling. These studies suggest a role for the IFIH1 risk allele in SLE in vivo.     

Evaluation of Schistosome Promoter Expression for Transgenesis and Genetic Analysis

Schistosome worms of the genus Schistosoma are the causative agents of schistosomiasis, a devastating parasitic disease affecting more than 240 million people worldwide. Schistosomes have complex life cycles, and have been challenging to manipulate genetically due to the dearth of molecular tools. Although the use of gene overexpression, gene knockouts or knockdowns are straight-forward genetic tools applied in many model systems, gene misexpression and genetic manipulation of schistosome genes in vivo has been exceptionally challenging, and plasmid based transfection inducing gene expression is limited. We recently reported the use of polyethyleneimine (PEI) as a simple and effective method for schistosome transfection and gene expression. Here, we use PEI-mediated schistosome plasmid transgenesis to define and compare gene expression profiles from endogenous and nonendogenous promoters in the schistosomula stage of schistosomes that are potentially useful to misexpress (underexpress or overexpress) gene product levels. In addition, we overexpress schistosome genes in vivo using a strong promoter and show plasmid-based misregulation of genes in schistosomes, producing a clear and distinct phenotype- death. These data focus on the schistosomula stage, but they foreshadow strong potential for genetic characterization of schistosome molecular pathways, and potential for use in overexpression screens and drug resistance studies in schistosomes using plasmid-based gene expression.