Extrachromosomal Recombinant Adeno-Associated Virus Vector Genomes Are Primarily Responsible for Stable Liver Transduction In Vivo

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Although the vector genomes are found both as extrachromosomes and as chromosomally integrated forms in hepatocytes, the relative proportion of each has not yet been clearly established. Using an in vivo assay based on the induction of hepatocellular regeneration via a surgical two-thirds partial hepatectomy, we have determined the proportion of integrated and extrachromosomal rAAV genomes in mouse livers and their relative contribution to stable gene expression in vivo. Plasma human coagulation factor IX (hF.IX) levels in mice originating from a chromosomally integrated hF.IX-expressing transposon vector remained unchanged with hepatectomy. This was in sharp contrast to what was observed when a surgical partial hepatectomy was performed in mice 6 weeks to 12 months after portal vein injection of a series of hF.IX-expressing rAAV vectors. At doses of 2.4 x 10(11) to 3.0 x 10(11) vector genomes per mouse (n = 12), hF.IX levels and the average number of stably transduced vector genomes per cell decreased by 92 and 86%, respectively, after hepatectomy. In a separate study, one of three mice injected with a higher dose of rAAV had a higher proportion (67%) of integrated genomes, the significance of which is not known. Nevertheless, in general, these results indicate that, in most cases, no more than approximately 10% of stably transduced genomes integrated into host chromosomes in vivo. Additionally, the results demonstrate that extrachromosomal, not integrated, genomes are the major form of rAAV in the liver and are the primary source of rAAV-mediated gene expression. This small fraction of integrated genomes greatly decreases the potential risk of vector-related insertional mutagenesis associated with all integrating vectors but also raises uncertainties as to whether rAAV-mediated hepatic gene expression can persist lifelong after a single vector administration.     

A novel class of dual mPGES-1/5-LO inhibitors based on the α-naphthyl pirinixic acid scaffold

Dual inhibition of microsomal prostaglandin E₂ synthase-1 (mPGES-1) and 5-lipoxygenase (5-LO) represents a promising strategy in the development of novel anti-inflammatory drugs targeting the arachidonic acid cascade. Herein, a class of α-naphthyl pirinixic acids is characterized as dual mPGES-1/5-LO inhibitors. Systematic structural variation was focused on the lipophilic backbone of the scaffold and yielded detailed structure-activity relationships (SAR) with compound 16 (IC₅₀ mPGES-1 = 0.94 μM; IC₅₀ 5-LO = 0.1 μM) showing the most favorable in vitro pharmacological profile.     

Milk-Based Nutraceutical for Treating Autoimmune Arthritis via the Stimulation of IL-10- and TGF-β-producing CD39+ Regulatory T Cells

Autoimmune diseases arise from the loss of tolerance to self, and because the etiologies of such diseases are largely unknown, symptomatic treatments rely on anti-inflammatory and analgesic agents. Tolerogenic treatments that can reverse disease are preferred, but again, often thwarted by not knowing the responsible auto-antigens (auto-Ags). Hence, a viable alternative to stimulating regulatory T cells (Tregs) is to induce bystander tolerance. Colonization factor antigen I (CFA/I) has been shown to evoke bystander immunity and to hasten Ag-specific Treg development independent of auto-Ag. To translate in treating human autoimmune diseases, the food-based Lactococcus was engineered to express CFA/I fimbriae, and Lactococcus-CFA/I fermented milk fed to arthritic mice proved highly efficacious. Protection occurred via CD39⁺ Tregs producing TGF-β and IL-10 to potently suppress TNF-α production and neutrophil influx into the joints. Thus, these data demonstrate the feasibility of oral nutraceuticals for treating arthritis, and potency of protection against arthritis was improved relative to that obtained with Salmonella-CFA/I.     

Symmetrical Dose-Dependent DNA-Methylation Profiles in Children with Deletion or Duplication of 7q11.23

Epigenetic dysfunction has been implicated in a growing list of disorders that include cancer, neurodevelopmental disorders, and neurodegeneration. Williams syndrome (WS) and 7q11.23 duplication syndrome (Dup7) are rare neurodevelopmental disorders with broad phenotypic spectra caused by deletion and duplication, respectively, of a 1.5-Mb region that includes several genes with a role in epigenetic regulation. We have identified striking differences in DNA methylation across the genome between blood cells from children with WS or Dup7 and blood cells from typically developing (TD) children. Notably, regions that were differentially methylated in both WS and Dup7 displayed a significant and symmetrical gene-dose-dependent effect, such that WS typically showed increased and Dup7 showed decreased DNA methylation. Differentially methylated genes were significantly enriched with genes in pathways involved in neurodevelopment, autism spectrum disorder (ASD) candidate genes, and imprinted genes. Using alignment with ENCODE data, we also found the differentially methylated regions to be enriched with CCCTC-binding factor (CTCF) binding sites. These findings suggest that gene(s) within 7q11.23 alter DNA methylation at specific sites across the genome and result in dose-dependent DNA-methylation profiles in WS and Dup7. Given the extent of DNA-methylation changes and the potential impact on CTCF binding and chromatin regulation, epigenetic mechanisms most likely contribute to the complex neurological phenotypes of WS and Dup7. Our findings highlight the importance of DNA methylation in the pathogenesis of WS and Dup7 and provide molecular mechanisms that are potentially shared by WS, Dup7, and ASD.     

Peptide-functionalized poly(ethylene glycol) star polymers: DNA delivery vehicles with multivalent molecular architecture

Exploring the development of nonviral nucleic acid delivery vectors with progressive, specific, and novel designs in molecular architecture is a fundamental way to investigate how aspects of chemical and physical structure impact the transfection process. In this study, macromolecules comprised of a four-arm star poly(ethylene glycol) and termini modified with one of five different heparin binding peptides have been investigated for their ability to bind, compact, and deliver DNA to mammalian cells in vitro. These new delivery vectors combine a PEG-derived stabilizing moiety with peptides that exhibit unique cell-surface binding ability in a molecular architecture that permits multivalent presentation of the cationic peptides. Five peptide sequences of varying heparin binding affinity were studied; each was found to sufficiently bind heparin for biological application. Additionally, the macromolecules were able to bind and compact DNA into particles of proper size for endocytosis. In biological studies, the PEG-star peptides displayed a range of toxicity and transfection efficiency dependent on the peptide identity. The vectors equipped with peptides of highest heparin binding affinity were found to bind DNA tightly, increase levels of cellular internalization, and display the most promising transfection qualities. Our results suggest heparin binding peptides with specific sequences hold more potential than nonspecific cationic polymers to optimize transfection efficiency while maintaining cell viability. Furthermore, the built-in multivalency of these macromolecules may allow simultaneous binding of both DNA at the core of the polyplex and heparan sulfate on the surface of the cell. This scheme may facilitate a bridging transport mechanism, tethering DNA to the surface of the cell and subsequently ushering therapeutic nucleic acids into the cell. This multivalent star shape is therefore a promising architectural feature that may be exploited in the design of future polycationic gene delivery vectors.     

Human C4 haplotypes with duplicated C4A or C4B

In the course of study of families for the sixth chromosome markers HLA-A, C, B, D/DR, BF, and C2, the two loci for C4, C4A, and C4B, and glyoxalase I, we encountered five examples of probable duplication of one or the other of the two loci for C4. In one of these, both parents and one sib expressed two different structural genes for C4B, one sib expressed one, and one sib expressed none, suggesting that two C4B alleles were carried on a single haplotype: HLA-A2, B7, DR3, BFS1, C2C, C4A2, C4B1, C4B2, GLO1. In a second case, two siblings inherited C4B*1 and C4B*2 from one parent and C4B*Q0 from the other. This duplication appeared on the chromosome as HLA-AW33, B14, DR1, BFS, C2C, C4A2, C4B1, C4B2, GLO2. In a third, very large family with 3 generations, a duplication of the C4B locus occurred which was followed in 2 generations. In one individual, there were three C4B alleles and two C4A alleles. One of the C4B alleles had a hemolytically active product with electrophoretic mobility near C4B2 and was designated C4B*22. It segregated with C4B1 in the family studied. The complete haplotype was HLA-A11, CW1, BW56, DR5, BFS, C2C, C4A3, C4B22, C4B1, GLO2. In another family with 12 siblings, one parent and eight children expressed two C4A alleles on the haplotype HLA-AW30, BW38, DR1, BFF, C2C, C4A3, C4A2, C4BQ0, GLO1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Outlier Analysis Defines Zinc Finger Gene Family DNA Methylation in Tumors and Saliva of Head and Neck Cancer Patients

Head and Neck Squamous Cell Carcinoma (HNSCC) is the fifth most common cancer, annually affecting over half a million people worldwide. Presently, there are no accepted biomarkers for clinical detection and surveillance of HNSCC. In this work, a comprehensive genome-wide analysis of epigenetic alterations in primary HNSCC tumors was employed in conjunction with cancer-specific outlier statistics to define novel biomarker genes which are differentially methylated in HNSCC. The 37 identified biomarker candidates were top-scoring outlier genes with prominent differential methylation in tumors, but with no signal in normal tissues. These putative candidates were validated in independent HNSCC cohorts from our institution and TCGA (The Cancer Genome Atlas). Using the top candidates, ZNF14, ZNF160, and ZNF420, an assay was developed for detection of HNSCC cancer in primary tissue and saliva samples with 100% specificity when compared to normal control samples. Given the high detection specificity, the analysis of ZNF DNA methylation in combination with other DNA methylation biomarkers may be useful in the clinical setting for HNSCC detection and surveillance, particularly in high-risk patients. Several additional candidates identified through this work can be further investigated toward future development of a multi-gene panel of biomarkers for the surveillance and detection of HNSCC.     

c-Fms Signaling Mediates Neurofibromatosis Type-1 Osteoclast Gain-In-Functions

Skeletal abnormalities including osteoporosis and osteopenia occur frequently in both pediatric and adult neurofibromatosis type 1 (NF1) patients. NF1 (Nf1) haploinsufficient osteoclasts and osteoclast progenitors derived from both NF1 patients and Nf1^+/− mice exhibit increased differentiation, migration, and bone resorptive capacity in vitro, mediated by hyperactivation of p21^Ras in response to limiting concentrations of macrophage-colony stimulating factor (M-CSF). Here, we show that M-CSF binding to its receptor, c-Fms, results in increased c-Fms activation in Nf1^{+/ −} osteoclast progenitors, mediating multiple gain-in-functions through the downstream effectors Erk1/2 and p90RSK. PLX3397, a potent and selective c-Fms inhibitor, attenuated M-CSF mediated Nf1^+/− osteoclast migration by 50%, adhesion by 70%, and pit formation by 60%. In vivo, we administered PLX3397 to Nf1 ^+/− osteoporotic mice induced by ovariectomy (OVX) and evaluated changes in bone mass and skeletal architecture. We found that PLX3397 prevented bone loss in Nf1^+/−-OVX mice by reducing osteoclast differentiation and bone resorptive activity in vivo. Collectively, these results implicate the M-CSF/c-Fms signaling axis as a critical pathway underlying the aberrant functioning of Nf1 haploinsufficient osteoclasts and may provide a potential therapeutic target for treating NF1 associated osteoporosis and osteopenia.     

Hypercholesterolemia promotes a CD36-dependent and endothelial nitric-oxide synthase-mediated vascular dysfunction

Numerous studies have implicated either the presence or absence of CD36 in the development of hypertension. In addition, hypercholesterolemia is associated with the loss of nitric oxide-induced vasodilation and the subsequent increase in blood pressure. In the current study, we tested the hypothesis that diet-induced hypercholesterolemia promotes the disruption of agonist-stimulated nitric oxide generation and vasodilation in a CD36-dependent manner. To test this, C57BL/6, apoE null, CD36 null, and apoE/CD36 null mice were maintained on chow or high fat diets. In contrast to apoE null mice fed a chow diet, apoE null mice fed a high fat diet did not respond to acetylcholine with a decrease in blood pressure. Caveolae isolated from in vivo vessels did not contain endothelial nitric-oxide synthase and were depleted of cholesterol. Age-matched apoE/CD36 null mice fed a chow or high fat diet responded to acetylcholine with a decrease in blood pressure. The mechanism underlying the vascular dysfunction was reversible because vessels isolated from apoE null high fat-fed mice regained responsiveness to acetylcholine when incubated with plasma obtained from chow-fed mice. Further analysis demonstrated that the plasma low density lipoprotein fraction was responsible for depleting caveolae of cholesterol, removing endothelial nitric-oxide synthase from caveolae, and preventing nitric oxide production. In addition, the pharmacological removal of caveola cholesterol with cyclodextrin mimicked the effects caused by the low density lipoprotein fraction. We conclude that the ablation of CD36 prevented the negative impact of hypercholesterolemia on agonist-stimulated nitric oxide-mediated vasodilation in apoE null mice. These studies provide a direct link between CD36 and the early events that underlie hypercholesterolemia-mediated hypertension and mechanistic linkages between CD36 function, nitric-oxide synthase activation, caveolae integrity, and blood pressure regulation.     

B and T lymphocyte attenuator exhibits structural and expression polymorphisms and is highly Induced in anergic CD4+ T cells

B and T lymphocyte attenuator (BTLA) was initially identified as expressed on Th1 cells and B cells, but recently reported to be expressed by macrophages, dendritic cells, and NK cells as well. To address this discrepancy we generated a panel of BTLA-specific mAbs and characterized BTLA expression under various activation conditions. We report the existence of three distinct BTLA alleles among 23 murine strains, differing both in Ig domain structure and cellular distribution of expression on lymphoid subsets. The BALB/c and MRL/lpr alleles differ at one amino acid residue, but C57BL/6 has nine additional differences and alters the predicted cysteine bonding pattern. The BALB/c BTLA allele is also expressed by B cells, T cells, and dendritic cells, but not macrophages or NK cells. However, C57BL/6 BTLA is expressed on CD11b+ macrophages and NK cells. Finally, in CD4+ T cells, BTLA is expressed most highly following Ag-specific induction of anergy in vivo, and unlike programmed death-1 and CTLA-4, not expressed by CD25+ regulatory T cells. These results clarify discrepancies regarding BTLA expression, suggest that structural and expression polymorphisms be considered when analyzing BTLA in various murine backgrounds, and indicate a possible role in anergic CD4+ T cells.