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A number of hypotheses regarding how anti-Abeta antibodies alter amyloid deposition have been postulated, yet there is no consensus as to how Abeta immunotherapy works. We have examined the in vivo binding properties, pharmacokinetics, brain penetrance, and alterations in Abeta levels after a single peripheral dose of anti-Abeta antibodies to both wild-type (WT) and young non-Abeta depositing APP and BRI-Abeta42 mice. The rapid rise in plasma Abeta observed after antibody (Ab) administration is attributable to prolongation of the half-life of Abeta bound to the Ab. Only a miniscule fraction of Ab enters the brain, and despite dramatic increases in plasma Abeta, we find no evidence that total brain Abeta levels are significantly altered. Surprisingly, cerebral spinal fluid Abeta levels transiently rise, and when Ab:Abeta complex is directly injected into the lateral ventricles of mice, it is rapidly cleared from the brain into the plasma where it remains stable. When viewed in context of daily turnover of Abeta, these data provide a framework to evaluate proposed mechanisms of Abeta attenuation mediated by peripheral administration of an anti-Abeta monoclonal antibody (mAb) effective in passive immunization paradigm. Such quantitative data suggest that the mAbs are either indirectly enhancing clearance of Abeta or targeting a low abundance aggregation intermediate.
Accumulation of amyloid  protein (A) aggregates is hypothesized to trigger a pathological cascade that causes Alzheimer's disease (AD). Active or passive immunizations targeting A are therefore of great interest as potential therapeutic strategies. We have evaluated the use of recombinant anti-A single-chain variable fragments (scFvs) as a potentially safer form of anti-A immunotherapy. We have generated and characterized three anti-A scFvs that recognize A1-16, Ax-40, or Ax-42. To achieve widespread brain delivery, constructs expressing these anti-A scFvs were packaged into adeno-associated virus (AAV) vectors and injected into the ventricles of postnatal day 0 (P0) amyloid precursor protein CRND8-transgenic mice. Intracranial delivery of AAV to neonatal mice resulted in widespread neuronal delivery. In situ expression of each of the anti-A scFvs after intracerebroventricular AAV serotype 1 delivery to P0 pups decreased A deposition by 25-50%. These data suggest that intracranial anti-A scFv expression is an effective strategy to attenuate amyloid deposition. As opposed to transgenic approaches, these studies also establish a "somatic brain transgenic" paradigm to rapidly and cost-effectively evaluate potential modifiers of AD-like pathology in AD mouse models.
Maternal obesity during pregnancy has been linked to an increased risk of obesity and cardiometabolic disease in the offspring, a phenomenon attributed to developmental programming. Programming effects may be transmissible across generations through both maternal and paternal inheritance, although the mechanisms remain unclear. Using a mouse model, we explored the effects of moderate maternal diet-induced obesity (DIO) on weight gain and glucose-insulin homeostasis in first-generation (F1) and second-generation offspring. DIO was associated with insulin resistance, hyperglycemia and dyslipidemia before pregnancy. Birth weight was reduced in female offspring of DIO mothers (by 6%, P = .039), and DIO offspring were heavier than controls at weaning (males by 47%, females by 27%), however there were no differences in glucose tolerance, plasma lipids, or hepatic gene expression at 6 months. Despite the relative lack of effects in the F1, we found clear fetal growth restriction and persistent metabolic changes in otherwise unmanipulated second-generation offspring with effects on birth weight, insulin levels, and hepatic gene expression that were transmitted through both maternal and paternal lines. This suggests that the consequences of the current dietary obesity epidemic may also have an impact on the descendants of obese individuals, even when the phenotype of the F1 appears largely unaffected.
Adenoviruses are common pathogens, mostly targeting ocular, gastrointestinal and respiratory cells, but in some cases infection disseminates, presenting in severe clinical outcomes. Upon dissemination and contact with blood, coagulation factor X (FX) interacts directly with the adenovirus type 5 (Ad5) hexon. FX can act as a bridge to bind heparan sulphate proteoglycans, leading to substantial Ad5 hepatocyte uptake. FX “coating” also protects the virus from host IgM and complement-mediated neutralisation. However, the contribution of FX in determining Ad liver transduction whilst simultaneously shielding the virus from immune attack remains unclear. In this study, we demonstrate that the FX protection mechanism is not conserved amongst Ad types, and identify the hexon hypervariable regions (HVR) of Ad5 as the capsid proteins targeted by this host defense pathway. Using genetic and pharmacological approaches, we manipulate Ad5 HVR interactions to interrogate the interplay between viral cell transduction and immune neutralisation. We show that FX and inhibitory serum components can co-compete and virus neutralisation is influenced by both the location and extent of modifications to the Ad5 HVRs. We engineered Ad5-derived HVRs into the rare, native non FX-binding Ad26 to create Ad26.HVR5C. This enabled the virus to interact with FX at high affinity, as quantified by surface plasmon resonance, FX-mediated cell binding and transduction assays. Concomitantly, Ad26.HVR5C was also sensitised to immune attack in the absence of FX, a direct consequence of the engineered HVRs from Ad5. In both immune competent and deficient animals, Ad26.HVR5C hepatic gene transfer was mediated by FX following intravenous delivery. This study gives mechanistic insight into the pivotal role of the Ad5 HVRs in conferring sensitivity to virus neutralisation by IgM and classical complement-mediated attack. Furthermore, through this gain-of-function approach we demonstrate the dual functionality of FX in protecting Ad26.HVR5C against innate immune factors whilst determining liver targeting.
BackgroundDrug-eluting stents reduce the incidence of in-stent restenosis, but they result in delayed arterial healing and are associated with a chronic inflammatory response and hypersensitivity reactions. Identifying novel interventions to enhance wound healing and reduce the inflammatory response may improve long-term clinical outcomes. Micro–ribonucleic acids (miRNAs) are noncoding small ribonucleic acids that play a prominent role in the initiation and resolution of inflammation after vascular injury.ObjectivesThis study sought to identify miRNA regulation and function after implantation of bare-metal and drug-eluting stents.MethodsPig, mouse, and in vitro models were used to investigate the role of miRNA in in-stent restenosis.ResultsWe documented a subset of inflammatory miRNAs activated after stenting in pigs, including the miR-21 stem loop miRNAs. Genetic ablation of the miR-21 stem loop attenuated neointimal formation in mice post-stenting. This occurred via enhanced levels of anti-inflammatory M2 macrophages coupled with an impaired sensitivity of smooth muscle cells to respond to vascular activation.ConclusionsMiR-21 plays a prominent role in promoting vascular inflammation and remodeling after stent injury. MiRNA-mediated modulation of the inflammatory response post-stenting may have therapeutic potential to accelerate wound healing and enhance the clinical efficacy of stenting.
Background:Although the prevalence of obesity is higher among women than men, they are somewhat protected from the associated cardiometabolic consequences. The increase in cardiovascular disease risk seen after the menopause suggests a role for estrogens. There is also growing evidence for the importance of estrogen on body fat and metabolism in males. We hypothesized that that estrogen administration would ameliorate the adverse effects of obesity on metabolic parameters in males.Methods:Male and female C57Bl/6 mice were fed control or obesogenic (DIO) diets from 5 weeks of age until adulthood. Glucose tolerance testing was performed at 13 weeks of age. Mice were killed at 15 weeks of age and liver and adipose tissue were collected for analysis of gene expression. A second cohort of male mice underwent the same experimental design with the addition of estradiol pellet implantation or sham surgery at 6 weeks.Results:DIO males had greater mesenteric adipose deposition and more severe increases in plasma glucose, insulin and lipids than females. Treatment of males with estradiol from 6 weeks of age prevented DIO-induced increases in adipose tissue mass and alterations in glucose–insulin homeostasis. We also identified sex differences in the transcript levels and activity of hepatic and adipose glucocorticoid metabolizing enzymes. Estrogen treatment feminized the pattern of DIO-induced changes in glucocorticoid metabolism, rendering males similar to females.Conclusions:Thus, DIO induces sex-specific changes in glucose–insulin homeostasis, which are ameliorated in males treated with estrogen, highlighting the importance of sex steroids in metabolism. Given that altered peripheral glucocorticoid metabolism has been observed in rodent and human obesity, our results also suggest that sexually dimorphic expression and activity of glucocorticoid metabolizing enzymes may have a role in the differential metabolic responses to obesity in males and females.
Neointima formation and vascular remodeling through vascular smooth muscle cell migration and proliferation can limit the long-term success of coronary interventions, for example, in coronary artery bypass grafting (CABG). Ex vivo gene therapy has the potential to reduce unnecessary cell proliferation and limit neointima formation in vascular pathologies. To date, the species C adenovirus serotype 5 has been commonly used for preclinical gene therapy; however, its suitability is potentially limited by relatively poor tropism for vascular cells and high levels of preexisting immunity in the population. To avoid these limitations, novel species of adenovirus are being tested; here we investigate the potential of adenovirus 49 (Ad49) for use in gene therapy. Transduction of primary human vascular cells by a range of adenovirus serotypes was assessed; Ad49 demonstrated highest transduction of both vascular smooth muscle and endothelial cells. Gene transfer with Ad49 in vascular smooth muscle and endothelial cells was possible following short exposure times (<1 hr) and with low MOI, which is clinically relevant. Ex vivo delivery to surplus CABG tissue showed efficient gene transfer with Ad49, consistent with the in vitro findings. Luminal infusion of Ad49GFP into intact CABG samples ex vivo resulted in efficient vessel transduction. In addition, no seroprevalence rates to Ad49 were observed in a Scottish cohort of patients from cardiovascular clinics, thus circumventing issues with preexisting immunity. Our results show that Ad49 has tropism for vascular cells in vitro and ex vivo and demonstrate that Ad49 may be an improved vector for local vascular gene therapy compared with current alternatives.
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