Abstract
Coagulation factors are produced from hepatocytes, whereas production of coagulation factor VIII (FVIII) from primary tissues and cell species is still controversial. Here, we tried to ...characterize primary FVIII-producing organ and cell species using genetically engineered mice, in which enhanced green fluorescent protein (EGFP) was expressed instead of the
F8
gene. EGFP-positive FVIII-producing cells existed only in thin sinusoidal layer of the liver and characterized as CD31
high
, CD146
high
, and lymphatic vascular endothelial hyaluronan receptor 1 (Lyve1)
+
. EGFP-positive cells can be clearly distinguished from lymphatic endothelial cells in the expression profile of the podoplanin
−
and C-type lectin-like receptor-2 (CLEC-2)
+
. In embryogenesis, EGFP-positive cells began to emerge at E14.5 and subsequently increased according to liver maturation. Furthermore, plasma FVIII could be abolished by crossing
F8
conditional deficient mice with Lyve1-Cre mice. In conclusion, in mice, FVIII is only produced from endothelial cells exhibiting CD31
high
, CD146
high
, Lyve1
+
, CLEC-2
+
, and podoplanin
−
in liver sinusoidal endothelial cells.
Background
Intravenous administration of adeno‐associated virus (AAV) vectors is a promising gene therapy approach for monogenic diseases. However, re‐administration of the same AAV serotype is ...impossible because of the induction of anti‐AAV neutralizing antibodies (NAbs). Here, we examined the feasibility of re‐administrating AAV vector serotypes different from the initial AAV vector serotype.
Methods
Liver‐targeting AAV3B, AAV5, and AAV8 vectors were intravenously injected in C57BL/6 mice, and the emergence of NAbs and the transduction efficacy following re‐administration were evaluated.
Results
For all serotypes, re‐administration of the same serotype was not possible. Although the highest neutralizing activity of NAb was induced by AAV5, anti‐AAV5 NAbs did not react with other serotypes, resulting in successful re‐administration with the other serotypes. AAV5 re‐administration was also successful in all mice treated with AAV3B and AAV8. Effective secondary administration of AAV3B and AAV8 was observed in most mice initially administrated AAV8 and AAV3B, respectively. However, few mice developed NAbs cross‐reacting with the other serotypes, especially those with close sequence homology.
Conclusions
In summary, AAV vector administration induced NAbs relatively specific to the administrated serotype. Secondary administration of AAVs targeting liver transduction could be successfully achieved by switching AAV serotypes in mice.
Systemic administration of AAVs targeting liver transduction induced high titer serotype‐specific neutralizing antibodies in mice. Here, the secondary administration was successfully achieved by switching AAV serotypes. Considering the re‐administration of AAV vectors to patients who have failed to respond to initial gene therapy or lost therapeutic effect over time, the development of several AAV preparations is crucial for targeting one disease.
Eosinophils are multifunctional leukocytes involved in allergic reactions as well as adipose tissue regulation. IL‐5 is required for eosinophil survival; however, the in vivo mechanisms of eosinophil ...regulation are not fully understood. A tg mouse model with il5 promoter‐driven EGFP expression was established for detecting the IL‐5‐producing cells in vivo. Il5‐egfp tg mice expressed high levels of EGFP in gonadal adipose tissue (GAT) cells. EGFP+ cells in GAT were mainly group 2 innate lymphoid cells (ILCs). IL‐33 preferentially expanded EGFP+ cells and eosinophils in GAT in vivo. EGFP+ ILCs were found to upregulate prg2 mRNA expression in GAT eosinophils. These results demonstrate that ILCs activate eosinophils in GAT. The blockage of IL‐33Rα, on the other hand, did not impair EGFP+ ILC numbers but did impair eosinophil numbers in vivo. GAT eosinophils expressed IL‐33Rα and IL‐33 expanded eosinophil numbers in CD90+ cell‐depleted mice. IL‐33 was further observed to induce the expression of retnla and epx mRNA in eosinophils. These findings demonstrate that IL‐33 directly activates eosinophils in GAT, and together with our other findings described above, our findings show that IL‐33 has dual pathways via which it activates eosinophils in vivo: a direct activation pathway and a group 2 ILC‐mediated pathway.
•APRIL and BAFF induce proliferation of PP B cells.•PP B cells are phenotypically distinct from splenic B cells.•Ig downregulation in intestine by APRIL and BAFF.
Intestinal immunoglobulins (Igs) ...protect against microbes. However, the regulation of intestinal Ig production is poorly understood. In this study, we have investigated the roles of APRIL (tumor necrosis factor superfamily member TNFSF 13) and BAFF (TNFSF13B) in intestinal Ig induction. Peyer’s patches (PPs) are, at least in part, an inductive site for Igs, including IgA. Introducing APRIL and BAFF in vivo lowered the frequency of IgG1+ or IgG2b+ B cells in PPs. Administration of TACI-Fc upregulated the frequency of IgG1+, IgG2b+, and IgA+ B cells in PPs, suggesting that APRIL and BAFF attenuate Ig production in these regions. TACI-Fc also upregulated intestinal IgA levels and expanded germinal center B cells in PPs. These results indicate that APRIL and BAFF paradoxically downregulate homeostatic Ig production in the intestines.
Most gene therapy clinical trials that systemically administered adeno-associated virus (AAV) vector enrolled only patients without anti-AAV-neutralizing antibodies. However, laboratory tests to ...measure neutralizing antibodies varied among clinical trials and have not been standardized. In this study, we attempted to improve the sensitivity and reproducibility of a cell-based assay to detect neutralizing antibodies and to determine the detection threshold to predict treatment efficacy. Application of the secreted type of NanoLuc and AAV receptor-expressing cells reduced the multiplicity of infection (MOI) for AAV transduction and improved the sensitivity to detect neutralizing antibodies with a low coefficient of variation, whereas the detection threshold could not be improved by the reduction of MOI to <100. After human immunoglobulin administration into mice at various doses, treatment with high-dose AAV8 vector enabled evasion of the inhibitory effect of neutralizing antibodies. Conversely, gene transduction was slightly influenced in the mice treated with low-dose AAV8 vector, even when neutralizing antibodies were determined to be negative in the assay. In conclusion, we developed a reliable and sensitive cell-based assay to measure neutralizing antibodies against AAV and found that the appropriate MOI to detect marginal neutralizing antibodies was 100. Other factors, including noninhibitory antibodies, marginally influence in vivo transduction at low vector doses.
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Application of secNanoLuc in a cell-based assay to detect AAV neutralizing antibody can improve its sensitivity, reproducibility, and versatility. Our improved method could appropriately select eligible patients for systemic AAV treatment and predict interindividual viability of the treatment effect.
Adeno-associated virus (AAV) vectors are attractive tools for gene transfer to the liver and are used as gene therapeutic drugs for inherited disorders. The intravenous injection of an AAV vector ...harboring the gene of interest driven by the hepatocyte-specific promoter could efficiently express the target gene in liver hepatocytes. The delivery of genome editing tools including Cas9 and gRNA, by the AAV vector, can efficiently disrupt the target gene expression in the liver in vivo by intravenous administration in mice. We can quickly obtain mice lacking specific gene expression in the liver only by administering the AAV vector. The method could be suitable for developing genome editing treatments for inherited disorders and basic research exploring the physiological role of the target gene produced from liver hepatocytes.
Congenital long- or short-QT syndrome may lead to life-threatening ventricular tachycardia and sudden cardiac death. Apart from the rare disease-causing mutations, common genetic variants in CAPON, a ...neuronal nitric oxide synthase (NOS1) regulator, have recently been associated with QT interval variations in a human whole-genome association study. CAPON had been unsuspected of playing a role in cardiac repolarization; indeed, its physiological role in the heart (if any) is unknown. To define the biological effects of CAPON in the heart, we investigated endogenous CAPON protein expression and protein-protein interactions in the heart and performed electrophysiological studies in isolated ventricular myocytes with and without CAPON overexpression. We find that CAPON protein is expressed in the heart and interacts with NOS1 to accelerate cardiac repolarization by inhibition of L-type calcium channel. Our findings provide a rationale for the association of CAPON gene variants with extremes of the QT interval in human populations.
Background and Purpose— We have previously shown that the sphingosine 1-phosphate (S1P)/S1P receptor-1 (S1P 1 R) axis contributes to the migration of transplanted neural progenitor cells (NPCs) ...toward areas of spinal cord injury. In the current study, we examined a strategy to increase endogenous NPC migration toward the injured central nervous system to modify S1PR. Methods— S1P concentration in the ischemic brain was measured in a mouse thrombosis model of the middle cerebral artery. NPC migration in vitro was assessed by a Boyden chamber assay. Endogenous NPC migration toward the insult was evaluated after ventricular administration of the S1P 2 R antagonist JTE-013. Results— The concentration of S1P in the brain was increased after ischemia and was maximal 14 days after the insult. The increase in S1P in the infarcted brain was primarily caused by accumulation of microglia at the insult. Mouse NPCs mainly expressed S1P 1 R and S1P 2 R as S1PRs, and S1P significantly induced the migration of NPCs in vitro through activation of S1P 1 R. However, an S1P 1 R agonist failed to have any synergistic effect on S1P-mediated NPC migration, whereas pharmacologic or genetic inhibition of S1P 2 R by JTE-013 or short hairpin RNA expression enhanced S1P-mediated NPC migration but did not affect proliferation and differentiation. Interestingly, administration of JTE-013 into a brain ventricle significantly enhanced endogenous NPC migration toward the area of ischemia. Conclusions— Our findings suggest that S1P is a chemoattractant for NPCs released from an infarcted area and regulation of S1P 2 R function further enhances the migration of NPCs toward a brain infarction.
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