Selective stimulation of caveolae-mediated endocytosis by an osmotic polymannitol-based gene transporter

Controlling the cellular uptake mechanism and consequent intracellular route of polyplexes is important to improve the transfection efficiency of the non-viral gene delivery. Here, we report a new non-viral vector, polymannitol-based gene transporter (PMT), generated by crosslinking low molecular weight polyethylenimine with mannitol diacrylate, which has low cytotoxicity and good transfection efficiency. Interestingly, the uptake pathway of PMT/DNA complexes was shifted into caveolae-mediated endocytosis, avoiding lysosomal degradation. The mechanism of increased caveolae-mediated endocytosis of PMT/DNA complexes was found to be correlated with mechanosensing signal transduction by the hyperosmotic polymannitol part. Our results suggested that PMT, polymannitol-based gene transporter, is a safe and efficient gene delivery system with a well-modulated uptake pathway and intracellular route for gene therapy.     

The role of endocytosis in renal dopamine D1 receptor signaling

Desensitization of G-protein-coupled receptors (GPCR) includes receptor endocytosis. This phenomenon is suggested, at least for some receptors, to be associated with receptor resensitization. Here, we examined the role of receptor endocytosis for two different GPCR, the dopamine-1 (D1) receptor and the 1-adrenoceptor (₁-AR) in renal tissue. The functional role of receptor endocytosis was examined on Na⁺, K⁺ -ATPase activity in microdissected proximal tubules from rat kidney. The spatial regulation of endogenous D1 receptors and ₁-AR was examined by confocal microscopy techniques in LLCPK cells. Phenylarsine oxide (PAO) an endocytosis inhibitor, attenuated isoproterenol-induced decrease in Na⁺, K⁺ -ATPase activity but had no such effect on dopamine-induced decrease in Na⁺, K⁺ -ATPase activity. We have previously shown that isoproterenol sensitizes the renal dopamine system, by recruiting silent D1 receptors from the interior of the cell towards the plasma membrane. This effect was attenuated by PAO as well as by cytochalasin D while these substances had no effect on dopamine-induced D1 receptor recruitment. The ₁-AR was localized to the plasma membrane in control cells. Isoproterenol induced a rapid internalization of the ₁-AR; which was prevented by PAO. The results suggest that endocytosis of ₁-AR in renal proximal tubular cells is an important step in signal generation, while endocytosis of proximal tubular D1 receptor is not.     

The role of adenosine receptor engagement in murine fetal thymocyte development.

The role of adenosine receptor engagement in murine T-cell development was evaluated by culturing day 15-16 fetal thymic lobes in the presence of various concentrations of the adenosine receptor agonist 5'-(N-ethyl)-carboxamidoadenosine (NECA) or the adenosine receptor antagonist 8-phenyl-theophylline (8-PT) using the fetal thymic organ culture (FTOC) system. Before and 8 days after culture, thymocytes were isolated, counted, and analyzed for the expression of CD4 and CD8 T-cell differentiation molecules. Analysis of fresh thymocytes prior to culture showed that the majority of cells were of the CD4 single-positive or CD4+ CD8+ immature phenotype. Eight days after culture with media alone, 44% of cells were CD4+ and 23% were CD8+, and the number of viable thymocytes had increased from 1.7 x 10(5) to 2.2 x 10(5) cells per thymic lobe. A dose-dependent inhibition of maturation was observed in cultures with 8-PT, with greater than 85% inhibition at 50 microM. The double-positive thymocyte subset was most severely depleted. The number of cells obtained from cultures with NECA was also reduced, with about 65% inhibition at 50 microM, especially the CD8+ subset that was most severely affected. These results suggest that adenosine receptor engagement is required for normal T-cell differentiation and that adenosine receptor agonists and antagonists have distinct effects on thymocyte differentiation. An understanding of the cell-type-specific and developmental expression of adenosine receptors will help elucidate the mechanisms by which adenosine receptor engagement influences T-cell development.     

The role of sperm-mediated gene transfer in genome mutation and evolution

Contradictory evidence surrounds the claim that sperm cells are able to introduce exogenous DNA into the oocyte at the time of fertilisation. Although strong natural barriers exist against sperm-mediated gene transfer, such barriers are unlikely to be absolutely inviolable. If sperm cells can act as vectors for exogenous DNA, it follows that the genome of sexually reproducing animals may be subject to alteration by exogenous DNA sequences carried by sperm cells. At present there are insufficient data to permit quantification of the rate of sperm-mediated gene transfer. The implications of sperm-mediated gene transfer are significant and include evolutionary effects on the mammalian genome and pathologies in humans from de novo mutations. Despite the absence of firm data, geneticists would be wise to be vigilant to the potential consequences of sperm-mediated gene transfer.     

The role of doxorubicin in non-viral gene transfer in the lung

Proteasome inhibitors have been shown to increase adeno-associated virus (AAV)-mediated transduction in vitro and in vivo. To assess if proteasome inhibitors also increase lipid-mediated gene transfer with relevance to cystic fibrosis (CF), we first assessed the effects of doxorubicin and N-acetyl-l-leucinyl-l-leucinal-l-norleucinal in non-CF (A549) and CF (CFTE29o-) airway epithelial cell lines. CFTE29o- cells did not show a response to Dox or LLnL; however, gene transfer in A549 cells increased in a dose-related fashion (p < 0.05), up to approximately 20-fold respectively at the optimal dose (no treatment: 9.3 × 10⁴ ± 1.5 × 10³, Dox: 1.6 × 10⁶ ± 2.6 × 10⁵, LLnL: 1.9 × 10⁶ ± 3.2 × 10⁵ RLU/mg protein). As Dox is used clinically in cancer chemotherapy we next assessed the effect of this drug on non-viral lung gene transfer in vivo. CF knockout mice were injected intraperitoneally (IP) with Dox (25–100 mg/kg) immediately before nebulisation with plasmid DNA carrying a luciferase reporter gene under the control of a CMV promoter/enhancer (pCIKLux) complexed to the cationic lipid GL67A. Dox also significantly (p < 0.05) increased expression of a plasmid regulated by an elongation factor 1α promoter (hCEFI) approximately 8-fold. Although administration of Dox before lung gene transfer may not be a clinically viable option, understanding how Dox increases lung gene expression may help to shed light on intracellular bottle-necks to gene transfer, and may help to identify other adjuncts that may be more appropriate for use in man.     

The role of specific and non-specific interactions in receptor-mediated endocytosis of nanoparticles

The role of specific and non-specific interactions in the receptor-mediated endocytosis of nanoparticles is analyzed. The characteristic time tau(w), the threshold R(th) and optimal R(opt) radii for particle endocytosis are estimated as a function of the binding energy factor C, bond elasticity factor G, and non-specific attractive/repulsive factor F at the cell-particle interface. It is shown that the contribution of F is as important as that of C and G. General and ready to use formulas are presented that can be a guide in designing nanoparticles with controlled endocytic performances.     

Interindividual variation in anxiety response to amphetamine: possible role for adenosine A2A receptor gene variants.

Amphetamine is thought to produce its stimulant effects mainly via the dopamine system, but its effects may also be influenced by other systems. Dopamine D1 and D2 receptors exist as heterodimers with adenosine A1 and A(2A) receptors, which modulate their responsiveness, suggesting that responses to amphetamine may also depend on adenosinergic function. We therefore studied the relevance of one adenosine A1 and three adenosine A(2A) receptor gene polymorphisms for the interindividual variability in amphetamine response in 99 healthy volunteers who received placebo or d-amphetamine (10 or 20 mg). The 1976C/T and 2592C/T(ins) polymorphisms of the adenosine receptor gene were associated with increases in anxiety at both doses. This is consistent with recent observations indicating a role for adenosine A(2A) receptor gene polymorphisms in anxiety.     

Emerging role of regulatory T cells in gene transfer

Induction and maintenance of immune tolerance to therapeutic transgene products are key requirements for successful gene replacement therapies. Gene transfer may also be used to specifically induce immune tolerance and thereby augment other types of therapies. Similarly, gene therapies for treatment of autoimmune diseases are being developed in order to restore tolerance to self-antigens. Regulatory T cells have emerged as key players in many aspects of immune tolerance, and a rapidly increasing body of work documents induction and/or activation of regulatory T cells by gene transfer. Regulatory T cells may suppress antibody formation and cytotoxic T cell responses and may be critical for immune tolerance to therapeutic proteins. In this regard, CD4(+)CD25(+) regulatory T cells have been identified as important components of tolerance in several gene transfer protocols, including hepatic in vivo gene transfer. Augmentation of regulatory T cell responses should be a promising new tool to achieve tolerance and avoid immune-mediated rejection of gene therapy. During the past decade, it has become obvious that immune regulation is an important and integral component of tolerance to self-antigens and of many forms of induced tolerance. Gene therapy can only be successful if the immune system does not reject the therapeutic transgene product. Recent studies provide a rapidly growing body of evidence that regulatory T cells (T(reg)) are involved and often play a crucial role in tolerance to proteins expressed by means of gene transfer. This review seeks to provide an overview of these data and their implications for gene therapy.     

The effect of B cell receptor signaling on antigen endocytosis and processing

B cell receptor (BCR)-mediated antigen processing and presentation involves both the BCR-mediated internalization and processing of cognate antigen as well as the formation and expression of antigenic peptide-MHC class II complexes. While BCR signaling is known to result in changes in the biosynthesis and intracellular trafficking of class II molecules, the effect of BCR signaling on the cell biology of antigen endocytosis and processing is less clear. Therefore, the effect of BCR signaling on the cell biology of fluid phase antigen endocytosis, processing and presentation was analyzed in both B cell lines or in normal splenic B cells. The results demonstrate that BCR signaling alters neither the global level of fluid phase antigen endocytosis nor the duration of intracellular persistence of fluid phase internalized antigen. Moreover, while BCR signal does result in an increase in the level of total cell surface MHC class II molecules as well as specific peptide-class II complexes, stimulation failed to alter the fraction of class II molecules loaded with antigen-derived peptide. These results indicate that while BCR-mediated signaling elicits an increase in the expression of antigenic peptide-class II complexes, signaling does not augment antigen presentation by profoundly altering the basic biology of antigen endocytosis and processing. These results also demonstrate that the high efficiency of BCR-mediated antigen processing (when compared to fluid phase antigen processing) is likely to occur independent of BCR signaling-induced global alterations in the biology of endocytosis, processing and presentation. This finding suggests that if BCR signaling augments the efficiency of processing of cognate antigen, it must impact unique aspects of BCR-mediated antigen processing, such as the intracellular persistence of internalized antigen-BCR complexes.     

The effect of micro and nanotopography on endocytosis in drug and gene delivery systems

Endocytosis is a fundamental biological process and is also the key mechanism for drug and non-viral gene delivery. The importance of topographical cues in modulating cell behaviors has become increasingly evident, but the influence of topography on endocytosis has however only been sparsely studied. We hypothesize that topography can enhance cellular endocytosis, and in turn the non-viral transfection efficiency. Nano- to microtopographical patterns were fabricated using nano-imprinting lithography (NIL). We first investigated if the substrate topographies could modulate endocytosis and in turn the cellular transfectability. Our results showed increased internalization of fluorescently labeled dextran by human mesenchymal stem cells (hMSCs) and monkey kidney fibroblasts (COS7) when they were cultured on micro- and nanopillars. When the hMSCs were introduced to green-fluorescent protein (GFP) encoding plasmid with Lipofectamine, highest transfection efficiency was observed in cells on nanopillars. Tunable detachable substrate topographies were also fabricated using NIL to promote endocytosis in different cell types, and our results show hMSCs phagocytosis of these polymeric structures. Besides being important in understanding the fundamental process of endocytosis, the current research results may also lead to applications utilizing nanotopography to enhance drug and gene delivery.