Optimization of nonviral transfection: variables influencing liposome-mediated gene transfer in proliferating vs. quiescent cells in culture and in vivo using a porcine restenosis model

Cationic liposomes/DNA complexes are widely used vectors for delivering genes in clinical and experimental trials. Relatively low transfer efficiencies in vivo compared with viral gene transfer may be improved using local application. In addition, markedly increased transfer efficiency may be achieved in vitro and in vivo via optimization of known variables influencing liposomal transfection. Lipofection under different conditions was performed in various cell lines and primary porcine smooth muscle cells. Optimized conditions found in vitro were verified in vivo using a porcine restenosis model. Toxicity was monitored analyzing cell metabolism. Transfer efficiency and safety were determined using morphometry, histology, galactosidase assays, PCR, and RT-PCR. The most important variables enabling maximum transfer efficiency were firstly the appropriate selection of cationic lipids for the cell type to be transfected, secondly the DNA/liposome ratio chosen, which depended on the cell type and cationic lipids used, and thirdly the state of proliferation of the targeted cells. Transfection in vivo demonstrated two- to fivefold higher transfer efficiencies when transfer conditions were extrapolated from optimization experiments in stationary cells compared with the use of conditions established in proliferating cells. Application of the therapeutic gene for cecropin using optimized transfer conditions resulted in a significantly reduced neointima formation compared with the transfection using a control gene for ss-galactosidase. Thus, in this vascular model, initial optimization of lipofection in stationary cells in culture followed by local delivery in vivo and with selection of a suitable therapeutic gene led to markedly improved transfer efficiencies, gene expression, and biological effect. Stationary cell cultures simulate more realistically the in vivo situation and may therefore represent a better model for future in vivo experiments. In addition, the advantages of liposomes are easy handling, low toxicity, and the lack of carcinogenicity or immunogenic reactions.     

Regulation of Th2 cell differentiation by mel-18, a mammalian polycomb group gene

Polycomb group (PcG) gene products regulate homeobox gene expression in Drosophila and vertebrates and also cell cycle progression of immature lymphocytes. In a gene-disrupted mouse for polycomb group gene mel-18, mature peripheral T cells exhibited normal anti-TCR-induced proliferation; however, the production of Th2 cytokines (IL-4, IL-5, and IL-13) was significantly reduced, whereas production of IFNgamma was modestly enhanced. Th2 cell differentiation was impaired, and the defect was associated with decreased levels in demethylation of the IL-4 gene. Significantly, reduced GATA3 induction was demonstrated. In vivo antigen-induced IgG1 production and Nippostrongylus brasiliensis-induced eosinophilia were significantly affected, reflecting the deficit in Th2 cell differentiation. Thus, the PcG gene products play a critical role in the control of Th2 cell differentiation and Th2-dependent immune responses.     

Mold filling of titanium alloys in two different wedge-shaped molds

Pure titanium and titanium alloys are potential materials for the fabrication of cast dental appliances. One important factor in producing sound castings is the capacity of the metal to fill the mold. This study used a wedge-shaped mold to compare the mold filling of titanium with that of conventional dental casting alloys. The metals used were CP Ti, Ti-6Al-7Nb, Ti-6Al-4V, Ti with 1 and 4wt% Cu and ADA Type III gold alloy and an Ni-Cr alloy. The castings were cut into four pieces parallel to the triangular surface. Mold filling was evaluated as the distance between the tip of the cast wedge and theoretical tip of the triangle. The mold filling of the gold alloy was superior compared to all the metals tested, while the mold filling of the Ni-Cr alloy was the worst. There were no statistical differences at the 30 degrees marginal angle for all the cast titanium metals. At the sharper 15 degrees angle, CP Ti and Ti-6Al-7Nb was superior to both the Ti-Cu alloys. Although the mold filling of titanium was inferior compared to the gold alloy, the data justify the use of titanium for the production of dental appliances.     

Development of a new calcium phosphate cement that contains sodium calcium phosphate

A cement powder consisting of sodium calcium phosphate, Na3Ca6(PO4)5, in addition to tetracalcium phosphate and beta-tricalcium phosphate was prepared by pulverizing blocks of 4 wt% sodium-, 11 wt% carbonate-containing apatite samples that were heated at 1700 degrees C for 5 h. When mixed with 30 wt% malic acid or citric acid at a powder liquid ratio of 3:1, the cement set in 3 or 7 min at room temperature with compressive strength being around 52 or 27 MPa. In HeLa-cell cultures, the cement mixed with malic acid was less cytotoxic than the cement mixed with citric acid, which was far less cytotoxic than a commercial carboxylate cement used as a negative control, suggesting malic acid to be superior to citric acid as a liquid in this regard. Similar findings were also obtained with osteoclasts, of which culture experiments clearly suggested that the number of osteoclasts on the cement mixed with malic acid was significantly greater than that on the cement mixed with citric acid. Since osteoclastic response to substrates could be used as a maker in evaluating their bioresorbability associated with osteoclasts, the above finding may suggest that the cement that is to be mixed with malic acid would be more useful as bone substitutes.     

Inflammatory pseudotumor of lymph nodes: clinicopathologic and immunohistological study of 11 Japanese cases

We report 11 Japanese cases of inflammatory pseudotumor (IPT) of the lymph node. There were 7 males and 4 females with ages ranging from 5 to 68 years (median; 48). Only 2 patients had systemic lymphadenopathy, and all others had involvement of only 1 lymph node group. Constitutional symptoms such as fever were present in 8 patients and laboratory abnormalities were detected in 5. All patients recovered and were alive and well after 2 to 180 months (median; 32 months). Histologically, the process mainly involved the connective tissue framework of the lymph node, secondarily spreading into the lymph node parenchyma and the perinodal tissue. It was characterized by a storiform growth pattern of myofibroblasts, marked vascularity with associated vascular lesions, and a polymorphous reactive cellular infiltrate in a collagen-rich stroma. An immunohistochemical study revealed numerous myofibroblasts, histiocytes, and vascular endothelial cells expressing vascular endothelial growth factor (VEGF) in 6 cases. It was suggested that VEGF may be involved, in part, in the induction of the angiogenesis of IPT. Moreover, the present study indicates that follicular dendritic cell sarcoma, nasal T/natural killer cell lymphoma, and anaplastic large cell lymphoma should be added to the differential diagnosis from IPT of the lymph node. Int J Surg Pathol 9(3):207-214, 2001     

The absence of DNA polymerase kappa does not affect somatic hypermutation of the mouse immunoglobulin heavy chain gene

During the immune response to T cell-dependent antigen, somatic hypermutation (SHM) is introduced into immunoglobulin (Ig) genes. The variable region is the target for SHM and it is here that DNA lesions are introduced and mutations are generated. It has been suggested that error-prone DNA polymerase(s) may play an important role in this mutagenesis phase. Recently, DNA polymerase kappa (Polkappa), which belongs to the Y-family of DNA polymerases, was identified. Since a hot spot of SHMs (RGYW motif) is also a hot spot of mutations by human Polkappa, this enzyme was suggested to be an SHM instigator. In order to address the question whether Polkappa is involved in SHM, we immunized Polkappa-deficient mice and analyzed the SHM of the Ig heavy chain gene. We found that the SHM frequency and spectrum were indistinguishable between the Polkappa knockout mice and control mice. These results suggested that Polkappa is not essential for this process.