In vivo gene transfer into the ocular ciliary muscle mediated by ultrasound and microbubbles.

This study aimed to assess application of ultrasound (US) combined with microbubbles (MB) to transfect the ciliary muscle of rat eyes. Reporter DNA plasmids encoding for Gaussia luciferase, β-galactosidase or the green fluorescent protein (GFP), alone or mixed with 50% Artison MB, were injected into the ciliary muscle, with or without US exposure (US set at 1 MHz, 2 W/cm(2), 50% duty cycle for 2 min). Luciferase activity was measured in ocular fluids at 7 and 30 days after sonoporation. At 1 week, the US+MB treatment showed a significant increase in luminescence compared with control eyes, injected with plasmid only, with or without MB (×2.6), and, reporter proteins were localized in the ciliary muscle by histochemical analysis. At 1 month, a significant decrease in luciferase activity was observed in all groups. A rise in lens and ciliary muscle temperature was measured during the procedure but did not result in any observable or microscopic damages at 1 and 8 days. The feasibility to transfer gene into the ciliary muscle by US and MB suggests that sonoporation may allow intraocular production of proteins for the treatment of inflammatory, angiogenic and/or degenerative retinal diseases.

Bystander activation of CD4+ T cells.

Bystander activation of T cells, i.e. the stimulation of unrelated (heterologous) T cells by cytokines during an Ag-specific T-cell response, has been best described for CD8(+) T cells. In the CD8(+) compartment, the release of IFN and IFN-inducers leads to the production of IL-15, which mediates the proliferation of CD8(+) T cells, notably memory-phenotype CD8(+) T cells. CD4(+) T cells also undergo bystander activation, however, the signals inducing this Ag-nonspecific stimulation of CD4(+) T cells are less well known. A study in this issue of the European Journal of Immunology sheds light on this aspect, suggesting that common gamma-chain cytokines including IL-2 might be involved in bystander activation of CD4(+) T cells.

Members of the PHO1 gene family show limited functional redundancy in phosphate transfer to the shoot, and are regulated by phosphate deficiency via distinct pathways.

The PHO1 family comprises 11 members in Arabidopsis thaliana. In order to decipher the role of these genes in inorganic phosphate (Pi) transport and homeostasis, complementation of the pho1 mutant, deficient in loading Pi to the root xylem, was determined by the expression of the PHO1 homologous genes under the control of the PHO1 promoter. Only PHO1 and the homologue PHO1;H1 could complement pho1. The PHO1;H1 promoter was active in the vascular cylinder of roots and shoots. Expression of PHO1;H1 was very low in Pi-sufficient plants, but was strongly induced under Pi-deficient conditions. T-DNA knock-out mutants of PHO1;H1 neither showed growth defects nor alteration in Pi transport dynamics, or Pi content, compared with wild type. However, the double mutant pho1/pho1;h1 showed a strong reduction in growth and in the capacity to transfer Pi from the root to the shoot compared with pho1. Grafting experiments revealed that phenotypes associated with the pho1 and pho1/pho1;h1 mutants were linked to the lack of gene expression in the root. The increased expression of PHO1;H1 under Pi deficiency was largely controlled by the transcription factor PHR1 and was suppressed by the phosphate analogue phosphite, whereas the increase of PHO1 expression was independent of PHR1 and was not influenced by phosphite. Together, these data reveal that although transfer of Pi to the root xylem vessel is primarily mediated by PHO1, the homologue PHO1;H1 also contributes to Pi loading to the xylem, and that the two corresponding genes are regulated by Pi deficiency by distinct signal transduction pathways.

The IL-4 rapidly produced in BALB/c mice after infection with Leishmania major down-regulates IL-12 receptor beta 2-chain expression on CD4+ T cells resulting in a state of unresponsiveness to IL-12.

Within 1 day of infection with Leishmania major, susceptible BALB/c mice produce a burst of IL-4 in their draining lymph nodes, resulting in a state of unresponsiveness to IL-12 in parasite-specific CD4+ T cells within 48 h. In this report we examined the molecular mechanism underlying this IL-12 unresponsiveness. Extinction of IL-12 signaling in BALB/c mice is due to a rapid down-regulation of IL-12R beta2-chain mRNA expression in CD4+ T cells. In contrast, IL-12R beta2-chain mRNA expression was maintained on CD4+ T cells from resistant C57BL/6 mice. The down-regulation of the IL-12R beta2-chain mRNA expression in BALB/c CD4+ T cells is a consequence of the early IL-4 production. In this murine model of infection, a strict correlation is shown in vivo between expression of the IL-12R beta2-chain in CD4+ T cells and the development of a Th1 response and down-regulation of the mRNA beta2-chain expression and the maturation of a Th2 response. Treatment of BALB/c mice with IFN-gamma, even when IL-4 has been produced for 48 h, resulted in maintenance of IL-12R beta2-chain mRNA expression and IL-12 responsiveness. The data presented here support the hypothesis that the genetically determined susceptibility of BALB/c mice to infection with L. major is primarily based on an up-regulation of IL-4 production, which secondarily induces extinction of IL-12 signaling.

A role for the alpha-chain connecting peptide motif in mediating TCR-CD8 cooperation.

To generate peripheral T cells that are both self-MHC restricted and self-MHC tolerant, thymocytes are subjected to positive and negative selection. How the TCR discriminates between positive and negative selection ligands is not well understood, although there is substantial evidence that the CD4 and CD8 coreceptors play an important role in this cell fate decision. We have previously identified an evolutionarily conserved motif in the TCR, the alpha-chain connecting peptide motif (alpha-CPM), which allows the TCR to deliver positive selection signals. Thymocytes expressing alpha-CPM-deficient receptors do not undergo positive selection, whereas their negative selection is not impaired. In this work we studied the ligand binding and receptor function of alpha-CPM-deficient TCRs by generating T cell hybridomas expressing wild-type or alpha-CPM-deficient forms of the T1 TCR. This K(d)-restricted TCR is specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide(252-260) IASA-YIPSAEK(ABA)I and is therefore amenable to TCR photoaffinity labeling. The experiments presented in this work show that alpha-CPM-deficient TCRs fail to cooperate with CD8 to enhance ligand binding and functional responses.

Gene transfer of programmed death ligand-1.Ig prolongs cardiac allograft survival.

BACKGROUND: The CD28 homologue programmed death-1 (PD-1) and its ligands, PD-L1 and PD-L2 (which are homologous to B7), constitute an inhibitory pathway of T cell costimulation. The PD-1 pathway is of interest for immune-mediated diseases given that PD-1-deficient mice develop autoimmune diseases. We have evaluated the effect of local overexpression of a PD-L1.Ig fusion protein on cardiac allograft survival. METHODS: Adenovirus-mediated PD-L1.Ig gene transfer was performed in F344 rat donor hearts placed in the abdominal position in Lewis recipients. Inflammatory cell infiltrates in the grafts were assessed by immunohistochemistry. RESULTS: Allografts transduced with the PD-L1.Ig gene survived for longer periods of time compared with those receiving noncoding adenovirus or virus dilution buffer alone: median survival time (MST), 17 (range: 16-20) days vs. 11 (8-14) and 9 (8-13) days, respectively (P < 0.001). PD-L1.Ig gene transfer combined with a subtherapeutic regimen of cyclosporin A (CsA) was superior to CsA alone: MST, 25 (15-42) vs. 15 (13-19) days (P < 0.05). PD-L1.Ig gene transfer was associated with decreased numbers of CD4 cells and monocytes/macrophages infiltrating the graft (P < 0.05). CONCLUSIONS: Localized PD-L1.Ig expression in donor hearts attenuates acute allograft rejection in a rat model. The effect is additive to that of a subtherapeutic regimen of CsA. These results suggest that targeting of PD-1 by gene therapy may inhibit acute cardiac allograft rejection in vivo.

Characterization of MAP1B heavy chain interaction with actin.

Microtubule-associated protein 1B is an essential protein during brain development and neurite outgrowth and was studied by several assays to further characterize actin as a major interacting partner. Tubulin and actin co-immunoprecipitated with MAP1B at similar ratios throughout development. Their identity was identified by mass spectrometry and was confirmed by Western blots. In contrast to previous reports, the MAP1B-actin interaction was not dependent on the MAP1B phosphorylation state, since actin was precipitated from brain tissue throughout development at similar ratios and equal amounts were precipitated before and after dephosphorylation with alkaline phosphatase. MAP1B heavy chain was able to bind actin directly and therefore the N-terminal part of MAP1B heavy chain must also contain an actin-binding site. The binding force of this interaction was measured by atomic force microscopy and values were in the same range as those of MAP1B binding to tubulin or that measured in MAP1B self-aggregation. Aggregation was confirmed by negative staining and electron microscopy. Experiments including COS-7 cells, PC12 cells, cytochalasin D and immunocytochemistry with subsequent confocal laser microscopy, suggested that MAP1B may bind to actin but has no obvious microfilament stabilizing effect. We conclude, that the MAP1B heavy chain has a microtubule-stabilization effect, and contains an actin-binding site that may play a role in the crosslinking of actin and microtubules, a function that may be important in neurite elongation.

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors.

Gene transfer-based therapeutic approaches have greatly benefited from the ability of some viral vectors to efficiently integrate within the cell genome and ensure persistent transmission of newly acquired transgenes to the target cell progeny. However, integration of provirus has been associated with epigenetic repercussions that may influence the expression of both the transgene and cellular genes close to vector integration loci. The exploitation of genetic insulator elements may overcome both issues through their ability to act as barriers that limit transgene silencing and/or as enhancer-blockers preventing the activation of endogenous genes by the vector enhancer. We established quantitative plasmid-based assay systems to screen enhancer-blocker and barrier genetic elements. Short synthetic insulators that bind to nuclear factor-I protein family transcription factors were identified to exert both enhancer-blocker and barrier functions, and were compared to binding sites for the insulator protein CTCF (CCCTC-binding factor). Gamma-retroviral vectors enclosing these insulator elements were produced at titers similar to their non-insulated counterparts and proved to be less genotoxic in an in vitro immortalization assay, yielding lower activation of Evi1 oncogene expression and reduced clonal expansion of bone marrow cells.

Optimizing the supply chain strategy of a multi-unit Finnish nursery company

Abstract

Laser scanning evaluation of atrophy after autologous free muscle transfer.

Denervated muscle tissue undergoes morphologic changes that result in atrophy. The amount of muscle atrophy after denervation following free muscle transfer has not been measured so far. Therefore, the amount of muscle atrophy in human free muscle transfer for lower extremity reconstruction was measured in a series of 10 patients. Three-dimensional laser surface scanning was used to measure flap volume changes 2 weeks as well as 6 and 12 months after the operation. None of the muscles transferred was re-innervated.All muscles healed uneventfully without signs of compromised perfusion resulting in partial flap loss. The muscle volume decreased to 30 ± 4% and 19 ± 4% 6 and 12 months, respectively, after the operation, ie, the volume decreased by approximately 80% within a 12-month period.Denervated free muscle flap tissue undergoes massive atrophy of approximately 80%, mostly within the first 6 months.

Combined effect of natural antimicrobials and high pressure processing to prevent Listeria monocytogenes growth after a cold chain break during storage of cooked ham

The effect of high pressure processing (400 MPa for 10 min) and natural antimicrobials 2 (enterocins and lactate-diacetate) on the behaviour of L. monocytogenes in sliced cooked ham 3 during refrigerated storage (1ºC and 6ºC) was assessed. The efficiency of the treatments after a 4 cold chain break was evaluated. Lactate-diacetate exerted a bacteriostatic effect against L. 5 monocytogenes during the whole storage period (3 months) at 1ºC and 6ºC, even after 6 temperature abuse. The combination of low storage temperature (1ºC), high pressure 7 processing (HPP) and addition of lactate-diacetate reduced the levels of L. monocytogenes 8 during storage by 2.7 log CFU/g. The most effective treatment was the combination of HPP, 9 enterocins and refrigeration at 1ºC, which reduced the population of the pathogen to final counts 10 of 4 MPN/g after 3 months of storage, even after the cold chain break.

Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses.

Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell-expressed CXCR6.