Dual AAV/IL-10 Plus STAT3 Anti-Inflammatory Gene Delivery Lowers Atherosclerosis in LDLR KO Mice, but without Increased Benefit

Both IL-10 and STAT3 are in the same signal transduction pathway, with IL-10-bound IL10 receptor (R) acting through STAT3 for anti-inflammatory effect. To investigate possible therapeutic synergism, we delivered both full-length wild-type human (h) STAT3 and hIL-10 genes by separate adenoassociated virus type 8 (AAV8) tail vein injection into LDLR KO on HCD. Compared to control Neo gene-treated animals, individual hSTAT3 and hIL-10 delivery resulted in significant reduction in atherogenesis, as determined by larger aortic lumen size, thinner aortic wall thickness, and lower blood velocity (all statistically significant). However, dual hSTAT3/hIL-10 delivery offered no improvement in therapeutic effect. Plasma cholesterol levels in dual hSTAT3/hIL-10-treated animals were statistically higher compared to hIL-10 alone. While no advantage was seen in this case, we consider that the dual gene approach has intrinsic merit, but properly chosen partnered genes must be used

High Reliability Pistons for Reciprocating Compressors with Validated Performance Modelling

LecturePiston ring leakage on reciprocating compressors is predictable due to defined leakage paths at the end gaps of piston rings. A new engineering approach quantifies the slippage and determines the dynamic pressure difference on each ring. With this approach the expected discharge gas temperature increase, expected capacity losses and the risks of rider bands activation due to piston ring slippage can be quantified. The piston design and ring styles can be iterated to find an optimized piston layout for a given application. 30% of the reciprocating compressors in the process gas industry show high sensitivity to piston ring leakage and subsequent performance related issues. This paper suggests quantifying piston performance as a standard when evaluating compressor reliability and efficiency. The industry managed to reduce compressor valve related problems due to more sophisticated modelling tools and smart design changes on valves. It is time to go that next step on pistons

AAV2/8-hSMAD3 gene delivery attenuates aortic atherogenesis, enhances Th2 response without fibrosis, in LDLR-KO mice on high cholesterol diet

BACKGROUND: Inflammation is a key etiologic component in atherogenesis and transforming growth factor beta 1 (TGFβ1) is a well known anti-inflammatory cytokine which potentially might be used to limit it. Yet TGFβ1 is pleiomorphic, causing fibrosis, cell taxis, and under certain circumstances, can even worsen inflammation. SMAD3 is an important member of TGFβ1′s signal transduction pathway, but is a fully intracellular protein. OBJECTIVES: With the hope of attenuating TGFβ1′s adverse systemic effects (eg. fibrosis) and accentuating its anti-inflammatory activity, we proposed the use of human (h)SMAD3 as an intracellular substitute for TGFβ1. STUDY DESIGN: To test this hypothesis adeno-associated virus type 2/8 (AAV)/hSMAD3 or AAV/Neo (control) was tail vein injected into the low density lipoprotein receptor knockout (LDLR-KO) mice, then placed on a high-cholesterol diet (HCD). RESULTS: The hSMAD3 delivery was associated with significantly lower atherogenesis as measured by larger aortic cross sectional area, thinner aortic wall thickness, and lower aortic systolic blood velocity compared with Neo gene-treated controls. HSMAD3 delivery also resulted in fewer aortic macrophages by immunohistochemistry for CD68 and ITGAM, and quantitative reverse transcriptase polymerase chain reaction analysis of EMR and ITGAM. Overall, aortic cytokine expression showed an enhancement of Th2 response (higher IL-4 and IL-10); while Th1 response (IL-12) was lower with hSMAD3 delivery. While TGFβ1 is often associated with increased fibrosis, AAV/hSMAD3 delivery exhibited no increase of collagen 1A2 or significantly lower 2A1 expression in the aorta compared with Neo-delivery. Connective tissue growth factor (CTGF), a mediator of TGFβ1/SMAD3-induced fibrosis, was unchanged in hSMAD3-delivered aortas. In the liver, all three of these genes were down-regulated by hSMAD3 gene delivery. CONCLUSION: These data strongly suggest that AAV/hSMAD3 delivery gave anti-atherosclerosis therapeutic effect without the expected undesirable effect of TGFβ1-associated fibrosis

Autocrine, Not Paracrine, Interferon-Gamma Gene Delivery Enhances Ex Vivo Antigen-Specific Cytotoxic T Lymphocyte Stimulation and Killing

The adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTL) shows promise in the treatment of cancer and infectious diseases. We utilize adeno-associated virus-(AAV-) based antigen gene-loaded dendritic cells (DCs) to stimulate such antigen-specific CTL. Yet further improvements in CTL stimulation and killing may result by gene delivery of various Th1-response interferons/cytokines, such as interferon γ (IFN-γ), as the delivered gene can continuously produce that interferon. However which immune cell type should optimally express IFN-γ is unclear as the phenotypes of both DC and T cells are enhanced by it. Here, we used AAV to compare and contrast IFN-γ gene delivery into DC or T cells, and versus the addition of exogenous IFN-γ, for stimulating carcinoembryonic antigen-(CEA-) specific CTL. It was found that AAV/IFN-γ delivery into T cells (autocrine) resulted in T cell populations with the highest CD8(+)/CD4(+) ratio, highest IFN-γ(+)/IL-4(+) ratio, highest CD69(+),CD8(+) levels, and lowest CD4(+)/CD25(+) levels, all consistent with the strongest Th1 response. Most importantly, AAV/IFN-γ transduction of T cells resulted in antigen-specific T cell populations with the highest killing capabilities, 49% above other treatments. These data strongly suggest that AAV/IFN-γ autocrine gene delivery into T cells is worthy of further study towards maximizing the generation of antigen-specific anticancer CTL killers

Cervical cancer isolate PT3, super-permissive for adeno-associated virus replication, over-expresses DNA polymerase δ, PCNA, RFC and RPA

<p>Abstract</p> <p>Background</p> <p>Adeno-associated virus (AAV) type 2 is an important virus due to its use as a safe and effective human gene therapy vector and its negative association with certain malignancies. AAV, a dependo-parvovirus, autonomously replicates in stratified squamous epithelium. Such tissue occurs in the nasopharynx and anogenitals, from which AAV has been clinically isolated. Related autonomous parvoviruses also demonstrate cell tropism and preferentially replicate in oncogenically transformed cells. Combining these two attributes of parvovirus tropism, squamous and malignant, we assayed if AAV might replicate in squamous cervical carcinoma cell isolates.</p> <p>Results</p> <p>Three primary isolates (PT1-3) and two established cervical cancer cell lines were compared to normal keratinocytes (NK) for their ability to replicate AAV. One isolate, PT3, allowed for high levels of AAV DNA replication and virion production compared to others. In research by others, four cellular components are known required for <it>in vitro </it>AAV DNA replication: replication protein A (RPA), replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and DNA polymerase delta (POLD1). Thus, we examined PT3 cells for expression of these components by DNA microarray and real-time quantitative PCR. All four components were over-expressed in PT3 over two representative low-permissive cell isolates (NK and PT1). However, this super-permissiveness did not result in PT3 cell death by AAV infection.</p> <p>Conclusion</p> <p>These data, for the first time, provide evidence that these four cellular components are likely important for AAV <it>in vivo </it>DNA replication as well as <it>in vitro</it>. These data also suggest that PT3 will be a useful reagent for investigating the AAV-permissive transcriptome and AAV anti-cancer effect.</p

Detection of adeno-associated virus type 2 genome in cervical carcinoma

Adeno-associated virus (AAV) can impair the replication of other viruses. Adeno-associated virus seroprevalences have been reported to be lower among women with cervical cancer. In-vitro, AAV can interfere with the production of human papillomavirus virions. Adeno-associated virus-2 DNA has also been detected in cervical cancer tissue, although not consistently. To evaluate the role of AAV infection in relation to invasive cervical cancer, we performed a nested case–control study within a retrospectively followed population-based cohort. A total of 104 women who developed invasive cervical cancer on average 5.6 years of follow-up (range: 0.5 months–26.2 years) and 104 matched control-women who did not develop cervical cancer during the same follow-up time were tested for AAV and human papillomavirus by polymerase chain reaction. At baseline, two (2%) case-women and three (3%) control-women were positive for AAV-2 DNA. At the time of cancer diagnosis, 12 (12%) case-women and 3 (3%) matched control-women were positive for AAV-2 DNA. Persisting AAV infection was not evident. In conclusion, AAV-2 DNA was present in a low proportion of cervical cancers and we found no evidence that the presence of AAV in cervical smears of healthy women would be associated with reduced risk of cervical cancer

Delivery of drugs, proteins and genes into cells using transferrin as a ligand for receptor-mediated endocytosis

Transferrin, an iron-transporting serum glycoprotein, is efficiently taken up into cells by the process of receptor-mediated endocytosis. Transferrin receptors are found on the surface of most proliferating cells, in elevated numbers on erythroblasts and on many kinds of tumors. The efficient cellular mechanism for uptake of transferrin has been subverted for the delivery of low-molecular-weight drugs, protein toxins, and liposomes by linkage of these agents to transferrin or to anti-transferrin receptor antibodies. Linkage may be via chemical conjugation procedures or by the generation of chimeric fusion proteins. Transferrin conjugated to DNA-binding compounds (e.g. polycations or intercalating agents) has been successfully used for the import of DNA molecules into cells. High-level gene expression is obtained only if endosome-disruptive agents such as influenza hemagglutinin peptides or adenovirus particles are included which release the DNA complex from intracellular vesicles into the cytoplasm

Synthesis and characterization of fourth generation polyester-based dendrimers with cationic amino acids-modified crown as promising water soluble biomedical devices

Dendrimers are nanostructured \u201carchitectural motifs\u201d which fascinate researchers fortheir several potentiality due to well\u2010tailored structure, symmetric tree\u2010like shape,and abilities in entrapping or binding hydrophilic or hydrophobic entities such asgenetic materials, drugs, and target molecules. Nowadays dendrimers inhabit thetop places among the materials suitable for biomedical applications as drug delivery,gene transfection, and imaging. In this work, we report the design and realization oftwo versatile successful procedures to decorate a fourth generation polyester\u2010baseddendrimer matrix with a mixture of four different amino acids. The hydrochloridedendrimers achieved after removal of protecting groups were characterized by acore\u2010shell structure. They harmonized a not charged hydrolysable inner matrixpotentially able to accommodate hydrophobic molecules and a cationic highlyhydrophilic crown conferred by biocompatible amino acids that provided very satis-factory buffer capacity and will allow easy host/guest electrostatic interactions.Their structures and peripheral composition were confirmed by NMR analysis andexperimental molecular weight computed by volumetric titration, while their buffercapacity was obtained by potentiometric titrations. Because in the inner matrix, theachieved hetero dendrimers do not present the high density of positive chargestypical of PAMAM, they ensure a lower level of toxicity. But thanks to the cationicperiphery, as preliminary investigations still in progress have already put in evidence, they were able to entrap not water soluble molecules by electrostatic inter-actions, with the result to increase their water solubility in a very satisfactory oramazing way. They therefore represent two new very promising devices for bio-medical applications