Promoter Attenuation in Gene Therapy: Interferon-γ and Tumor Necrosis Factor-α Inhibit Transgene Expression

Overview summary Transgene expression can be eliminated even in the presence of substantial amounts of vector DNA in the transduced cells, which suggests that mechanisms other than the antigen-specific immune response may mediate non-cytodestructive events that determine the presence of transgene expression. Our data indicate that the cytokines interferon-γ) (IFN-γ) and tumor necrosis factor-α (TNF-α) inhibit transgene expression from certain widely used viral promoters/enhancers (human cytomegalovirus immediate early, Rous sarcoma virus long terminal repeat, simian virus 40, Moloney murine leukemia virus long terminal repeat) delivered by adenoviral, retroviral, or plasmid vectors in vivo. Inhibition is at the mRNA level and cytokines do not cause vector DNA degradation, inhibit total cellular protein synthesis, or kill infected/transfected cells. Thus, cytokine-regulated promoter function rather than specific immune destruction could limit transgene expression. These results have significant implications for the construction of transfer vectors for human gene therapy because gene transfer vectors could be exposed to a cytokine-rich environment when they are administered in vivo.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63157/1/hum.1997.8.17-2019.pd

Adenovirus-Mediated Gene Transfer of Viral Interleukin-10 Inhibits the Immune Response to Both Alloantigen and Adenoviral Antigen

Overview summary Adenoviral vectors are efficient for in vivo delivery of genes to a wide variety of tissue types, whereas the duration of expression is limited by the potent adenovirus-specific immune response directed to the infected cell. In this study, we demonstrate that adenovirus-mediated gene transfer and expression of viral interleukin-10 (vIL-10) not only prolongs murine cardiac allograft survival, but also inhibits the immune response toward adenoviral antigens, and thereby improves the persistence of the vector and extends transgene expression. These findings could be used to design a new generation of adenoviral vector that expresses both an immunosuppressive cytokine gene and another gene of interest. This strategy should have general application in many gene therapy settings other than transplantation. Nonetheless, although the efficacy of adenoviral vectors can be improved by incorporating immunosuppressive genes into the vector, there are also nonimmune mechanisms serving to limit vector gene expression.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63260/1/hum.1997.8.11-1365.pd

Efficient Transfer of Genes into Murine Cardiac Grafts by Starburst Polyamidoamine Dendrimers

Overview summary Plasmid-mediated gene therapy has been used to deliver immunosuppressive molecules into allografts to prolong graft survival. However, direct injection of naked plasmid DNA is inefficient because transgene expression is low and transient. This study investigated the ability of Starburst dendrimers to augment plasmid-mediated gene transfer efficiency in a murine cardiac transplantation model. The results demonstrate that dendrimers increased the efficiency of transfer and expression of exogenous DNA in cardiac grafts. Improved expression of an immunosuppressive cytokine viral interleukin-10 (vIL-10) by dendrimers significantly prolonged allograft survival. The dose of DNA, the charge ratio of DNA to dendrimer, and the size generation of the dendrimers were all critical for prolongation of allograft survival. Thus, the use of the Starburst dendrimer as a carrier molecule for plasmid-mediated gene transfer improved the efficiency of transfer and expression, providing further therapeutic value for treatment of cardiac allograft rejection.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63156/1/hum.1998.9.4-553.pd

Evolutionary Analysis of Structural Protein Gene VP1 of Foot-and-Mouth Disease Virus Serotype Asia 1

Foot-and-mouth disease virus (FMDV) serotype Asia 1 was mostly endemic in Asia and then was responsible for economically important viral disease of cloven-hoofed animals, but the study on its selection and evolutionary process is comparatively rare. In this study, we characterized 377 isolates from Asia collected up until 2012, including four vaccine strains. Maximum likelihood analysis suggested that the strains circulating in Asia were classified into 8 different groups (groups I–VIII) or were unclassified (viruses collected before 2000). On the basis of divergence time analyses, we infer that the TMRCA of Asia 1 virus existed approximately 86.29 years ago. The result suggested that the virus had a high mutation rate (5.745 × 10−3 substitutions/site/year) in comparison to the other serotypes of FMDV VP1 gene. Furthermore, the structural protein VP1 was under lower selection pressure and the positive selection occurred at many sites, and four codons (positions 141, 146, 151, and 169) were located in known critical antigenic residues. The remaining sites were not located in known functional regions and were moderately conserved, and the reason for supporting all sites under positive selection remains to be elucidated because the power of these analyses was largely unknown

Подбор оборудования для эксплуатации нефтяной скважины

Результат исследования: Был подобран погружной электроцентробежный насос, для данной нефтяной скважины, достоинствами которого является простота конструкции, так же унифицированная база запасных частей и комплектующих изделий отечественного производства.Result of the research: A submersible electric centrifugal pump was selected, for this oil well, whose advantages are simplicity of designs, as well as a unified base of spare parts and components of domestic production

Analysis of synonymous codon usage in Hepatitis A virus

<p>Abstract</p> <p>Background</p> <p>Hepatitis A virus is the causative agent of type A viral hepatitis, which causes occasional acute hepatitis. Nevertheless, little information about synonymous codon usage pattern of HAV genome in the process of its evolution is available. In this study, the key genetic determinants of codon usage in HAV were examined.</p> <p>Results</p> <p>The overall extent of codon usage bias in HAV is high in <it>Picornaviridae</it>. And the patterns of synonymous codon usage are quite different in HAV genomes from different location. The base composition is closely correlated with codon usage bias. Furthermore, the most important determinant that results in such a high codon bias in HAV is mutation pressure rather than natural selection.</p> <p>Conclusions</p> <p>HAV presents a higher codon usage bias than other members of <it>Picornaviridae</it>. Compositional constraint is a significant element that influences the variation of synonymous codon usage in HAV genome. Besides, mutation pressure is supposed to be the major factor shaping the hyperendemic codon usage pattern of HAV.</p

Lymphangiogenesis Is Required for Pancreatic Islet Inflammation and Diabetes

Lymphangiogenesis is a common phenomenon observed during inflammation and engraftment of transplants, but its precise role in the immune response and underlying mechanisms of regulation remain poorly defined. Here we showed that in response to injury and autoimmunity, lymphangiogenesis occurred around islets and played a key role in the islet inflammation in mice. Vascular endothelial growth factors receptor 3 (VEGFR3) is specifically involved in lymphangiogenesis, and blockade of VEGFR3 potently inhibited lymphangiogenesis in both islets and the draining LN during multiple low-dose streptozotocin (MLDS) induced autoimmune insulitis, which resulted in less T cell infiltration, preservation of islets and prevention of the onset of diabetes. In addition to their well-known conduit function, lymphatic endothelial cells (LEC) also produced chemokines in response to inflammation. These LEC attracted two distinct CX3CR1hi and LYVE-1+ macrophage subsets to the inflamed islets and CX3CR1hi cells were influenced by LEC to differentiate into LYVE-1+ cells closely associated with lymphatic vessels. These observations indicate a linkage among lymphangiogenesis and myeloid cell inflammation during insulitis. Thus, inhibition of lymphangiogenesis holds potential for treating insulitis and autoimmune diabetes

Molecular mechanisms of interleukin -10 -mediated immune regulation.

Cellular IL-10s (cIL-10) of human and murine origin have extensive sequence and structural homology to the Epstein-Barr Virus BCRF I gene product, known as viral IL-10 (vIL-10). Although these cytokines share many immunosuppressive properties, vIL-10 lacks several of the immunostimulatory activities of cIL-10 on certain cell types. The molecular and cellular basis for this dichotomy is not currently defined. In this thesis, we show that the single amino acid isoleucine at position 87 of cIL-10 is required for its immunostimulatory function. Substitution of isoleucine in cIL-10 with alanine, which corresponds to the vIL-10 residue, abrogates immunostimulatory activity for thymocytes, mast cells and alloantigenic responses, while preserving immunosuppressive activity for inhibition of IFNgamma production and prolongation of cardiac allograft survival. Conversely, substitution of alanine with isoleucine in vIL-10 converts it to a cIL-10 like molecule with immunostimulatory activity. We characterized the receptor binding specificity and affinity for hIL-10 and vIL-10 using CHO cells expressing different IL-10 receptor component(s). IL-10R1 alone binds to hIL-10 or vIL-10, and the binding affinity of hIL-10 is 1000-fold higher than that of vL-10. Although IL-10R2 alone does not bind to IL-10, it is essential for both hIL-10 and vIL-10 mediated signal transduction and immune regulation. Further studies on previously identified single amino acid mutants demonstrated that the amino acid at position 87 of IL-10 plays a critical role in determining its receptor binding affinity, suggesting that receptor binding affinity rather than specificity might determine the spectrum of the biological activities of various IL-10s. The signal transduction initiated by hIL-10 and vIL-10 were compared in B cell and mast cell lines, and we demonstrated that the inability of vIL-10 to stimulate immune responses as compared to hIL-10 is largely due to the inability to initiate signaling. The absent signal transduction is due to low level expression of cell surface IL-10R1 expression, as overexpressing IL-10R1 on these cells allows vIL-10 to initiate hIL-10-like signals and subsequent biological responses. Further more, the intensity of these responses depends on the density of IL-10R1 expression. These data demonstrate that expression of IL-10R1 plays a critical role in determining whether cells respond to IL-10s. Taken together, our results demonstrate that ligand-receptor interactions play a critical role in determining whether certain cells are activated by various IL-10s. Alteration of receptor binding affinity or modulation of cell surface IL-10R1 expression can lead an IL-10 molecule to have stimulatory or suppressive immune responses in vivo.Ph.D.Health and Environmental SciencesImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/132332/2/9963767.pd