In vivo engineering of mobilized stem cell grafts with the immunomodulatory drug FTY720 for allogeneic transplantation

The immunological attributes of stem cell grafts play an important role in the outcome of allogeneic stem cell transplants. Currently, ex vivo manipulation techniques such as bulk T cell depletion or positive selection of CD34(+) cells are utilized to improve the immunological attributes of grafts and minimize the potential for graft-versus-host disease (GvHD). Here, we demonstrate a novel graft engineering technique, which utilizes the immunomodulatory drug FTY720 for in vivo depletion of naïve T (TN ) cells from donor G-CSF-mobilized grafts without ex vivo manipulation. We show that treatment of donor mice with FTY720 during mobilization depletes grafts of TN cells and prevents lethal GvHD following transplantation in a major mismatch setting. Importantly, both stem cells and NK cells are retained in the FTY720-treated grafts. FTY720 treatment does not negatively affect the engraftment potential of stem cells as demonstrated in our congenic transplants or the functionality of NK cells. In addition, potentially useful memory T cells may be retained in the graft. These findings suggest that FTY720 may be used to optimize the immunological attributes of G-CSF-mobilized grafts by removing potentially deleterious TN cells which can contribute to GvHD, and by retaining useful cells which can promote immunity in the recipient

UPF1, a Conserved Nonsense-Mediated mRNA Decay Factor, Regulates Cyst Wall Protein Transcripts in Giardia lamblia

The Giardia lamblia cyst wall is required for survival outside the host and infection. Three cyst wall protein (cwp) genes identified to date are highly up-regulated during encystation. However, little is known of the molecular mechanisms governing their gene regulation. Messenger RNAs containing premature stop codons are rapidly degraded by a nonsense-mediated mRNA decay (NMD) system to avoid production of non-functional proteins. In addition to RNA surveillance, NMD also regulates thousands of naturally occurring transcripts through a variety of mechanisms. It is interesting to know the NMD pathway in the primitive eukaryotes. Previously, we have found that the giardial homologue of a conserved NMD factor, UPF1, may be functionally conserved and involved in NMD and in preventing nonsense suppression. In this study, we tested the hypothesis that NMD factors can regulate some naturally occurring transcripts in G. lamblia. We found that overexpression of UPF1 resulted in a significant decrease of the levels of CWP1 and cyst formation and of the endogenous cwp1-3, and myb2 mRNA levels and stability. This indicates that NMD could contribute to the regulation of the cwp1-3 and myb2 transcripts, which are key to G. lamblia differentiation into cyst. Interestingly, we also found that UPF1 may be involved in regulation of eight other endogenous genes, including up-regulation of the translation elongation factor gene, whose product increases translation which is required for NMD. Our results indicate that NMD factor could contribute to the regulation of not only nonsense containing mRNAs, but also mRNAs of the key encystation-induced genes and other endogenous genes in the early-diverging eukaryote, G. lamblia

Structural and functional insights into the human Upf1 helicase core

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance pathway that recognizes and degrades aberrant mRNAs containing premature stop codons. A critical protein in NMD is Upf1p, which belongs to the helicase super family 1 (SF1), and is thought to utilize the energy of ATP hydrolysis to promote transitions in the structure of RNA or RNA–protein complexes. The crystal structure of the catalytic core of human Upf1p determined in three states (phosphate-, AMPPNP- and ADP-bound forms) reveals an overall structure containing two RecA-like domains with two additional domains protruding from the N-terminal RecA-like domain. Structural comparison combined with mutational analysis identifies a likely single-stranded RNA (ssRNA)-binding channel, and a cycle of conformational change coupled to ATP binding and hydrolysis. These conformational changes alter the likely ssRNA-binding channel in a manner that can explain how ATP binding destabilizes ssRNA binding to Upf1p

Chandipura Virus infection in mice: the role of toll like receptor 4 in pathogenesis

<p>Abstract</p> <p>Background</p> <p>The susceptibility of mice and humans to Chandipura virus infection is age-dependent. Upon experimental infection, mice secrete significant amounts of proinflammatory cytokines. Similarly, children who recover from natural infection with the virus show significant amounts of TNF-α production, suggesting that innate immunity plays a major role in the response to Chandipura virus. Toll-like receptors (TLR) are key host molecules involved in innate immune responses in infections. Therefore, the aim of this study was to examine the role of TLR in the response to Chandipura virus infection.</p> <p>Methods</p> <p>The mouse monocyte-macrophage cell line, RAW 264.7, and C3H/HeJ mice were used as models. Micro array techniques were used to identify the type of TLR involved in the response to infection. The results were validated by examining TLR expression using flow cytometry and by measuring the levels of proinflammatory cytokines and nitric oxide (NO) in the culture supernatants using bead assays and the Griess method, respectively. The pathogenic role of Toll-like receptor 4 (TLR4) was studied in a TLR4 mutant strain of mice -C3H/HeJ and the results compared with those from wild-type mice- C3H/CaJ. The pathogenic effects of NO were studied by treating experimentally infected mice with the NO inhibitor, aminoguanidine (AG).</p> <p>Results</p> <p>The micro array results showed that TLR4 was regulated after Chandipura virus infection. At high multiplicities of infection (10 MOI), RAW cells up- regulated cell surface expression of TLR4 and secreted significant amounts of TNF-α, MCP-1, IL-10 and IL-12 and NO. The survival rate of C3H/HeJ mice was higher than those of wild-type C3H/CaJ mice. The survived C3H/HeJ mice secreted significant quantity of MCP-1 and IFN-γ cytokines and cleared virus from brain. Similarly, the survival rate of AG-treated mice was higher than those of the untreated controls.</p> <p>Conclusions</p> <p>Chandipura virus regulates TLR4, which leads to the secretion of proinflammatory cytokines and NO by infected RAW cells. Difference in survival rate in TLR4 mutant mice and nitric oxide inhibitor treated mice, confirmed the role of these molecules in disease pathogenesis. The result is significant in clinical management and designing antiviral intervention for Chandipura virus infection.</p