Overcoming GNA/RNA base-pairing limitations using isonucleotides improves the pharmacodynamic activity of ESC+ GalNAc-siRNAs

We recently reported that RNAi-mediated off-target effects are important drivers of the hepatotoxicity observed for a subset of GalNAc–siRNA conjugates in rodents, and that these findings could be mitigated by seed-pairing destabilization using a single GNA nucleotide placed within the seed region of the guide strand. Here, we report further investigation of the unique and poorly understood GNA/RNA cross-pairing behavior to better inform GNA-containing siRNA design. A reexamination of published GNA homoduplex crystal structures, along with a novel structure containing a single (S)-GNA-A residue in duplex RNA, indicated that GNA nucleotides universally adopt a rotated nucleobase orientation within all duplex contexts. Such an orientation strongly affects GNA-C and GNA-G but not GNA-A or GNA-T pairing in GNA/RNA heteroduplexes. Transposition of the hydrogen-bond donor/acceptor pairs using the novel (S)-GNA-isocytidine and -isoguanosine nucleotides could rescue productive base-pairing with the complementary G or C ribonucleotides, respectively. GalNAc-siRNAs containing these GNA isonucleotides showed an improved in vitro activity, a similar improvement in off-target profile, and maintained in vivo activity and guide strand liver levels more consistent with the parent siRNAs than those modified with isomeric GNA-C or -G, thereby expanding our toolbox for the design of siRNAs with minimized off-target activity.     

Roles of Residues Arg-61 and Gln-38 of Human DNA Polymerase η in Bypass of Deoxyguanosine and 7,8-Dihydro-8-oxo-2′-deoxyguanosine

Like the other Y-family DNA polymerases, human DNA polymerase η (hpol η) has relatively low fidelity and is able to tolerate damage during DNA synthesis, including 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG), one of the most abundant DNA lesions in the genome. Crystal structures show that Arg-61 and Gln-38 are located near the active site and may play important roles in the fidelity and efficiency of hpol η. Site-directed mutagenesis was used to replace these side chains either alone or together, and the wild type or mutant proteins were purified and tested by replicating DNA past deoxyguanosine (G) or 8-oxoG. The catalytic activity of hpol η was dramatically disrupted by the R61M and Q38A/R61A mutations, as opposed to the R61A and Q38A single mutants. Crystal structures of hpol η mutant ternary complexes reveal that polarized water molecules can mimic and partially compensate for the missing side chains of Arg-61 and Gln-38 in the Q38A/R61A mutant. The combined data indicate that the positioning and positive charge of Arg-61 synergistically contribute to the nucleotidyl transfer reaction, with additional influence exerted by Gln-38. In addition, gel filtration chromatography separated multimeric and monomeric forms of wild type and mutant hpol η, indicating the possibility that hpol η forms multimers in vivo.     

Tolerance induced by apoptotic antigen-coupled leukocytes is induced by PD-L1+ and IL-10-producing splenic macrophages and maintained by T regulatory cells

Ag-specific tolerance is a highly desired therapy for immune-mediated diseases. Intravenous infusion of protein/peptide Ags linked to syngeneic splenic leukocytes with ethylene carbodiimide (Ag-coupled splenocytes [Ag-SP]) has been demonstrated to be a highly efficient method for inducing peripheral, Ag-specific T cell tolerance for treatment of autoimmune disease. However, little is understood about the mechanisms underlying this therapy. In this study, we show that apoptotic Ag-SP accumulate in the splenic marginal zone, where their uptake by F4/80(+) macrophages induces production of IL-10, which upregulates the expression of the immunomodulatory costimulatory molecule PD-L1 that is essential for Ag-SP tolerance induction. Ag-SP infusion also induces T regulatory cells that are dispensable for tolerance induction but required for long-term tolerance maintenance. Collectively, these results indicate that Ag-SP tolerance recapitulates how tolerance is normally maintained in the hematopoietic compartment and highlight the interplay between the innate and adaptive immune systems in the induction of Ag-SP tolerance. To our knowledge, we show for the first time that tolerance results from the synergistic effects of two distinct mechanisms, PD-L1-dependent T cell-intrinsic unresponsiveness and the activation of T regulatory cells. These findings are particularly relevant as this tolerance protocol is currently being tested in a Phase I/IIa clinical trial in new-onset relapsing-remitting multiple sclerosis.     

Fatty acid transport protein 1 and long-chain acyl coenzyme A synthetase 1 interact in adipocytes

The fatty acid transport proteins (FATP) and long-chain acyl coenzyme A synthetase (ACSL) proteins have been shown to play a role in facilitating long-chain fatty acid (LCFA) transport in mammalian cells under physiologic conditions. The involvement of both FATP and ACSL proteins is consistent with the model of vectorial acylation, in which fatty acid transport is coupled to esterification. This study was undertaken to determine whether the functions of these proteins are coordinated through a protein-protein interaction that might serve as a point of regulation for cellular fatty acid transport. We demonstrate for the first time that FATP1 and ACSL1 coimmunoprecipitate in 3T3-L1 adipocytes, indicating that these proteins form an oligomeric complex. The efficiency of FATP1 and ACSL1 coimmunoprecipitation is unaltered by acute insulin treatment, which stimulates fatty acid uptake, or by treatment with isoproterenol, which decreases fatty acid uptake and stimulates lipolysis. Moreover, inhibition of ACSL1 activity in adipocytes impairs fatty acid uptake, suggesting that esterification is essential for fatty acid transport. Together, our findings suggest that a constitutive interaction between FATP1 and ACSL1 contributes to the efficient cellular uptake of LCFAs in adipocytes through vectorial acylation.     

Inhibition of T cell responses to alloantigens and polyclonal mitogens by Ly-5 antisera

The effects of Ly-5 alloantisera on the generation of cytotoxic T cells (CTL), on the effector phase of CTL killing, and on polyclonal mitogenesis were studied. Ly-5 antisera added at the beginning of mixed lymphocyte culture (MLC) suppressed the production of CTL in an allele-specific manner. Neither Ly-5.1 nor Ly-5.2 antisera inhibited the generation of cytotoxic effectors by Ly-5.1/Ly-5.2 heterozygous spleen cells; however, a combination of Ly-5.1 and Ly-5.2 antisera markedly suppressed the appearance of Ly-5 heterozygous CTL. Similarly, Ly-5 antisera inhibited the effector phase of CTL killing in an allele-specific manner. In addition, Ly-5 alloantisera specifically blocked concanavalin A and oxidative mitogenesis of splenocytes carrying the appropriate Ly-5 alloantigen. The results are discussed in light of a possible functional role of Ly-5 molecules in immune processes.     

DNA sequence patterns in precisely positioned nucleosomes

Several investigators have recognized the importance of non-periodic DNA sequence information in determining the translational position of precisely positioned nucleosomes. The purpose of this study is to determine the extent of such information, in addition to the character of periodic information present. This is accomplished by examining the half-nucleosome DNA sequences of a considerable number of precisely positioned nucleosomes, and determining the probability of occurrence of each dinucleotide type as a function of position from the nucleosome center to the terminus (positions 0 to 72). By the nature of this procedure, no assumptions of periodicity are made. The results show the importance of several DNA sequence periodicities including 6-7, 10, and 21 base pairs, in addition to significant nonperiodic information. The results demonstrate that each dinucleotide type is unique in terms of its positional preference in precisely positioned nucleosomes (for example AA not equal to TT). The probabilities of occurrence for the dinucleotide types can be used to predict the translational positions of a number of observed nucleosomes.     

Cryptosporidium parvum-induced inflammatory bowel disease of TCR-beta- x TCR-delta-deficient mice

Experimental inoculation of neonatal immunocompetent strains of mice with Cryptosporidium parvum results in a transient, noninflammatory enteric infection. In the present study, we show that inoculation of mice deficient in alphabeta and gammadelta T cells (TCR-beta- x TCR-delta-deficient mice) with C. parvum results in persistent infection and severe inflammatory bowel disease-like lesions. The most severe lesions in these mice were in the cecum with similar yet less severe lesions in the ileum and proximal colon. The most notable aspect of the histopathology was glandular hyperplasia with abscess formation, extensive fibrosis of the lamina propria with infiltrates of predominately polymorphonuclear cells and macrophages, and a few small aggregates of B cells. Persistently infected mice also developed extensive hepatic periportal fibrosis in association with C. parvum colonization of bile ducts. Lesions observed in TCR-beta- x TCR-delta-deficient mice were markedly different than previously described lesions detected in C. parvum-infected TCR-alpha-deficient mice. Cryptosporidium parvum-infected TCR-alpha-deficient mice have extensive infiltrations of B cells, whereas TCR-beta- x TCR-delta-deficient mice had only a few small aggregates of B cells. These findings indicate that although gammadelta T cells are not necessary for induction of intestinal inflammation in C. parvum-infected alphabeta T-cell-deficient mice, their presence does alter the morphology of the ensuing lesion.