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We previously showed that the 12/15-lipoxygenase (12/15-LO) pathway of arachidonate acid metabolism is involved in multiple events related to diabetic nephropathy (DN), including glomerular hypertrophy and extracellular matrix deposition (Kang SW, Adler SG, Nast CC, LaPage J, Gu JL, Nadler JL, Natarajan R. Kidney Int 59: 1354-1362, 2001; Kang SW, Natarajan R, Shahed A, Nast CC, LaPage J, Mundel P, Kashtan C, Adler SG. J Am Soc Nephrol 14: 3178-3187, 2003; Kim YS, Lanting L, Adler SG, Natarajan R. Kindney Int 64: 1702-1714, 2003; Reddy MA, Adler SG, Kim YS, Lanting L, Rossi JJ, Kang SW, Nadler JL, Shahed A, Natarajan R. Am J Physiol Renal Physiol 283: F985-F994, 2002). In this study, we investigated whether in vivo delivery of small interfering RNAs (siRNAs) targeting 12/15-LO can ameliorate renal injury and DN in a streptozotocin-injected mouse model of type 1 diabetes. To achieve greater in vivo access and siRNA expression in the kidney, we used double-stranded 12/15-LO siRNA oligonucleotides conjugated with cholesterol. Diabetic DBA/2J mice were injected subcutaneously with either cholesterol-tagged 12/15-LO siRNA, mismatched control siRNA, or vehicle alone, twice weekly for 7 wk. Relative to controls, mice that received 12/15-LO siRNA showed significant reduction in albuminuria, kidney-to-body weight ratios, glomerular mesangial matrix expansion, renal structural damage, and monocyte/macrophage infiltration. These effects were associated with lower renal cortical or glomerular levels of profibrotic markers transforming growth factor-[beta], connective tissue growth factor, type I and type IV collagens, plasminogen activator inhibitor 1, and fibronectin. The diabetes-induced increase in glomerular cyclin-dependent kinase inhibitors that are associated with hypertrophy was also prevented by siRNA administration. Our results show for the first time that systemic delivery of cholesterol-tagged siRNAs targeting 12/15-LO has renoprotective effects under diabetic conditions and therefore could be a novel therapeutic approach for DN. gene delivery

Gene transfer and drug selection systems that enforce ongoing transgene expression in vitro and in vivo which are compatible with human pharmaceutical drugs are currently underdeveloped. Here, we report on the utility of incorporating human enzyme muteins that confer resistance to the lymphotoxic/immunosuppressive drugs methotrexate (MTX) and mycophenolate mofetil (MMF) in a multicistronic lentiviral vector for in vivo T lymphocyte selection. We found that co-expression of human dihydrofolate reductase (DHFRFS; L22F, F31S) and inosine monophosphate dehydrogenase II (IMPDH2IY; T333I, S351Y) conferred T cell resistance to the cytocidal and anti-proliferative effects of these drugs at concentrations that can be achieved clinically (up to 0.1 µM MTX and 1.0 µM MPA). Furthermore, using a immunodeficient mouse model that supports the engraftment of central memory derived human T cells, in vivo selection studies demonstrate that huEGFRt+DHFRFS+IMPDH2IY+ T cells could be enriched following adoptive transfer either by systemic administration of MTX alone (4.4 -fold), MMF alone (2.9-fold), or combined MTX and MMF (4.9-fold). These findings demonstrate the utility of both DHFRFS/MTX and IMPDH2IY/MMF for in vivo selection of lentivirally transduced human T cells. Vectors incorporating these muteins in combination with other therapeutic transgenes may facilitate the selective engraftment of therapeutically active cells in recipients. [ABSTRACT FROM AUTHOR]

We previously showed that the 12/15-lipoxygenase (12/15-LO) pathway of arachidonate acid metabolism is involved in multiple events related to diabetic nephropathy (DN), including glomerular hypertrophy and extracellular matrix deposition (Kang SW, Adler SG, Nast CC, LaPage J, Gu JL, Nadler JL, Natarajan R. Kidney mt 59: 1354-1362, 2001; Kang SW, Natarajan R, Shahed A, Nast CC, LaPage J, Mundel P, Kashtan C, Adler SG. JAm Soc Nephrol 14: 3 178-3187, 2003; Kim YS, Lanting L, Adler SG, Natarajan R. Kindney mt 64: 1702-1714, 2003; Reddy MA, Adler SG, Kim YS, Lanting L, Rossi JJ, Kang SW, Nadler JL, Shahed A, Natarajan R. Am J Physiol Renal Physiol 283: F985-F994, 2002). In this study, we investigated whether in vivo delivery of small interfering RNAs (siRNAs) target- ing 12/15-LO can ameliorate renal injury and DN in a streptozotocin- injected mouse model of type I diabetes. To achieve greater in vivo access and siRNA expression in the kidney, we used double-stranded 12/15-LO siRNA oligonucleotides conjugated with cholesterol. Diabetic DBAI2J mice were injected subcutaneously with either cholesterol-tagged 12/15-LO s1RNA, mismatched control siRNA, or vehicle alone, twice weekly for 7 wk. Relative to controls, mice that received 12/15-LO siRNA showed significant reduction in albuminuria, kidney-to-body weight ratios, glomerular mesangial matrix expansion, renal structural damage, and monocyte/macrophage infiltration. These effects were associated with lower renal cortical or glomerular levels of profibrotic markers transforming growth factor-13, connective tissue growth factor, type I and type IV collagens, plasminogen activator inhibitor 1, and fibronectin. The diabetes-induced increase in glomerular cyclin-dependent kinase inhibitors that are associated with hypertrophy was also prevented by siRNA administration. Our results show for the first time that systemic delivery of cholesterol-tagged siRNAs target- ing 12/15-LO has renoprotective effects under diabetic conditions and therefore could be a novel therapeutic approach for DN. [ABSTRACT FROM AUTHOR]

Introduction: Achieving a secure airway in rabbits is generally considered more difficult than in cats or dogs. Their relatively large tongue, small oropharyngeal cavity and glottis limit direct visualization. A rabbit-specific supraglottic airway device (SGAD) may offer benefits over blind orotracheal intubation., Animals and Methods: Fifteen adult New Zealand white rabbits were randomized to SGAD or orotracheal intubation (ETT). All animals were sedated with dexmedetomidine (0.1 mg kg -1 IM) and midazolam (0.5 mg kg -1 IM), followed by induction with alfaxalone (0.3 mg kg -1 IV). Two CT scans of the head and neck were performed, following sedation and SGAD/ETT placement. The following were recorded: time to successful device insertion, smallest cross-sectional airway area, airway sealing pressure, and histological score of tracheal tissue. Data were analyzed with a Mann-Whitney test., Results: Two rabbits were excluded following failed ETT. Body masses were similar [ETT; n  = 6, 2.6 (2.3-4.5) kg, SGAD; n  = 7, 2.7 (2.4-5.0) kg]. SGAD placement was significantly faster [33 (14-38) s] than ETT [59 (29-171) s]. Cross-sectional area (CSA) was significantly reduced from baseline [12.2 (6.9-3.4) mm 2 ] but similar between groups [SGAD; 2.7 (2.0-12.3) mm 2 , ETT; 3.8 (2.3-6.6) mm 2 ]. In the SGAD group, the device tip migrated into the laryngeal vestibule in 6/7 rabbits, reducing the CSA. ETT airway seals were higher [15 (10-20) cmH 2 O], but not significant [SGAD; 5 (5-20) cmH 2 O, p  = 0.06]. ETT resulted in significantly more mucosal damage [histological score 3.3 (1.0-5.0)], SGAD; 0.67 (0.33-3.67)., Conclusion: The SGAD studied was faster to place and caused less damage than orotracheal intubation, but resulted in a similar CSA.