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Non-tuberculous mycobacteria (NTM) cause pulmonary infections in patients with structural lung damage, impaired immunity, or other risk factors. Delivering antibiotics to the sites of these infections is a major hurdle of therapy because pulmonary NTM infections can persist in biofilms or as intracellular infections within macrophages. Inhaled treatments can improve antibiotic delivery into the lungs, but efficient nebulization delivery, distribution throughout the lungs, and penetration into biofilms and macrophages are considerable challenges for this approach. Therefore, we developed amikacin liposome inhalation suspension (ALIS) to overcome these challenges. Nebulization of ALIS has been shown to provide particles within the respirable size range that distribute to both central and peripheral lung compartments in humans. The in vitro and in vivo efficacy of ALIS against NTM has been demonstrated previously. The key mechanistic questions are whether ALIS penetrates NTM biofilms and enhances amikacin uptake into macrophages. We found that ALIS effectively penetrated throughout NTM biofilms and concentration-dependently reduced the number of viable mycobacteria. Additionally, we found that ALIS improved amikacin uptake by ∼4-fold into cultured macrophages compared with free amikacin. In rats, inhaled ALIS increased amikacin concentrations in pulmonary macrophages by 5- to 8-fold at 2, 6, and 24 h post-dose and retained more amikacin at 24 h in airways and lung tissue relative to inhaled free amikacin. Compared to intravenous free amikacin, a standard-of-care therapy for refractory and severe NTM lung disease, ALIS increased the mean area under the concentration-time curve in lung tissue, airways, and macrophages by 42-, 69-, and 274-fold. These data demonstrate that ALIS effectively penetrates NTM biofilms, enhances amikacin uptake into macrophages, both in vitro and in vivo, and retains amikacin within airways and lung tissue. An ongoing Phase III trial, adding ALIS to guideline based therapy, met its primary endpoint of culture conversion by month 6. ALIS represents a promising new treatment approach for patients with refractory NTM lung disease.

Activation of mammalian sterile 20–like kinase 1 (Mst1) by genotoxic compounds is known to stimulate apoptosis in some cell types. The importance of Mst1 in cell death caused by clinically relevant pathologic stimuli is unknown, however. In this study, we show that Mst1 is a prominent myelin basic protein kinase activated by proapoptotic stimuli in cardiac myocytes and that Mst1 causes cardiac myocyte apoptosis in vitro in a kinase activity–dependent manner. In vivo, cardiac-specific overexpression of Mst1 in transgenic mice results in activation of caspases, increased apoptosis, and dilated cardiomyopathy. Surprisingly, however, Mst1 prevents compensatory cardiac myocyte elongation or hypertrophy despite increased wall stress, thereby obscuring the use of the Frank-Starling mechanism, a fundamental mechanism by which the heart maintains cardiac output in response to increased mechanical load at the single myocyte level. Furthermore, Mst1 is activated by ischemia/reperfusion in the mouse heart in vivo. Suppression of endogenous Mst1 by cardiac-specific overexpression of dominant-negative Mst1 in transgenic mice prevents myocyte death by pathologic insults. These results show that Mst1 works as both an essential initiator of apoptosis and an inhibitor of hypertrophy in cardiac myocytes, resulting in a previously unrecognized form of cardiomyopathy

Recent developments in high-density genotyping and statistical analysis methods that have enabled genome-wide association studies in humans can also be applied to outbred mouse populations. Increased recombination in outbred populations is expected to provide greater mapping resolution than traditional inbred line crosses, improving prospects for identifying the causal genes. We carried out genome-wide association mapping by using 288 mice from a commercially available outbred stock; NMRI mice were genotyped with a high-density single-nucleotide polymorphism array to map loci influencing high-density lipoprotein cholesterol, systolic blood pressure, triglyceride levels, glucose, and urinary albumin-to-creatinine ratios. We found significant associations (P < 10−5) with high-density lipoprotein cholesterol and identified Apoa2 and Scarb1, both of which have been previously reported, as candidate genes for these associations. Additional suggestive associations (P < 10−3) identified in this study were also concordant with published quantitative trait loci, suggesting that we are sampling from a limited pool of genetic diversity that has already been well characterized. These findings dampen our enthusiasm for currently available commercial outbred stocks as genetic mapping resources and highlight the need for new outbred populations with greater genetic diversity. Despite the lack of novel associations in the NMRI population, our analysis strategy illustrates the utility of methods that could be applied to genome-wide association studies in humans.