RESUMO
Background: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition resulting from mutations in the alpha-1 antitrypsin (AAT) protein, a major systemic antiproteinase, resulting in reduced/no release of AAT, disrupting the proteinase/antiproteinase balance. A sustained imbalance can cause structural changes to the lung parenchyma, leading to emphysema. Predicting and assessing human responses to potential therapeutic candidates from preclinical animal studies have been challenging. Our aims were to develop a more physiologically relevant in vitro model of the proteinase/antiproteinase balance and assess whether the data generated could better predict the efficacy of pharmacological candidates to inform decisions on clinical trials, together with expected biomarker responses. Methods: We developed an in vitro model assessing the proteinase/antiproteinase balance by the changes in the fibrinogen cleavage products of neutrophil elastase (NE) and proteinase 3 (PR3). This allowed the assessment of physiological and pharmaceutical neutrophil serine proteinase (NSP) inhibitors to determine the putative threshold at which the maximal effect is achieved. Results: AAT significantly reduced NE and PR3 activity footprints, with the maximal reduction achieved at concentrations above 10 µM. The inhibitor MPH966 alone also significantly reduced NE footprint generation in a concentration-dependent manner, leveling out above 100 nM but had no effect on the PR3 footprint. At levels of AAT consistent with AATD, MPH966 had an additive effect, reducing the NE activity footprint more than either inhibitor alone. Conclusion: Our results support an inhibitor threshold above which the activity footprint generation appears resistant to increasing dosage. Our model can support the testing of inhibitors, confirming activity biomarkers as indicators of likely pharmaceutical efficacy, the assessment of NSP activity in the pathophysiology of emphysema, and the likely function of biological or pharmacological inhibitors in disease management.
RESUMO
Four major types of resistant starch (RS1-4) are present in foods, all of which can alter the microbiome and are fermented in the cecum and colon to produce short-chain fatty acids (SCFAs). Type 4 RSs are chemically modified starches, not normally found in foods, but have become a popular food additive as their addition increases fiber content. Multiple studies, in humans and rodents, have explored how different RS4 affect post-prandial glucose metabolism, but fewer studies have examined the effects of RS4 consumption on the microbiome. In addition, many RS studies conducted in rodents use high-fat diets that do not approximate what is typically consumed by humans. To address this, mice were fed a Total Western Diet (TWD), based on National Health and Nutrition Examination Survey (NHANES) data that mimics the macro and micronutrient composition of a typical American diet, for six weeks, and then supplemented with 0, 2, 5, or 10% of the RS4, Versafibe 1490™ (VF), a phosphorylated and cross-linked potato starch, for an additional three weeks. The cecal contents were analyzed for SCFA content and microbiota composition. Butyrate production was increased while branched chain SCFA production decreased. The alpha-diversity of the microbiome decreased in mice fed the TWD with 10% VF 1490 added while the beta-diversity plot showed that the 5% and 10% VF groups were distinct from mice fed the TWD. Similarly, the largest changes in relative abundance of various genera were greatest in mice fed the 10% VF diet. To examine the effect of VF consumption on tissue gene expression, cecal and distal colon tissue mRNA abundance were analyzed by RNASeq. Gene expression changes were more prevalent in the cecum than the colon and in mice fed the 10% VF diet, but the number of changes was substantially lower than we previously observed in mice fed the TWD supplemented with native potato starch (RPS). These results provide additional evidence that the structure of the RS is a major factor determining its effects on the microbiome and gene expression in the cecum and colon.