Optimizing tylosin dosage for co-infection of Actinobacillus pleuropneumoniae and Pasteurella multocida in pigs using pharmacokinetic/pharmacodynamic modeling.

Formulating a therapeutic strategy that can effectively combat concurrent infections of Actinobacillus pleuropneumoniae (A. pleuropneumoniae) and Pasteurella multocida (P. multocida) can be challenging. This study aimed to 1) establish minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time kill curve, and post-antibiotic effect (PAE) of tylosin against A. pleuropneumoniae and P. multocida pig isolates and employ the MIC data for the development of epidemiological cutoff (ECOFF) values; 2) estimate the pharmacokinetics (PKs) of tylosin following its intramuscular (IM) administration (20 mg/kg) in healthy and infected pigs; and 3) establish a PK-pharmacodynamic (PD) integrated model and predict optimal dosing regimens and PK/PD cutoff values for tylosin in healthy and infected pigs. The MIC of tylosin against both 89 and 363 isolates of A. pleuropneumoniae and P. multocida strains spread widely, ranging from 1 to 256 µg/mL and from 0.5 to 128 µg/mL, respectively. According to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) ECOFFinder analysis ECOFF value (≤64 µg/mL), 97.75% (87 strains) of the A. pleuropnumoniae isolates were wild-type, whereas with the same ECOFF value (≤64 µg/mL), 99.72% (363 strains) of the P. multicoda isolates were considered wild-type to tylosin. Area under the concentration time curve (AUC), T1/2, and Cmax values were significantly greater in healthy pigs than those in infected pigs (13.33 h × µg/mL, 1.99 h, and 5.79 µg/mL vs. 10.46 h × µg/mL, 1.83 h, and 3.59 µg/mL, respectively) (p < 0.05). In healthy pigs, AUC24 h/MIC values for the bacteriostatic activity were 0.98 and 1.10 h; for the bactericidal activity, AUC24 h/MIC values were 1.97 and 1.99 h for A. pleuropneumoniae and P. multocida, respectively. In infected pigs, AUC24 h/MIC values for the bacteriostatic activity were 1.03 and 1.12 h; for bactericidal activity, AUC24 h/MIC values were 2.54 and 2.36 h for A. pleuropneumoniae and P. multocida, respectively. Monte Carlo simulation lead to a 2 µg/mL calculated PK/PD cutoff. Managing co-infections can present challenges, as it often demands the administration of multiple antibiotics to address diverse pathogens. However, using tylosin, which effectively targets both A. pleuropneumoniae and P. multocida in pigs, may enhance the control of bacterial burden. By employing an optimized dosage of 11.94-15.37 mg/kg and 25.17-27.79 mg/kg of tylosin can result in achieving bacteriostatic and bactericidal effects in 90% of co-infected pigs.

Angiotensin II receptor type 1 blockade regulates Klotho expression to induce TSC2-deficient cell death.

Lymphangioleiomyomatosis (LAM) is a multisystem disease occurring in women of child-bearing age manifested by uncontrolled proliferation of smooth muscle-like "LAM" cells in the lungs. LAM cells bear loss-of-function mutations in tuberous sclerosis complex (TSC) genes TSC1 and/or TSC2, causing hyperactivation of the proliferation promoting mammalian/mechanistic target of Rapamycin complex 1 pathway. Additionally, LAM-specific active renin-angiotensin system (RAS) has been identified in LAM nodules, suggesting this system potentially contributes to neoplastic properties of LAM cells; however, the role of this renin-angiotensin signaling is unclear. Here, we report that TSC2-deficient cells are sensitive to the blockade of angiotensin II receptor type 1 (Agtr1). We show that treatment of these cells with the AGTR1 inhibitor losartan or silencing of the Agtr1 gene leads to increased cell death in vitro and attenuates tumor progression in vivo. Notably, we found the effect of Agtr1 blockade is specific to TSC2-deficient cells. Mechanistically, we demonstrate that cell death induced by Agtr1 inhibition is mediated by an increased expression of Klotho. In TSC2-deficient cells, we showed overexpression of Klotho or treatment with recombinant (soluble) Klotho mirrored the cytocidal effect of angiotensin blockade. Furthermore, Klotho treatment decreased the phosphorylation of AKT, potentially leading to this cytocidal effect. Conversely, silencing of Klotho rescued TSC2-deficient cells from cell death induced by Agtr1 inhibition. Therefore, we conclude that Agtr1 and Klotho are important for TSC2-deficient cell survival. These findings further illuminate the role of the RAS in LAM and the potential of targeting Agtr1 inhibition in TSC2-deficient cells.

Identification of novel epigenetic abnormalities as sputum biomarkers for lung cancer risk among smokers and COPD patients.

OBJECTIVES: Smoking is a common risk factor for chronic obstructive pulmonary disease (COPD) and lung cancer. Although COPD patients have higher risk of lung cancer compared to non-COPD smokers, the molecular links between these diseases are not well-defined. This study aims to identify genes that are downregulated by cigarette smoke and commonly repressed in COPD and lung cancer. MATERIALS AND METHODS: Primary human airway epithelial cells (HAEC) were exposed to cigarette-smoke-extract (CSE) for 10-weeks and significantly suppressed genes were identified by transcriptome array. Epigenetic abnormalities of these genes in lung adenocarcinoma (LUAD) from patients with or without COPD were determined using genome-wide and gene-specific assays and by in vitro treatment of cell lines with trichostatin-A or 5-aza-2-deoxycytidine. RESULTS: The ten most commonly downregulated genes following chronic CSE exposure of HAEC and show promoter hypermethylation in LUAD were selected. Among these, expression of CCNA1, SNCA, and ZNF549 was significantly reduced in lung tissues from COPD compared with non-COPD cases while expression of CCNA1 and SNCA was further downregulated in tumors with COPD. The promoter regions of all three genes were hypermethylated in LUAD but not normal or COPD lungs. The reduced expression and aberrant promoter hypermethylation of these genes in LUAD were independently validated using data from the Cancer Genome Atlas project. Importantly, SNCA and ZNF549 methylation detected in sputum DNA from LUAD (52% and 38%) cases were more prevalent compared to cancer-free smokers (26% and 15%), respectively (p < 0.02). CONCLUSIONS: Our data show that suppression of CCNA1, SNCA, and ZNF549 in lung cancer and COPD occurs with or without promoter hypermethylation, respectively. Detecting methylation of these and previously identified genes in sputum of cancer-free smokers may serve as non-invasive biomarkers for early detection of lung cancer among high risk smokers including COPD patients.

Extent of allergic inflammation depends on intermittent versus continuous sensitization to house dust mite.

OBJECTIVE: House dust mite (HDM) exposure is used to model experimental asthma in mice. However, a direct comparison of inflammatory responses following continuous versus intermittent HDM exposure has not been reported. Therefore, we investigated whether the HDM dose at sensitization or challenge affects extent of inflammation in mice that were either continuously or intermittently sensitized with HDM. MATERIALS AND METHODS: C57BL/6 mice received either 10 continuous exposures with 10 µg HDM per exposure or two intermittent HDM exposures over a period of two weeks and were subsequently challenged by three instillations with HDM during the third week. For the intermittent model, mice were sensitized with 1 or 10 µg HDM and challenged on three consecutive days with 1 or 10 µg HDM. Inflammatory cells in the bronchoalveolar lavage fluid and epithelial cell hyperplasia and mucous cell metaplasia were quantified. RESULTS: Significantly higher levels of inflammation and mucous cell metaplasia were observed when mice were sensitized intermittently compared with continuously. Intermittent sensitization and challenge with 10 µg HDM caused maximum inflammation, mucous cell metaplasia, and epithelial cell hyperplasia. However, sensitization with 1 µg HDM only also showed increased inflammation when challenged with 10 µg HDM. DISCUSSION: These findings suggest major differences in adaptive immunity, depending on the sensitization protocol. CONCLUSIONS: Because of significant differences, the HDM sensitization protocol should be carefully considered when designing studies to investigate the underlying mechanisms of immunity in mouse models of asthma.