Mesenchymal stromal/stem cells promote intestinal epithelium regeneration after chemotherapy-induced damage.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for leukemia and a range of non-malignant disorders. The success of the therapy is hampered by occurrence of acute graft-versus-host disease (aGvHD); an inflammatory response damaging recipient organs, with gut, liver, and skin being the most susceptible. Intestinal GvHD injury is often a life-threatening complication in patients unresponsive to steroid treatment. Allogeneic mesenchymal stromal/stem cell (MSC) infusions are a promising potential treatment for steroid-resistant aGvHD. Data from our institution and others demonstrate rescue of approximately 40-50% of aGvHD patients with MSCs in Phase I, II studies and minor side effects. Although promising, better understanding of MSC mode of action and patient response to MSC-based therapy is essential to improve this lifesaving treatment. METHODS: Single cell human small intestine organoids were embedded in Matrigel, grown for 5 days and treated with busulfan for 48 h. Organoids damaged by treatment with busulfan or control organoids were co-cultured with 5000, 10,000, and 50,000 MSCs for 24 h, 48 h or 7 days and the analyses such as surface area determination, proliferation and apoptosis assessment, RNA sequencing and proteomics were performed. RESULTS: Here, we developed a 3D co-culture model of human small intestinal organoids and MSCs, which allows to study the regenerative effects of MSCs on intestinal epithelium in a more physiologically relevant setting than existing in vitro systems. Using this model we mimicked chemotherapy-mediated damage of the intestinal epithelium. The treatment with busulfan, the chemotherapeutic commonly used as conditioning regiment before the HSCT, affected pathways regulating epithelial to mesenchymal transition, proliferation, and apoptosis in small intestinal organoids, as shown by transcriptomic and proteomic analysis. The co-culture of busulfan-treated intestinal organoids with MSCs reversed the effects of busulfan on the transcriptome and proteome of intestinal epithelium, which we also confirmed by functional evaluation of proliferation and apoptosis. CONCLUSIONS: Collectively, we demonstrate that our in vitro co-culture system is a new valuable tool to facilitate the investigation of the molecular mechanisms behind the therapeutic effects of MSCs on damaged intestinal epithelium. This could benefit further optimization of the use of MSCs in HSCT patients.

Evaluation of polymyxin B in combination with 13 other antibiotics against carbapenemase-producing Klebsiella pneumoniae in time-lapse microscopy and time-kill experiments.

OBJECTIVES: This study aimed to explore the interactions of polymyxin B in combination with 13 other antibiotics against carbapenemase-producing Klebsiella pneumoniae. METHODS: Five clinical isolates of multidrug-resistant K. pneumoniae producing KPC-2, KPC-3, NDM-1, OXA-48 and VIM-1 carbapenemases were used. Polymyxin B was tested alone and in combination with amikacin, aztreonam, cefepime, chloramphenicol, ciprofloxacin, fosfomycin, linezolid, meropenem, minocycline, rifampicin, temocillin, thiamphenicol and trimethoprim. Inhibition of growth during antibiotic exposure was evaluated in 24-hr automated time-lapse microscopy experiments. Combinations that showed positive interactions were subsequently evaluated in static time-kill experiments. RESULTS: All strains carried multiple (≥9) resistance genes as determined by whole-genome sequencing. In the initial screening the combination of polymyxin B and minocycline was most active with enhanced activity compared with the single antibiotics detected against all strains. Positive interactions were also observed with polymyxin B in combination with rifampicin and fosfomycin against four of five strains and less frequently with other antibiotics. Time-kill experiments demonstrated an additive or synergistic activity (1-2 log10 or ≥2 log10 CFU/mL reduction, respectively, compared with the most potent single antibiotic) with 21 of 23 tested combinations. However, because of regrowth, only 13 combinations were synergistic at 24 hr. Combinations with minocycline or rifampicin were most active, each showing synergy and bacteriostatic or bactericidal effects resulting in 1.93-3.97 and 2.55-5.91 log10 CFU/mL reductions, respectively, after 24 hr against four strains. DISCUSSION: Polymyxin B in combination with minocycline, rifampicin or fosfomycin could be of potential clinical interest. Time-lapse microscopy showed some discrepancy in results compared with the time-kill data but was useful for screening purposes.