Microbiota-derived 3-IAA influences chemotherapy efficacy in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is expected to be the second most deadly cancer by 2040, owing to the high incidence of metastatic disease and limited responses to treatment1,2. Less than half of all patients respond to the primary treatment for PDAC, chemotherapy3,4, and genetic alterations alone cannot explain this5. Diet is an environmental factor that can influence the response to therapies, but its role in PDAC is unclear. Here, using shotgun metagenomic sequencing and metabolomic screening, we show that the microbiota-derived tryptophan metabolite indole-3-acetic acid (3-IAA) is enriched in patients who respond to treatment. Faecal microbiota transplantation, short-term dietary manipulation of tryptophan and oral 3-IAA administration increase the efficacy of chemotherapy in humanized gnotobiotic mouse models of PDAC. Using a combination of loss- and gain-of-function experiments, we show that the efficacy of 3-IAA and chemotherapy is licensed by neutrophil-derived myeloperoxidase. Myeloperoxidase oxidizes 3-IAA, which in combination with chemotherapy induces a downregulation of the reactive oxygen species (ROS)-degrading enzymes glutathione peroxidase 3 and glutathione peroxidase 7. All of this results in the accumulation of ROS and the downregulation of autophagy in cancer cells, which compromises their metabolic fitness and, ultimately, their proliferation. In humans, we observed a significant correlation between the levels of 3-IAA and the efficacy of therapy in two independent PDAC cohorts. In summary, we identify a microbiota-derived metabolite that has clinical implications in the treatment of PDAC, and provide a motivation for considering nutritional interventions during the treatment of patients with cancer.

Covalent Linkage and Macrocylization Preserve and Enhance Synergistic Interactions in Catalytic Amyloids.

The self-assembly of short peptides into catalytic amyloid-like nanomaterials has proven to be a powerful tool in both understanding the evolution of early proteins and identifying new catalysts for practically useful chemical reactions. Here we demonstrate that both parallel and antiparallel arrangements of ß-sheets can accommodate metal ions in catalytically productive coordination environments. Moreover, synergistic relationships, identified in catalytic amyloid mixtures, can be captured in macrocyclic and sheet-loop-sheet species, that offer faster rates of assembly and provide more complex asymmetric arrangements of functional groups, thus paving the way for future designs of amyloid-like catalytic proteins. Our findings show how initial catalytic activity in amyloid assemblies can be propagated and improved in more-complex molecules, providing another link in a complex evolutionary chain between short, potentially abiotically produced peptides and modern-day enzymes.

CD45-Directed CAR-T Cells with CD45 Knockout Efficiently Kill Myeloid Leukemia and Lymphoma Cells In Vitro Even after Extended Culture.

BACKGROUND: CAR-T cell therapy has shown impressive results and is now part of standard-of-care treatment of B-lineage malignancies, whereas the treatment of myeloid diseases has been limited by the lack of suitable targets. CD45 is expressed on almost all types of blood cells including myeloid leukemia cells, but not on non-hematopoietic tissue, making it a potential target for CAR-directed therapy. Because of its high expression on T and NK cells, fratricide is expected to hinder CD45CAR-mediated therapy. Due to its important roles in effector cell activation, signal transduction and cytotoxicity, CD45 knockout aimed at preventing fratricide in T and NK cells has been expected to lead to considerable functional impairment. METHODS: CD45 knockout was established on T and NK cell lines using CRISPR/Cas9-RNPs and electroporation, and the successful protocol was transferred to primary T cells. A combined protocol was developed enabling CD45 knockout and retroviral transduction with a third-generation CAR targeting CD45 or CD19. The functionality of CD45ko effector cells, CD45ko/CD45CAR-T and CD45ko/CD19CAR-T cells was studied using proliferation as well as short- and long-term cytotoxicity assays. RESULTS: As expected, the introduction of a CD45-CAR into T cells resulted in potent fratricide that can be avoided by CD45 knockout. Unexpectedly, the latter had no negative impact on T- and NK-cell proliferation in vitro. Moreover, CD45ko/CD45CAR-T cells showed potent cytotoxicity against CD45-expressing AML and lymphoma cell lines in short-term and long-term co-culture assays. A pronounced cytotoxicity of CD45ko/CD45CAR-T cells was maintained even after four weeks of culture. In a further setup, we confirmed the conserved functionality of CD45ko cells using a CD19-CAR. Again, the proliferation and cytotoxicity of CD45ko/CD19CAR-T cells showed no differences from those of their CD45-positive counterparts in vitro. CONCLUSIONS: We report the efficient production of highly and durably active CD45ko/CAR-T cells. CD45 knockout did not impair the functionality of CAR-T cells in vitro, irrespective of the target antigen. If their activity can be confirmed in vivo, CD45ko/CD45CAR-T cells might, for example, be useful as part of conditioning regimens prior to stem cell transplantation.

How gastrin-releasing peptide receptor (GRPR) and αvß3 integrin expression reflect reorganization features of tumors after hyperthermia treatments.

The outcome of tumor treatment via hyperthermia in the clinic has been reported to be heterogeneous. Here, we assessed how the presence of gastrin-releasing peptide receptor (GRPR) and αvß3 integrin together with the morphology of the vascularization reflects the growth behavior of tumors after hyperthermia treatment. MDA-MB-231 tumor bearing mice were treated either with high (46 °C) or low dose (42 °C) water hyperthermia for 60 min. Changes of GRPR and αvß3 integrin expression were assessed via multiplexed optical imaging. Vascularization was reconstructed and quantified by µCT imaging after contrast agent injection. We found that high dose hyperthermia is capable of increasing the expression of GRPR, αvß3 integrin, CD31, and Ki67 in tumors. Also the morphology of tumor vasculature changed (increased relative blood volume and small-diameter vessel density, decreased expression of α-SMA). Low dose hyperthermia induced comparatively moderate effects on the investigated protein expression pattern and vascular remodeling. We conclude that under defined circumstances, specific temperature doses affect the reorganization of tumor regrowth, which is triggered by residual "dormant" cells even though tumor volumes are transiently decreasing. Further on, GRPR, αvß3 integrin expression are versatile tools to surveil potential tumor regrow during therapy, beyond the conventional determination of tumor volumes.

Free flap reconstruction for patients with bisphosphonate related osteonecrosis of the jaws after mandibulectomy.

INTRODUCTION: Bisphosphonate related osteonecrosis of the jaws (BRONJ) is a recognised unwanted effect of these drugs which affect bone remodelling. Treatment options range from conservative approaches through local bone debridement to free flap reconstruction following segmental resection. This current study aims to evaluate clinical outcomes after microvascular tissue transfer in BRONJ patients. MATERIAL AND METHODS: A total of 212 BRONJ patients were included in this prospective investigation. Those who met defined inclusion criteria and received a surgical intervention were reviewed regularly during a follow-up period of at least 6 months. RESULTS: Twenty-five patients (11.8%) received free flap reconstructions. A mean of 2.12 local debridements were performed before microvascular tissue transfer. A mean of 29.25% showed BRONJ recurrence after minimalist surgical intervention, compared to significantly less in patients after resection and free flap reconstruction. The postoperative fistula rate was significantly higher in patients, who received mucoperiosteal flaps. DISCUSSION: This study underlines the importance and effectiveness radical resection and free flap reconstruction in the complex and challenging surgical treatment of BRONJ patients in a large patient cohort study. Nevertheless, all patients received radical intervention after failure of minimally invasive treatment. An individualized analysis and planning is necessary to identify appropriate patients for free flap reconstructions.

The role of mylohyoid flap in the treatment of bisphosphonate-related osteonecrosis of the jaws.

INTRODUCTION: Surgical treatment of bisphosphonate-related osteonecrosis of the jaws (BRONJ) combines excision of adequate damaged bone and watertight coverage by appropriate vascularized tissue. Local tissues are preferred when possible. This study compares local mucoperiosteal flaps with mylohyoid flaps with special emphasis on their influence on wound healing. MATERIAL AND METHODS: A total of 195 patients with BRONJ in the mandible were included in this prospective study. The control group (n = 169) were treated with a mucoperiosteal flap, whereas patients of the study group (n = 26) received a mylohyoid flap. RESULTS: Recurrence of BRONJ was significantly reduced (p = 0.023) as was extent of necrosis (p = 0.001) in patients with mylohyoid flaps. DISCUSSION: This study demonstrates the importance of a sufficient mucosal coverage in surgical treatment of BRONJ. The mylohyoid flap provides an additional tissue coverage, which seems to account for the significantly reduced rate of disease recurrence. CONCLUSION: The vascularized mylohyoid flap is an important tool in the complex and challenging surgical care of BRONJ.

Do measurements of inflammatory mediators in blood predict recurrence in patients with bisphosphonate-related osteonecrosis of the jaws?

Bisphosphonate-related osteonecrosis of the jaws (BRONJ) is well-recognised, difficult to manage, and often recurs. The aim of this study was to examine the value of preoperative measurements of inflammatory mediators in blood in 212 patients with BRONJ who were studied prospectively. Multiple logistic regression analysis was used to assess the importance of the amounts of substances in the blood that are independently associated with the dependent variable "recurrence of BRONJ". The only factor that significantly influenced the development of recurrent BRONJ was reduction in the white cell count (p<0.0001; hazard ratio 5.324; 95% CI 2.373 to 11.945). Neither white cell counts nor C-reactive protein concentrations within or above the reference ranges were significantly associated with recurrent BRONJ. Patients whose white cell counts were lower than the reference range were at increased risk of recurrent BRONJ. This may be a marker of reduced immunocompetence, and additional prophylactic measures or treatment should be considered for these patients.