Dual targeting of CD19 and CD22 with bicistronic CAR-T cells in patients with relapsed/refractory large B-cell lymphoma.

Relapse after CD19-directed chimeric antigen receptor T-cell (CAR-T) therapy for large B-cell lymphoma (LBCL) is commonly ascribed to antigen loss or CAR-T exhaustion. Multiantigen targeting and programmed cell death protein-1 blockade are rational approaches to prevent relapse. Here, we test CD19/22 dual-targeting CAR-T (AUTO3) plus pembrolizumab in relapsed/refractory LBCL (NCT03289455). End points include toxicity (primary) and response rates (secondary). Fifty-two patients received AUTO3 and 48/52 received pembrolizumab. Median age was 59 years (range, 27-83), 46/52 had stage III/ IV disease and median follow-up was 21.6 months. AUTO3 was safe; grade 1-2 and grade 3 cytokine release syndrome affected 18/52 (34.6%) and 1/52 (1.9%) patients, neurotoxicity arose in 4 patients (2/4, grade 3-4), and hemophagocytic lymphohistiocytosis affected 2 patients. Outpatient administration was tested in 20 patients, saving a median of 14 hospital days per patient. Overall response rates were 66% (48.9%, complete response [CR]; 17%, partial response). Median duration of remission (DOR) for CR patients was not reached and for all responding patients was 8.3 months (95% confidence interval [CI]: 3.0-not evaluable). 54.4% (CI: 32.8-71.7) of CR patients and 42.6% of all responding patients were projected to remain progression-free at ≥12 months. AUTO3 ± pembrolizumab for relapsed/refractory LBCL was safe and delivered durable remissions in 54.4% of complete responders, associated with robust CAR-T expansion. Neither dual-targeting CAR-T nor pembrolizumab prevented relapse in a significant proportion of patients, and future developments include next-generation-AUTO3, engineered for superior expansion in vivo, and selection of CAR binders active at low antigen densities.

High-Grade Limbs Open Fractures: Time to Find Milestones in the Emergency Setting.

(1) Background: The Gustilo-Anderson (G/A) grading system is a universally accepted tool used to classify high-grade limb open fractures. The purpose of this study is to find early independent predictors of amputation in emergency settings. (2) Methods: A retrospective analysis involving patients treated at our center between 2010 and 2016 was conducted. Patients with at least one G/A grade III fracture or post-traumatic amputation were included. Three groups were identified: G/A IIIA (A group), G/A IIIB-C (BC group), and Amputation group (AMP group). Each group was further divided into two subgroups considering timing of coverage (early vs. delayed). Univariate and multivariate logistic regression models were developed to identify independent predictors of the limb's outcome. (3) Results: One-hundred-six patients with G/A III A-B-C fractures or amputation of the affected limb were selected from the Niguarda Hospital Trauma Registry. The patients were divided into the A group (26), BC group (66), and AMP group (14). The rate of infectious complications following early or delayed coverage was evaluated: A group, 9.1% vs. 66.7% (p > 0.05); BC group, 32% vs. 63.6% (p = 0.03); and AMP group, 22% vs. 18.5% (p > 0.05). After further recategorization, the BC subgroups were analyzed: multivariate logistic regression model identified systolic blood pressure (SBP) <90 mmHg (p = 0.03) and Mangled Extremity Severity Score MESS ≥ 7 (p = 0.001) were determined to be independent predictors of limb amputation. (4) Conclusion: MESS and SBP serve as predictors of amputation. Based on the results, we propose a new management algorithm for mangled extremities. Early coverage is related to lower rates of infectious complications. Referral to high-volume centers with specific expertise is mandatory to guarantee the best results.

Dissecting the early steps of MLL induced leukaemogenic transformation using a mouse model of AML.

Leukaemogenic mutations commonly disrupt cellular differentiation and/or enhance proliferation, thus perturbing the regulatory programs that control self-renewal and differentiation of stem and progenitor cells. Translocations involving the Mll1 (Kmt2a) gene generate powerful oncogenic fusion proteins, predominantly affecting infant and paediatric AML and ALL patients. The early stages of leukaemogenic transformation are typically inaccessible from human patients and conventional mouse models. Here, we take advantage of cells conditionally blocked at the multipotent haematopoietic progenitor stage to develop a MLL-r model capturing early cellular and molecular consequences of MLL-ENL expression based on a clear clonal relationship between parental and leukaemic cells. Through a combination of scRNA-seq, ATAC-seq and genome-scale CRISPR-Cas9 screening, we identify pathways and genes likely to drive the early phases of leukaemogenesis. Finally, we demonstrate the broad utility of using matched parental and transformed cells for small molecule inhibitor studies by validating both previously known and other potential therapeutic targets.

A systematic review on the use of topical hemostats in trauma and emergency surgery.

BACKGROUND: A wide variety of hemostats are available as adjunctive measures to improve hemostasis during surgical procedures if residual bleeding persists despite correct application of conventional methods for hemorrhage control. Some are considered active agents, since they contain fibrinogen and thrombin and actively participate at the end of the coagulation cascade to form a fibrin clot, whereas others to be effective require an intact coagulation system. The aim of this study is to provide an evidence-based approach to correctly select the available agents to help physicians to use the most appropriate hemostat according to the clinical setting, surgical problem and patient's coagulation status. METHODS: The literature from 2000 to 2016 was systematically screened according to PRISMA [Preferred Reporting Items for Systematic Reviews and Meta-Analyses] protocol. Sixty-six articles were reviewed by a panel of experts to assign grade of recommendation (GoR) and level of evidence (LoE) using the GRADE [Grading of Recommendations Assessment, Development and Evaluation] system, and a national meeting was held. RESULTS: Fibrin adhesives, in liquid form (fibrin glues) or with stiff collagen fleece (fibrin patch) are effective in the presence of spontaneous or drug-induced coagulation disorders. Mechanical hemostats should be preferred in patients who have an intact coagulation system. Sealants are effective, irrespective of patient's coagulation status, to improve control of residual oozing. Hemostatic dressings represent a valuable option in case of external hemorrhage at junctional sites or when tourniquets are impractical or ineffective. CONCLUSIONS: Local hemostatic agents are dissimilar products with different indications. A knowledge of the properties of each single agent should be in the armamentarium of acute care surgeons in order to select the appropriate product in different clinical conditions.

RNA Nanotherapeutics for the Amelioration of Astroglial Reactivity.

In response to injuries to the CNS, astrocytes enter a reactive state known as astrogliosis, which is believed to be deleterious in some contexts. Activated astrocytes overexpress intermediate filaments including glial fibrillary acidic protein (GFAP) and vimentin (Vim), resulting in entangled cells that inhibit neurite growth and functional recovery. Reactive astrocytes also secrete inflammatory molecules such as Lipocalin 2 (Lcn2), which perpetuate reactivity and adversely affect other cells of the CNS. Herein, we report proof-of-concept use of the packaging RNA (pRNA)-derived three-way junction (3WJ) motif as a platform for the delivery of siRNAs to downregulate such reactivity-associated genes. In vitro, siRNA-3WJs induced a significant knockdown of Gfap, Vim, and Lcn2 in a model of astroglial activation, with a concomitant reduction in protein expression. Knockdown of Lcn2 also led to reduced protein secretion from reactive astroglial cells, significantly impeding the perpetuation of inflammation in otherwise quiescent astrocytes. Intralesional injection of anti-Lcn2-3WJs in mice with contusion spinal cord injury led to knockdown of Lcn2 at mRNA and protein levels in vivo. Our results provide evidence for siRNA-3WJs as a promising platform for ameliorating astroglial reactivity, with significant potential for further functionalization and adaptation for therapeutic applications in the CNS.

Dysregulation of haematopoietic stem cell regulatory programs in acute myeloid leukaemia.

Haematopoietic stem cells (HSC) are situated at the apex of the haematopoietic differentiation hierarchy, ensuring the life-long supply of mature haematopoietic cells and forming a reservoir to replenish the haematopoietic system in case of emergency such as acute blood loss. To maintain a balanced production of all mature lineages and at the same time secure a stem cell reservoir, intricate regulatory programs have evolved to control multi-lineage differentiation and self-renewal in haematopoietic stem and progenitor cells (HSPCs). Leukaemogenic mutations commonly disrupt these regulatory programs causing a block in differentiation with simultaneous enhancement of proliferation. Here, we briefly summarize key aspects of HSPC regulatory programs, and then focus on their disruption by leukaemogenic fusion genes containing the mixed lineage leukaemia (MLL) gene. Using MLL as an example, we explore important questions of wider significance that are still under debate, including the importance of cell of origin, to what extent leukaemia oncogenes impose specific regulatory programs and the relevance of leukaemia stem cells for disease development and prognosis. Finally, we suggest that disruption of stem cell regulatory programs is likely to play an important role in many other pathologies including ageing-associated regenerative failure.

Is there a role for homeopathy in breast cancer surgery? A first randomized clinical trial on treatment with Arnica montana to reduce post-operative seroma and bleeding in patients undergoing total mastectomy.

AIM: This study aimed to evaluate the benefits of Arnica montana on post-operative blood loss and seroma production in women undergoing unilateral total mastectomy by administering Arnica Montana 1000 Korsakovian dilution (1000 K). MATERIALS AND METHODS: From 2012 to 2014, 53 women were randomly assigned to A. montana or placebo and were followed up for 5 days. The main end point was the reduction in blood and serum volumes collected in drainages. Secondary end points were duration of drainage, a self-evaluation of pain, and the presence of bruising or hematomas. RESULTS: The per-protocol analysis revealed a lower mean volume of blood and serum collected in drainages with A. montana (-94.40 ml; 95% confidence interval [CI]: 22.48-211.28; P = 0.11). A regression model including treatment, volume collected in the drainage on the day of surgery, and patient weight showed a statistically significant difference in favor of A. montana (-106.28 ml; 95% CI: 9.45-203.11; P = 0.03). Volumes collected on the day of surgery and the following days were significantly lower with A. montana at days 2 (P = 0.033) and 3 (P = 0.0223). Secondary end points have not revealed significant differences. CONCLUSIONS: A. montana 1000 K could reduce post-operative blood and seroma collection in women undergoing unilateral total mastectomy. Larger studies are needed with different dilutions of A. montana to further validate these data.

An experimentally validated network of nine haematopoietic transcription factors reveals mechanisms of cell state stability.

Transcription factor (TF) networks determine cell-type identity by establishing and maintaining lineage-specific expression profiles, yet reconstruction of mammalian regulatory network models has been hampered by a lack of comprehensive functional validation of regulatory interactions. Here, we report comprehensive ChIP-Seq, transgenic and reporter gene experimental data that have allowed us to construct an experimentally validated regulatory network model for haematopoietic stem/progenitor cells (HSPCs). Model simulation coupled with subsequent experimental validation using single cell expression profiling revealed potential mechanisms for cell state stabilisation, and also how a leukaemogenic TF fusion protein perturbs key HSPC regulators. The approach presented here should help to improve our understanding of both normal physiological and disease processes.