Amphotericin B lipid complex represents a valuable therapeutic option in the treatment of fungal infections but improved strategies for the management of infusion-related adverse events are required.

We describe a patient who had chronic lymphocytic leukaemia (CLL) Binet stage A at presentation with further evidence of disease at multiple sites but who initially required no treatment. However, several years later, her peripheral blood lymphocyte count started to increase, and soon after that she suffered an acute myocardial infarct (in the absence of coronary atheroma) together with proteinuric renal failure due to membranoproliferative glomerulonephritis. Her renal function improved markedly following anti-CLL chemotherapy. We postulate that her cardiac and renal disease were both complications of her CLL. In patients with CLL who develop new clinical signs or symptoms (even if apparently unrelated), consideration should be given as to whether these may be disease complications as this may serve as an indication to commence anti-CLL therapy; close liaison between different specialties is vital.

Multiple myeloma (MM) is a tumor characterised by the generation of large quantities of immunoglobulin which undergoes protein folding and secretion through the endoplasmic reticulum (ER). Many studies have shown that primary MM cells have high ER stress (Lee et al, 2003 PNAS, Obeng et al 2006 Blood and Mimura et al, 2012 Blood,). Moreover, it has also been shown that serum from MM contains significantly more extracellular vesicles (EVs) than serum from normal patients (Caivano et al, 2015 Tumour Biol). Here we explore if these two observations are connected and determine whether primary MM cells export ER inside EVs and the impact this has on the tumor microenvironment. Primary MM and primary MM bone marrow stromal cells (BMSC) were isolated from the bone marrow of patients. To determine if MM secrete ER inside EV, patient derived MM and MM cell lines were transduced with rLV.EF.mCherry-ER lentivirus which fluorescently tags the ER. High-resolution imaging combined with image cytometry shows that CD38+ vesicles containing ER are formed by budding from the MM cell surface. Analysis of MM derived EVs using a combination of proteomics, confocal microscopy, image cytometry and dynamic light scattering shows that MM actively export ER in the large EVs, approximately 0.6micron-1.5micron in diameter. To track the recipient cell for large EV packaged ER in vivo, U266 MM cell line (transduced with rLV.EF.mCherry-ER9 lentivirus) was injected into the tail of NSG mice. BM was extracted from engrafted mice and various cell populations were analysed for increases in mCherry fluorescence, as evidence of MM derived ER uptake. Murine CD45-/CD31-Ter119-/CD105+/CD140a+ BMSC had increased mCherry fluorescence but not F4/80+/GR1-/CD115int BM macrophages or CD45-/Ter119-/CD31+ endothelial cells or CD45+ leukocytes. Two proteins detected at high levels in the proteomics analysis of large EV were endoplasmic reticulum oxidoreductin 1 (ERO1) and protein disulfide-isomerase (P4B4) which functionally catalyze the formation, breakage and rearrangement of disulfide bonds resulting in the production of superoxide. Next, we analysed large EVs containing ER for superoxide. MM derived ER+ and ER- large EVs were isolated by sorting for mCherry fluorescence (MM transduced with rLV.EF.mCherry-ER9 lentivirus) and Amplex Red assay confirmed that ER+ large EVs had increased levels of H2O2. In vivo analysis of the BMSC from MM engrafted NSG mice confirmed high oxidative stress as measured by increased H2DCFDA fluorescence. To determine the impact of MM derived ER containing EVs on the function of the BMSC isolated ER containing EVs were incubated with BMSC repeatedly for up to 7 days and senescent markers were assessed. Beta-galactosidase staining, p16ink4a gene expression and a senescence associated secretary phenotype (SASP) were all upregulated in BMSC cultured with MM derived ER+ large EV and not ER- large EV. To determine if MM induced BMSC senescence in vivo we injected U266 and primary MM into NSG mice, humanised NSG mice were used as a control. Post MM engraftment, animals were sacrificed and BMSC were isolated by cell sorting for CD45-/CD31-Ter119-/CD105+/CD140a+ cells and senescent markers were analysed by real-time PCR. p16ink4a and p21 were both upregulated in BMSC from U266 and primary MM engrafted NSG mice and not from humanised NSG mice. Knockdown of p16 in BMSC prevents ER+ large EV from inducing a SASP and conditioned media had no effect on MM proliferation compared to conditioned media from ER+ large EV treated BMSC. Finally, we used an NSG mouse model whereby we transplanted p16ink4a KD BMSC or control KD BMSC with MM cells subcutaneously into the flank. MM combined with p16ink4a KD BMSC has reduced tumor volume compared with animals with control KD BMSC. Data indicates that MM secrete ER in large EV and that MM derived EVs containing ER function as a signal which then changes the physiology of BMSC, towards a senescent phenotype which in turn promotes malignant plasma cell survival and proliferation. Disclosures Bowles: Janssen: Research Funding; Abbvie: Research Funding. Rushworth:Abbvie: Research Funding; Janssen: Research Funding.

Se presenta la revisión del tema código sepsis. El objetivo del artículo es identificar áreas de oportunidad para el diagnóstico y tratamiento de los pacientes con sepsis, así como proponer la creación de un código de respuesta rápida ante los futuros casos de sepsis en nuestro hospital. La implementación de un código sepsis en este centro hospitalario tiene como objetivo asegurar una atención oportuna y de calidad optimizando la detección de complicaciones con la finalidad de mejorar el pronóstico de los pacientes con esta enfermedad.

Bloqueadores neuromusculares en el síndrome de insuficiencia respiratoria progresiva aguda: metaanálisis Neuromuscular blockers in acute respiratory distress syndrome: meta-analysis Bloqueadores neuromusculares na síndrome de insuficiência respiratória progressiva aguda: metanálise Abreviaturas: BNM = agentes bloqueadores neuromusculares. ECA = estudio controlado aleatorizado. OR = razón de momios. RR = razón de riesgo. UCI = Unidad de Cuidados Intensivos. SIRPA = síndrome de insuficiencia respiratoria progresiva aguda.Recepción: 18/09/2019. Aceptación: 02/12/2019. Este artículo puede ser consultado en versión completa en www.medigraphic.com/medicinacritica Methods: In this systematic review and meta-analysis, different databases were searched, comparing the administration of neuromuscular blocking agents versus placebo or non-treatment in patients with acute respiratory distress syndrome. Titles, abstracts and full texts of the articles were selected in duplicate by two researchers. The data for the study design, patient characteristics, interventions and results were summarized independently and in duplicate. For additional information, the authors of the selected studies were contacted by email. The GRADE guides were used to rate the quality of the evidence. We calculate the risk ratios (RR) and odds ratios (OR) with 95% confidence intervals (95% CI) for dichotomous variables, while for the continuous variables we obtained the difference in means and performed a meta-analysis of random effects. The primary outcome was the evaluation of any-cause mortality, mortality in the Intensive Care Unit, the incidence of adverse effects and the evolution of respiratory parameters. Results: Six randomized controlled studies (RCTs) were included. Compared to the placebo group or no treatment, neuromuscular blocking agents were associated with a significant reduction in any-cause mortality (603 [35.7%] of 1,691 patients versus 673 [40.5%] of 1,660 patients;] p = 0.005 I2 33%); as well as decreased mortality in the ICU ). Compared to the placebo group or no treatment, the neuromuscular blocking agents group was associated with a significant reduction in adverse events (RR 0.72 [95% CI 0.52 to 0.99], four RCTs, 3,621 patients; p = 0.15 I2 64%) and a significant improvement in the PaO 2 /FiO 2 ratio (11.02 mmHg [95% CI 5.38 to 16.66]; four RCTs, 3,637 patients; p = 0.0001 I2 24%). Conclusions: The use of neuromuscular blocking agents in adults with acute respiratory distress syndrome was associated with a significant reduction in mortality from any cause. There were fewer adverse events and a significant improvement in the PaO 2 /FiO 2 ratio in the neuromuscular blocking agents' group. Based on our results, we recommend the use of neuromuscular blocking agents for patients with moderate to severe ARDS who need mechanical ventilation. Due to the moderate to low quality of the evidence, new randomized studies with sufficient statistical power are required to confirm these findings.

Background Multiple myeloma (MM) is malignancy highly reliant on its microenvironment. In this study, we investigated whether mitochondrial transfer occurred between bone marrow stromal cells (BMSC) and malignant plasma cells. We then used our observations as a platform to investigate the mechanisms controlling pro-tumoral mitochondrial transfer with a view to identifying druggable targets. Methods Primary MM cells were obtained from patients' bone marrow after informed consent and under approval from the United Kingdom Health Research Authority. Animal experiments were conducted under approvals from the UK Home Office and the University of East Anglia Animal Welfare and Ethics Review Board. Primary BMSC were also obtained from patient bone marrow, using adherence and characterised using flow cytometry. Mitochondrial transfer was assessed using two methods; a MitoTracker Green based staining of the BMSC (in-vitro), rLV.EF1.AcGFP-Mem9 labelling of the MM plasma membrane with MitoTracker CMXRos staining of the BMSC (in-vitro) and an in vivo MM NSG xenograft model. CD38 expression on MM cells was tested after ATRA treatment, using RT-qPCR and flow cytometry. Mitochondrial transfer levels were assessed when CD38 was over expressed using ATRA or inhibited using lentivirus targeted shRNA. Results We report that mitochondria are transferred from BMSC to MM cells. First, we cultured MM cells on MitoTracker Green labelled BMSC and found increased MitoTracker Green fluorescence in the MM cells. We then transduced MM with rLV.EF1.AcGFP-Mem9 lentivirus and stained BMSC with MitoTracker CMXRos and used wide field microscopy to show MM derived tunnelling nanotubles (TNT) formed between MM cells and BMSC, with red mitochondria located within the GFP-tagged TNT. Next, we engrafted the MM cell lines MM1S and U266 into NSG mouse, after isolation we detected the presence of mouse mitochondrial DNA in the purified MM population. Together, these data show that mitochondria are transferred from BMSC to MM cells. We next analysed OXPHOS levels in MM cells grown on BMSC, using the seahorse extracellular flux assay. We found that the MM cells had increased levels of OXPHOS after culture with BMSC, which was also the case for MM cell lines analysed after isolation from NSG mice, showing the micro-environment of MM can alter the metabolism of the malignant cell. To examine whether the mitochondrial transfer process was controlled by CD38, we knocked down CD38 in MM cells using lentiviral targeted shRNA. We found reduced levels of mitochondrial transfer in CD38KD MM cells, with a consequent reduction of OXPHOS in the malignant cells. Finally, as ATRA has previously been shown to increase CD38 expression in AML, we next quantified CD38 mRNA and surface glycoprotein level on malignant plasma cells with and without ATRA treatment. We found ATRA increased CD38 expression at the mRNA and protein levels and this resulted in an increase in mitochondrial transfer from BMSC to MM cells. Conclusion Here we show that CD38 mediated mitochondrial transfer in the MM micro-environment forms part of the malignant phenotype of multiple myeloma. This finding develops our understanding of the mechanisms which underpin the efficacy of CD38 directed therapy in MM. Disclosures No relevant conflicts of interest to declare.

Multiple myeloma (MM) is reported to cause 85,000 global deaths per year which is estimated to double by 2040. MM is an incurable malignancy of antibody (Ig) secreting differentiated B cells (plasma cells) characterized by the accumulation and localisation of tumor cells in the bone marrow microenvironment (BMM). Mitochondrial genome (mtDNA) contains CpG DNA repeats and CpG DNA has been shown to activate and induce proliferation of memory B cells to secrete Ig. Here we determine if malignant plasma cells maintain their proliferative advantage by releasing mtDNA into the tumour microenvironment which acts as feedback mechanism to induce further proliferation and Ig production by the malignant plasma cell. Human MM cell lines U266 and MM1S were engrafted into NSG immunocompromised mice. Blood sample from control and engrafted mice were collected at various times and plasma was isolated and spun at 15,000g to remove cell debris and large extracellular vesicles. Human and mouse mtDNA was determine by real-time PCR. Results show that human mtDNA can be detected in the plasma from mice engrafted with U266 and MM1S at 2 weeks and 3 weeks post injection of cells. Next, CpG oligonucleotides were used to determine if MM cells can be activated by mtDNA CpG repeats. Primary MM, U226 and MM1S cells were all treated with CpG and CpG control for 4 and 24 hours. Real-time PCR was then used to detect the pro-survival signature showing that IL-1beta, IL-6 and IL-8 were all upregulated by CpG. We then analysed primary MM and MM cell lines for TLR9 expression by flow cytometry and showed that all samples tested had high levels of cytosolic TLR9 expression. Using TLR9 antagonist (ODN TTAGGG (A151)) we show that primary MM cells and MM cell lines have reduced IL-6 and IL-8 expression and decreased cell proliferation. Finally, U266 and MM1S transduced with TLR9 knockdown lentivirus had reduced engraftment, as measured by bioluminescence, in NSG mice. Here we show that MM release mtDNA in vivo and this can induce a tumour directed proliferative response through the engagement of TLR9. This process is necessary for optimum tumor growth and forms part of the malignant phenotype of MM. Citation Format: Aisha Jibril, Jayna J. Mistry, Prakrit Kumar, Jamie A. Moore, Charlotte Hellmich, Cesar Gomez, Kristian Bowles, Stuart A. Rushworth. Myeloma derived mitochondrial DNA activates TLR9 driven pro-tumoral expansion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 397.