RB1 gene. Both alleles of the RB1 gene must be mutated for tumour development. The initial RB1 mutation may be constitutional germline or somatic . Distinguishing between these alternative mechanisms is crucial, with wider implications for management of the patient and family members. Bilateral retinoblastoma is nearly always due to a constitutional mutation; however, approximately 15% of unilateral cases also carry a germline mutation, and identifying these cases is important. This can be achieved by identifying both mutation types in tumour tissue and excluding their presence in blood. Modern eye-saving chemotherapy treatment  has resulted in fewer enucleations. As a result, tumour tissue required to identify sporadic RB1 mutation(s) is not always available. Modern intravitreal chemotherapeutic techniques for retinoblastoma involve aspiration of aqueous humour (AH), providing a novel sample source for analysis. By analysing cell-free DNA present in the AH fluid of eyes affected with retinoblastoma, we have developed a screening test capable of detecting somatic RB1 mutations that is comparable to current tests on enucleated tumour tissue. The results obtained with fluid from enucleated eyes were concordant with tumour tissue in all 10 cases analysed. In addition, AH analysis from two patients undergoing intravitreal chemotherapy successfully identified somatic variants in both cases. Our findings suggest that AH fluid is a promising source of tumour-derived DNA in retinoblastoma for analysis.Retinoblastoma is the most common eye malignancy in childhood caused by mutations in the  RB1 gene, with non-germline cases being caused by two somatic hits, and inherited cases caused by an initial germline hit and a further somatic hit. Determining the genetic cause of RB is important for planning management of individual cases and prognostic counselling. Patients with RB1 germline mutations are at risk of bilateral disease and non-ocular cancers. There is also the additional risk that other family members may carry the same mutation. Analysis of peripheral blood samples along with tumour DNA (tDNA) accessed through enucleation allows for the complete genetic picture to be uncovered. The possibility that a patient has inherited RB can only be discounted by the detection of two mutations within the tumour sample, which are not present in the blood. Increased treatment success with modalities such as systemic or locally delivered chemotherapy suggests that tDNA is often unavailable rendering it impossible to identify somatic RB1 mutations.Retinoblastoma (RB) is the most common childhood eye cancer with an incidence of ~1:20 000 live births  in peripheral blood was noted in 1989Following ethical approval, subjects were recruited through the national referral retinoblastoma unit. Approximately 100 μL of AH fluid was taken from chemonaive primary enucleated eyes by anterior chamber tap prior to opening the eye for tumour dissection, or prior to injection during IVC treatment in eyes with recurrent disease following prior systemic chemotherapy and local treatment. Venous blood was collected at the same time for genetic testing.RB1 gene , highly heterozygous single nucleotide polymorphisms and non-polymorphic regions across chromosome 13. Bioinformatic analysis performed for single-nucleotide variant (SNV), copy number variant and loss of heterozygosity (LOH) detection using an in-house pipeline (information available on request).DNA extracted from blood, AH fluid and tumour (enucleated samples only) was sequenced by MPS following targeted capture enrichment for the promoter and exonic regions of the AH samples were obtained from 10 eyes post enucleation and from two patients with retinoblastoma undergoing IVC treatment. cfDNA concentrations from the enucleated eyes were found to be highly variable . The AH samples from the patients treated with intravitreous injection had lower concentrations of cfDNA and could not be accurately quantified (<0.100 ng/μL); however, we were able to detect the presence of cfDNA. The DNA size distribution peaked at 133 bp in comparison to the plasma cfDNA which peaked at 167 bp . PrevioRB1 SNVs, two large RB1 deletions and seven regions of LOH  or >45% where two RB1 alleles (one mutant and one wild type) were present, suggesting that >90% of the cfDNA present in AH of enucleated eyes is of tumour origin. This result supports our size profile results.In order to validate our assay, we sequenced cfDNA and tDNA extracted from 10 enucleated eyes. Analysis of the tDNA from the enucleated eyes identified 11 s of LOH . This cor tissue . In the le types . InteresAs a proof of principle, we sequenced cfDNA from two IVC samples. Diagnostic testing had previously identified one germline mutation in each patient but the somatic variants were unknown due to lack of tDNA. Germline mutations in both gDNA and cfDNA samples were detected in addition to the somatic variants in AH cfDNA samples . These wThe introduction of new techniques in the conservative management of retinoblastoma, including IVC, has dramatically reduced enucleation rates.RB1 gene mutations and LOH alterations in AH cfDNA. This extends the findings of Berry et al and demonstrates the clinical utility of our approach although further development is required to increase the accuracy in detecting larger exon-spanning RB1 mutations. Our data also suggests that the majority of cfDNA in the AH is from the tumour due to the compartmentalised nature of ocular fluids, a factor which facilitates this test.This study demonstrates the feasibility of testing AH samples in non-enucleated eyes, with AH cfDNA enabling detection of somatic variants in patients undergoing IVC where tumour is not available. Capture-based technology was used to identify previously undetected Among the enucleated eyes tested, higher levels of cfDNA were seen in eyes undergoing primary enucleation. Adequate but lower DNA levels were present in eyes treated with systemic chemotherapy and local treatment such as laser and cryotherapy. This would suggest the cfDNA load varies with the tumour load.The distinction between germline and somatic mutations is vital, as germline cases need close monitoring with short-term risk of new ocular tumours and long-term risk of second systemic cancers. The risks of both are greatly reduced if there is evidence to indicate a constitutional mutation is highly unlikely. Furthermore, there are important implications for screening examinations in the wider family (siblings and offspring). Confirming the non-heritable nature of the retinoblastoma can avoid screening examinations in many family members, with significant cost savings and avoid potential morbidity in unaffected family members, previously screened unnecessarily.et al, there appears to be a clear clinical benefit of diagnostic AH taps in apparent non-genetic retinoblastoma where the affected eye is retained.In view of the extensive safety record of carefully performed intraocular procedures in eyes with retinoblastoma (


To determine the feasibility of Fluids in Shock, a randomised controlled trial (RCT) of restricted fluid bolus volume (10 mL/kg) versus recommended practice (20 mL/kg).Nine-month pilot RCT with embedded mixed-method perspectives study.13 hospitals in England.Children presenting to emergency departments with suspected infection and shock after 20 mL/kg fluid.Patients were randomly allocated (1:1) to further 10 or 20 mL/kg fluid boluses every 15 min for up to 4 hours if still in shock.These were based on progression criteria, including recruitment and retention, protocol adherence, separation, potential trial outcome measures, and parent and staff perspectives.Seventy-five participants were randomised; two were withdrawn. 23 (59%) of 39 in the 10 mL/kg arm and 25 (74%) of 34 in the 20 mL/kg arm required a single trial bolus before the shock resolved. 79% of boluses were delivered per protocol in the 10 mL/kg arm and 55% in the 20 mL/kg arm. The volume of study bolus fluid after 4 hours was 44% lower in the 10 mL/kg group (mean 14.5 vs 27.5 mL/kg). The Paediatric Index of Mortality-2 score was 2.1 (IQR 1.6–2.7) in the 10 mL/kg group and 2.0 (IQR 1.6–2.5) in the 20 mL/kg group. There were no deaths. Length of hospital stay, paediatric intensive care unit (PICU) admissions and PICU-free days at 30 days did not differ significantly between the groups. In the perspectives study, the trial was generally supported, although some problems with protocol adherence were described.Participants were not as unwell as expected. A larger trial is not feasible in its current design in the UK.ISRCTN15244462. Rapid, liberal fluid bolus resuscitation is integral to the management of children presenting to emergency departments with septic shock.No trials have compared a more restricted fluid bolus resuscitation strategy with the currently recommended strategy in high-income countries.The optimal amount of fluid for resuscitation for children presenting with septic shock in high-income countries is an important unanswered question.The Fluids in Shock (FiSh) pilot compared a restricted fluid bolus volume (10 mL/kg) with the current recommendation (20 mL/kg) to determine the feasibility of a large-scale trial.A larger FiSh trial is not feasible; participants had a lower severity of illness than expected.Further observational work is required to determine the epidemiology of severe childhood infection in the UK in the postvaccine era.Rapid, bolus fluid resuscitation is integral to the management of children presenting with septic shock. The 2009 American College of Critical Care Medicine-Pediatric Advanced Life Support (ACCM-PALS) clinical guideline recommended fluid resuscitation with boluses of 20 mL/kg, up to a total of 200 mL/kg in the first hour.In Africa, a multicentre randomised controlled trial (RCT), the Fluid Expansion as Supportive Therapy (FEAST) trial, compared bolus fluid resuscitation of 20 mL/kg with maintenance fluid in over 3000 children with severe infection.No trial has compared restricted bolus fluid resuscitation strategy with recommended bolus fluid resuscitation in children with septic shock in high-income countries. In the UK, children presenting to emergency departments (EDs) with severe sepsis and discussed with a paediatric intensive care retrieval service have a reported mortality of up to 17%.To address this problem, the Fluids in Shock (FiSh) trial was developed, which aimed to evaluate whether a restricted fluid bolus volume (10 mL/kg), compared with currently recommended fluid bolus volume (20 mL/kg), is associated with improved outcomes for children presenting to UK EDs with presumed septic shock. Our initial qualitative feasibility study results have been reported previously.https://www.journalslibrary.nihr.ac.uk/programmes/hta/1304105/#/.The study design was a pragmatic, open, multicentre pilot RCT. The pilot trial was sponsored by the Imperial College Healthcare NHS Trust and coordinated by the Intensive Care National Audit & Research Centre Clinical Trials Unit (CTU). The ISRCTN trial registration number is 15244462. The protocol is available at Sites were set up in a ‘hub and spoke’ model in three regions in England. The ‘hubs’ were four regional hospitals with PICUs , three of which also had an integrated ED. The ‘spokes’ were nine district general hospitals with an ED but not a PICU linked to the ‘hub’ PICU by the regional PICU retrieval service. Participating EDs were research-active sites and part of the Paediatric Emergency Research in the United Kingdom & Ireland network.The inclusion criteria were age older than 37 weeks  and younger than 16 years; clinical suspicion of infection; and signs of shock—defined as age-adjusted hypotension (less than fifth centile systolic blood pressure (BP) for age or capillary refill time (CRT) greater than or equal to 3 s)—after receipt of 20 mL/kg of bolus fluid. Fifth centile systolic BP was provided to sites in age bands on wallet-sized cards for quick reference . The exclusion criteria were prior receipt of more than 20 mL/kg of bolus fluid; conditions in which bolus fluid resuscitation should be curtailed; or full active resuscitation not within the current goals of care.Eligible patients were randomised while in an acute assessment area  and allocated 1:1 to either 10 mL/kg or 20 mL/kg boluses over a 4-hour resuscitation period, without prior consent. The resuscitation period was divided into 15 min cycles, with one bolus of either 10 mL/kg or 20 mL/kg to be delivered in each cycle. The maximum amount of fluid that could be given per bolus was either 500 mL  or 1000 mL . Fluid type and other interventions were left to the discretion of the treating clinician. At the end of each cycle, if age-adjusted signs of shock persisted, then another bolus of the same size was given within the next 15 min cycle.In participants whose shock resolved or who showed signs of fluid overload , delivery of further fluid boluses was withheld. If, within the 4-hour resuscitation period, fluid boluses were indicated, that is, signs of shock were present in the absence of signs of fluid overload, cycles were recommenced with allocated boluses until the end of the 4-hour intervention period. After this period, any further treatment was at the discretion of the treating clinician.The maximum amount of fluid that could be given within the pilot trial protocol, regardless of allocation, was 120 mL/kg (excluding the 20 mL/kg bolus prerandomisation).A member of the site research team approached parents/legal representatives as soon as appropriate after randomisation to take consent for use of study data, according to the guidance developed in the FiSh feasibility studyThe objectives of the pilot trial were to test if the processes worked together, and to inform the design and conduct of the full FiSh trial (should this be the recommendation from the pilot). Outcome measures were driven by the progression criteria to be assessed by the pilot, and included recruitment and retention, protocol adherence and demonstration of separation between the groups, distribution of potential trial outcome measures, and parent and staff perspectives.These were determined by the proportion of eligible participants recruited, number of participants recruited per site per month, proportion of parents/legal representatives refusing consent, proportion of fluid boluses delivered at the correct volume and time during the intervention period (at least 80% of bolus fluid resuscitation delivered at correct volume and timing ±10%), total volume of fluid received during the intervention period in each treatment group  in the 10 mL/kg group), characteristics of potential outcome measures and observed adverse events, and parent and staff perspectives.Data collection was via a secure, dedicated, electronic database. Sites collected data throughout each patient’s hospital admission on the inclusion criteria, baseline—including the Paediatric Index of Mortality The trial was set up as a small pilot RCT without a defined primary outcome, and hence without a usual power calculation to determine sample size. Instead, sample size was determined to be adequate to estimate the parameters to be tested.https://www.icnarc.org/Our-Research/Studies/Fish/Study-Documents). Statistical analyses were based on the intention-to-treat principle. All tests used were two-sided with significance levels set at p<0.05 and with no adjustment for multiplicity. Final analyses were conducted using Stata/SE V.14.0.All statistical analyses were documented a priori in a Statistical Analysis Plan  received a fluid bolus of less than 20 mL/kg, while 108 (51.7%) did not meet the defined clinical signs of shock after receiving 20 mL/kg. There were 18 (17.8%) patients who met one or more exclusion criteria.Overall, 75 (90.4%) out of 83 eligible patients were recruited, 40 into the 10 mL/kg group and 35 into the 20 mL/kg group. Two were withdrawn  as the pBaseline characteristics were well matched, although there was some imbalance in age and consequently in weight . The majThirty-seven participants  had infection confirmed by the site team, that is, positive bacterial, viral or fungal microscopy, culture, PCR or immunofluorescence test. However, in five of these, the organism was not recorded. Of the remaining 32, 13 had bacterial infections in sterile sites , only onAll participants randomised to the 20 mL/kg group received their first bolus. Three participants randomised to the 10 mL/kg group did not receive their first bolus, although correctly identified as in shock, because it was deemed they no longer required fluid postrandomisation. Of the subsequent boluses, one participant in the 20 mL/kg group was administered a bolus when the shock criteria were not met. Five participants in total (three from the 10 mL/kg group and two from the 20 mL/kg group) who met the shock criteria did not receive the designated fluid bolus .The majority of participants in both groups received one fluid bolus, 23 (59%) in the 10 mL/kg group and 25 (74%) in the 20 mL/kg group. During the total 4-hour intervention period, only four patients received four or more fluid boluses. Seventy per cent of participants required only one study fluid bolus and only 11% required more than two study boluses.Assessment of the separation progression criteria showed that the mean total volume of study fluid given during the first hour was 38% lower (13.5 vs 20.7 mL/kg) and during the entire 4-hour intervention period was 44% lower (mean 14.5 vs 27.5 mL/kg) in the 10 mL/kg group compared with the 20 mL/kg group. At the end of the 4-hour intervention period, this corresponded to a statistically significant mean difference of −11.2 mL/kg . With regard to the adherence progression criteria, overall, 37 (78.7%) of 47 boluses were delivered at the correct volume and within 15 min in the 10 mL/kg group, whereas 24 (54.5%) of 44 boluses were delivered at the correct volume and within 15 min in the 20 mL/kg group .There were no deaths and no serious adverse events reported. Two patients in the 20 mL/kg group developed signs of fluid overload after the first study bolus, and consequently did not receive a second bolus despite remaining in shock. Overall, 29% participants were admitted to PICU. As expected in this small pilot trial, length of hospital stay, transfers to PICU, length of stay in PICU and days alive and free of PICU to 30 days did not differ significantly between the groups .A total of 52 (69%) of 75 parents of randomised participants provided consent to complete a questionnaire or take part in a telephone interview. Of these, 45 (87%) parents  from 44 families completed a questionnaire before leaving the hospital. Data saturation was reached at 20 (38%) of 52 interviews with parents  days since child’s admission). There were three pilot trial ‘hub’ site focus groups (20 staff) and telephone interviews with 7 (35%) of 20 invited staff (until data saturation point), including 14 (52%) of 27 nurses and 13 (48%) of 27 doctors from 7 (58%) of 12 sites (for thematic analysis, see online 10.1136/archdischild-2018-314924.supp1Supplementary file 1Interview and questionnaire data indicated some parents were surprised to discover that their child had been enrolled into the pilot trial without prior informed consent online . HoweverSite training had prepared staff for recruitment and RWPC. The randomisation method was viewed as straightforward. However, some clinical staff found it difficult to complete the case report form while treating a child. Staff also described problems adhering to the protocol, including administering 20 mL/kg boluses within the 15 min cycles and a lack of equipoise among a minority of clinicians when a child had been randomised to a 20 mL/kg allocation due to concerns about fluid overload.The pilot trial was successfully conducted. Screening logs showed that over 90% of eligible patients were randomised across the study sites over the study period with a recruitment rate of 0.9 participants per site per month, very close to the anticipated recruitment rate of 1 per site per month.However, the sites opened over a 4-month period rather than all at once, reducing the anticipated recruitment total from 108 to 84. Recruitment was driven mainly by three study sites, which recruited 38 of the 75 participants. A staggered opening of sites would be needed in the design for a larger FiSh trial, targeting sites most likely to see a high number of eligible patients.The process of RWPC worked smoothly, with no parents refusing consent. This is likely due to the bespoke FiSh RWPC methodology, developed during the feasibility study.The intervention was delivered according to protocol in the majority of participants. Adherence to volume and timing was close to achieving the 80% progression criteria target in the 10 mL/kg group but was not as good in the 20 mL/kg group. This appeared to be due to the difficulty in delivering the 20 mL/kg bolus within the 15 min time frame, especially in the period immediately postrandomisation. There was also some suggestions from the embedded perspectives study, indicating that some clinicians lacked equipoise, favouring 10 mL/kg over 20 mL/kg fluid boluses, despite the recently updated ACCM-PALS guidance continuing to recommend 20 mL/kg boluses.Though the trial processes were deemed feasible, the population had lower severity of illness than expected from previous data A recent systematic reviewA larger FiSh trial, with the current design, and in the UK, is not feasible. Further observational research is required to determine the epidemiology of severe childhood infection in the UK in the postvaccine era.


Accumulating evidence highlights the importance of interactions between tumour cells and stromal cells for tumour initiation, progression, and metastasis. In tumours that contain adipocyte in their stroma, adipocytes contribute to modification of tumour microenvironment and affect metabolism of tumour and tumour progression by production of cytokines and adipokines from the lipids. The omentum and bone marrow (BM) are highly adipocyte-rich and are also common metastatic and primary tumour developmental sites. Omental adipocytes exhibit metabolic cross-talk, immune modulation, and angiogenesis. BM adipocytes secrete adipokines, and participate in solid tumour metastasis through regulation of the CCL2/CCR2 axis and metabolic interactions. BM adipocytes also contribute to the progression of hematopoietic neoplasms. Here, we here provide an overview of research progress on the cross-talks between omental/BM adipocytes and tumour cells, which may be pivotal modulators of tumour biology, thus highlighting novel therapeutic targets.Abbreviations: MCP-1, monocyte chemoattractant protein 1IL, interleukinSTAT3, signal transducer and activator of transcription 3FABP4, fatty acid binding protein 4PI3K/AKT, phosphoinositide 3-kinase/protein kinase BPPAR, peroxisome proliferator-activated receptorPUFA, polyunsaturated fatty acidTAM, tumour-associated macrophagesVEGF, vascular endothelial growth factorVEGFR, vascular endothelial growth factor receptorBM, bone marrowBMA, bone marrow adipocytesrBMA, regulated BMAcBMA, constitutive BMAUCP-1, uncoupling protein-1TNF-α, tumour necrosis factor-alphaRANKL, receptor activator of nuclear factor kappa-Β ligandVCAM-1, vascular cell adhesion molecule 1JAK2, Janus kinase 2CXCL (C–X–C motif) ligandPGE2, prostaglandin E2COX-2, cyclooxygenase-2CCL2, C-C motif chemokine ligand 2NF-κB, nuclear factor-kappa BMM, multiple myelomaALL, acute lymphoblastic leukemiaAML, acute myeloid leukemiaGDF15, growth differentiation factor 15AMPK, AMP-activated protein kinaseMAPK, mitogen-activated protein kinaseAPL, acute promyelocytic leukemiaCCR2, C-C motif chemokine receptor 2SDF-1α, stromal cell-derived factor-1 alphaFFA, free fatty acidsLPrA, leptin peptide receptor antagonistMCD, malonyl-CoA decarboxylase. The tumour microenvironment (TME) affects tumour biology through various biological processes. Adipocytes are a particularly important component of the TME exerting both systemic and local effects on tumour growth and progression when tumours contain adipocyte in their stroma ,2. AmongThe omentum is a visceral adipose tissue, mostly composed white adipose tissue that consisted of vascularized connective tissue, and doubled mesothelial layered membranous and translucent tissue. The omeOmental adipocytes have distinct characteristics from adipocytes derived from other sites. In particular, the lipolysis action of catecholamine is increased in omental adipocytes compared with subcutaneous adipocytes, whereas the anti-lipolytic action of insulin and prostaglandin is more prominent in subcutaneous adipocytes than in omental adipocytes. ExpressMost of the tumours found in the omentum are metastatic tumours, and primary omental tumours are extremely rare. Malignant mesothelioma is a representative example of an omental primary tumour. It is suggested that adipocytes can play a key role in tumour promotion during asbestos-induced mesothelial carcinogenesis. Upon exposure to asbestos, the levels of proinflammatory cytokines such as monocyte chemoattractant protein 1 (MCP-1) increase, while anti-inflammatory cytokines such as adiponectin decrease in the adipocytes, resulting in an inflammatory environment. Since MCP-1 promotes mesothelial cell proliferation, these inflammatory stimuli could trigger a carcinogenesis process under a proliferative state Figure .10.1080/The most common carcinomas that metastasize to the omentum include ovarian cancer, colorectal cancer, gastric cancer, and pancreatic cancer. In partAdipocyte-derived metabolites and bioactive peptides are collectively referred to as adipokines, with more than 600 identified to date. In genein vitro study with ovarian cancer cells demonstrated the lipid transfer from adipocytes to tumour cells, which was enhanced for omental adipocytes compared with subcutaneous and mesenteric adipocytes[Omental adipocytes supply lipids to tumour cells and also support their survival and proliferation in the omentum. An dipocytes. Lipids dipocytes. To meetdipocytes. FABP4 [dipocytes and CD36dipocytes are the dipocytes In gastrdipocytes,38.As mentioned above, a milky spot, the functional unit of the omentum, is composed of diverse cell types, including immune cells such as macrophages, mast cells, and B- and T-lymphocytes, that are important in the immune response of the omentum ,40. The VEGFR1, VEGFR2, CD31, and CD34 in omental metastatic cancer tissues than in the primary ovarian cancer[Milky spots exist along with the vascular network of the omentum and at sites of active angiogenesis. In general, angiogenesis in milky spots occurs by vascular endothelial growth factor A (VEGFA) secreted from omental mesothelial cells and macrophages. In a hypoxic condition, the omental adipocytes secrete VEGF, vascular endothelial growth factor receptor (VEGFR) 3, and CD105 ,48,49 toan cancer.BMA are an important component of the BM with diverse roles, and the proportion of BMA in the BM fluctuates under various conditions. The number of adipocytes tends to increase with age, obesity, malnutrition, and stimulation of drugs or radiation –56. ApprThe bone is one of the most common metastatic sites of cancers, especially for prostate, breast, and lung cancers. The incBMA secrete various adipocytokines such as leptin, adiponectin, IL-1β, IL-6, vascular cell adhesion molecule 1 (VCAM-1), TNF-α, and VEGF,  which inFABP4 and CD36 in metastatic prostate cancer compared to those of the primary cancer[in vivo study also supported the lipid transfer from BMA to tumour cells: in the early phase of bone metastasis, the number of BMA increased by adipogenesis, but the number of BMA with abundant lipid droplets decreased during tumour progression[ENO2, LDHa, PDK1, HK2, and GLUT1 were upregulated in prostate cancer cells that were co-cultured with adipocytes[BMA provide the lipid source required for the proliferation, migration, and invasion of solid cancer cells ,105. In ry cancer. In addiogression. Bone meogression. Glycolydipocytes. Exposurdipocytes.The lipid droplets of BMA are composed of large-sized saturated and unsaturated fatty acids, particularly oleic, palmitic, and omega-6 PUFAs, and AA. Some ofThe studies reviewed above clearly demonstrate the roles of interactions between solid tumours and BMA in bone metastasis. However, hematologic neoplasms such as multiple myeloma (MM) and leukaemia are primarily derived from the BM, which could be the primary niche of these neoplasms. In solid tumours, elevated leptin level is associated with cancer risk. Also, iIn vitro, myeloma cells co-cultured with adipocytes showed enhanced proliferation and migration, and leptin was found to clearly play a role in this process[in vitro and in vivo[An epidemiologic study showed that low adiponectin and high leptin levels are associated with an increased tumour risk in multiple myeloma. In vitrs process. Leptin s process. Autophas process. Adipones process. Decreass process. Adipsins process. Vistafis process. IL-6 prd in vivo, and thed in vivo. TNF-α id in vivo, and indd in vivo. CCL2 led in vivo.Cytokines and chemokines secreted by BMA induce proliferation of AML cells. Leptin increases survival of leukemic cells ,132, andPPARɣ, FABP4, CD36, and BCL2 genes, which collectively inhibit the apoptosis of acute monocytic leukaemia cells[in vitro[Blast cells of AML induce the phosphorylation of hormone-sensitive lipase in BMA, and activate lipolysis, resulting in the increased production of fatty acids. The fatty acids are transferred to AML blasts via FABP4, which help to promote tumour cell proliferation. BMA alsmia cells. Thus, amia cells. Furthermia cells. When ALmia cells. Fatty amia cells,144, whe[in vitro, and PUF[in vitro.Given the evident roles of omental and BM adipocytes in the various pathways of tumour biology, targeting the interaction between tumour cells and adipocytes could be an effective new cancer treatment strategy .10.1080/in vivo and in vitro[in vitro: breast cancer cell line, MDA-MB231[Adipokine modulators have shown promising tumour-suppressive effects. Leptin has pro-tumorigenic effects as well as induces in chemoresistance via NF-κB and TGF-β signalling pathways ,148. Lep in vitro. LPrA2 p in vitro. Allo-acMDA-MB231, leptin-MDA-MB231. AMD3100MDA-MB231. NOX-A12MDA-MB231. CarlumaMDA-MB231. Also knMDA-MB231. AlthougMDA-MB231,161. L-4MDA-MB231, breast MDA-MB231, and ovaMDA-MB231. APO866,MDA-MB231.Given the metabolic interactions between adipocytes and tumour cells, targeting of metabolic pathways has been explored as a treatment target. Fatty acids released during lipolysis are transferred to tumour cells and used for energy production via mitochondrial β-oxidation, which enhances tumour progression. Hence, fatty acid oxidation in cancer cells is a promising therapeutic target. A malonyl-CoA decarboxylase (MCD) inhibitor inhibits fatty acid oxidation by increasing the malonyl-CoA level, which is a key inhibitory enzyme of fatty acid uptake in mitochondria, and in turn could reduce the proliferation of human breast cancer cells. In addiThe omentum and BM are highly enriched in adipocytes and are the main sites of metastasis for various types of solid tumours. The BM is also a primary site of hematologic tumour development. Adipocytes are a component of the TME that dictates tumour development, survival, and progression; thus, targeting adipocytes can be an important strategy for suppressing tumour development, cancer cell survival, and progression. Adipocytes of omentum and BM differ in their origin and location, but both serve as endocrine organs secreting adipokines and are involved in tumour biology. Adipocytes of the omentum and BM secrete various adipokines with pro-tumour effects on growth signalling, angiogenesis, and immune modulation. Furthermore, they transfer lipids to adjacent tumour cells to influence tumour metabolism, and enhance tumour proliferation and survival. Thus, targeting the interaction between tumour cells and adipocytes of the omentum and BM could be an effective tumour treatment. In this regard, IL-6, TNF-α, CXCL12, and CCL2 are therapeutically targetable adipokines, while FABP4 and CD36 are potential targets regarding the metabolic interaction. Further study is required to uncover the detailed relationships between adipocytes of the omentum and BM and their influence on tumour biology, and to identify and validate new potential treatment targets.


E. coli surface display of streptavidin for directed evolution of an allylic deallylase’ by Tillmann Heinisch et al., Chem. Sci., 2018, 9, 5383–5388.Correction for ‘ In the original manuscript, funding support from the ERC  was mistakenly omitted from the acknowledgements section. The corrected acknowledgements section is given below:TRW thanks the SNF (grant number grant 200020_162348) and the EU  and SP and TRW thank the NCCR Molecular Systems Engineering for generous support. VBP and EC also acknowledge support from the ERC . Juliane Klehr is thanked for help with vector cloning, protein expression and purification, Dr Rosario Vanella for help with flow cytometry and fluorescence microscopy and Eleonore Schmidt for activity measurement of purified ADAse mutants.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


Eight dogs that bore spontaneous tumours were enrolled and were subsequently treated with a single dose of intravenous CA4P. Perfusion of tumours was evaluated by power Doppler ultrasound (PDUS) pre-treatment (0 h), during the injection  and after CA4P infusion (24 and 72 h). Vascularity index (VI) of the tumour tissue was quantitatively analysed and accuracy was verified by correlation analysis with the results of immunohistochemical evaluation of microvessel density (MVD). Central and peripheral perfusion was evaluated by contrast-enhanced ultrasound (CEUS) pre-treatment and at 72 h post-treatment. Post-treatment, PDUS demonstrated a significant decrease in VI within 10 min of CA4P infusion. CEUS parameters demonstrated a significant decrease in blood velocity and volume in the central aspect of the tumour. Histology revealed a 4.4-fold reduction  in MVD and a 4.1-fold increase  in necrotic tumour tissue. A strong correlation between PDUS results and immunohistochemical results was found . Furthermore, the findings of PDUS were supported by the objective results of the CEUS analyses. These data suggest a role for ultrasound in real-time, non-invasive monitoring of tumour vascular response as an early indicator of CA4P treatment efficacy.Combretastatin A4-phosphate (CA4P) is an anti-vascular agent which selectively shuts down blood supply in tumours, resulting in extensive tumour necrosis. The aim of this study was to assess  Vascular targeting exploits the morphological differences between tumour and normal vasculature9. Whereas antiangiogenic agents (AAA’s) prevent formation of new blood vessels, vascular disrupting agents (VDA’s) destroy the tumour neovasculature11.Tumour vasculature is crucial for survival, growth, and spread of tumours and is a promising pharmacological target in anti-cancer strategies13. This drug has been subjected to a range of combined-therapy trials, including surgery, anti-angiogenic therapy, chemotherapy, and radiotherapy21. Considering the success of anti-vascular therapies and their future prospects, efficient screening methods of treatment response are highly sought-after.Combretastatin A4-phosphate (CA4P) is the best known VDA. It depolymerizes the microtubules of immature vessel endothelial cells, inducing vessel occlusion and resulting in shutdown of tumour perfusion within an hour of administration22. Given the invasive nature of sequential biopsies and the repeated need for anaesthesia, immunohistochemical MVD assessment has limited utility in a clinical setting. Thus, there is a clinical interest in non-invasive yet accurate imaging techniques that allow serial assessment of tumour perfusion in situ, enabling visualization of therapy response.The gold standard to assess the therapeutic effect of anti-vascular therapy on tumour vascularisation is immunohistochemical determination of microvessel density (MVD)  in pre- and post-treatment surgical biopsies28, diffusion-weighted imaging29, perfusion computed tomography30, positron emission tomography (PET)31, bioluminescence imaging (BLI)33, photoacoustic imaging (PAI)35, and dynamic contrast-enhanced (DyCE) fluorescent imaging36. Recently, contrast-enhanced ultrasound (CEUS) assessment in tumour-bearing mice was added to this list39. Power Doppler ultrasound (PDUS) has been used to assess vascularity in a range of animal and human tumours43 and for detection of blood flow changes following anti-vascular treatment46. It was recently found to be a reliable technique for the in vivo assessment of tumour angiogenesis41, but it has not yet been evaluated if this modality can be used as a reliable, non-invasive alternative to biopsies to asses CA4P treatment response. Considering that CA4P has been shown to reduce tumour blood flow within the first hour of therapy, well before vessels can regress, PDUS might provide a more veracious assessment of early response than immunohistochemistry.Current non-invasive imaging modalities to effectively evaluate the vascular response to CA4P and other VDA’s in rodents, rabbits, and men include dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI)In this study, we performed PDUS to assess the vascularity index (VI) before, during, and after a single dose of intravenous CA4P in a variety of spontaneous superficial solid tumours in dogs. Additionally, we used CEUS to quantitatively evaluate the intratumoural perfusion, both in the central and peripheral regions of the tumours. To verify the accuracy of PDUS, a correlation analysis was performed on the PDUS results and the results of conventional MVD determined by immunohistochemistry, at 72 h post-treatment.The aim of this work was to conduct a proof-of-principle study to assess the feasibility of using PDUS and CEUS as methods for assessing early tumour vascular inhibition by CA4P in clinical studies involving spontaneous, superficial solid tumours.45. The study cohort consisted of four males and four females, belonging to six different breeds, and included five mesenchymal tumours, two epithelial tumours, and one round-cell tumour.Eight dogs entered the study. Baseline characteristics of participating patients are summarized in Table Pre-treatment (pre-CA4P), there appeared to be a marked variability in the tumour perfusion as evaluated by PDUS Table . The sarTumour perfusion parameters assessed by CEUS also revealed the sarcomas to be less well vascularized pre-treatment than the carcinomas and the mastocytoma Table . A diffePre-treatment and post-CA4P results for all patients are listed in Table Areas of necrosis were characterized by loss of tissue architecture, eosinophilic cellular debris, pyknosis of nuclei and/or nuclear fragmentation. The estimated overall ratio for necrosis was 0.08 (95% CI [0.02 to 0.19]) before treatment and 0.33 (95% CI [0.01 to 0.82]) 72 h after treatment, corresponding to an overall 4.1-fold increase in necrosis of the tumour after a single dose of CA4P . Several tumours demonstrated a heterogeneous response within one biopsy sample. Areas of necrotic cells were interspersed with areas of mitotic cells. In some tumours, large areas of the tumour had undergone complete cellular degeneration and only a few viable cells remained. These viable cells were arranged concentrically around the remaining patent blood vessels. In contrast, vessels that were blocked, resulting in complete vascular occlusion, were surrounded by necrotic tumour cells. The endothelial cells of these obstructed vessels were fragmented and in several of the blocked tumour vessels, thrombi were observed.  staining, was observed to have decreased significantly 72 h after treatment Fig. . The est2 = 0.957, p < 0.001). There was no significant correlation between any of the CEUS parameters and the MVD: PE , WiAUC , TTP , RT , and mTT .For both the treated and pre-treated dogs, correlations between PDUS and MVD and between CEUS and MVD are illustrated in Fig. in vivo assessment of spontaneous tumour vascular response to CA4P treatment. Additionally, the reduction in tumour blood velocity was assessed by means of CEUS. Power Doppler ultrasound (US), CEUS and histopathology each revealed vascular shutdown with considerable necrosis within a 72 h period post-treatment.In this study, PDUS was compared with histopathology and assessed as a technique for the 22. To improve blood vessel detection on histological slides, immunohistochemical staining of endothelial cell markers is performed. However, in addition to tissue biopsy being an invasive monitoring technique, early treatment response of anti-vascular agents such as CA4P can be misjudged in post-treatment biopsies; as several VDA’s induce changes in tumour blood perfusion well before vessel regression becomes apparent on histopathological tissue sections22.Ideally, clinical monitoring tools should be non-invasive, allowing for serial assessments of the same tumour mass without risk for the patient. Currently, the gold standard to assess the therapeutic effect of anti-vascular therapy on tumour vascularisation is microscopic determination of MVD in pre- and post-treatment histological tissue sections47. However, these techniques are not always readily available and they are expensive, and some require ionizing radiation (PET) and/or heavy sedation or even general anaesthesia of the patient . In contrast, US is assumed to be a promising angiogenesis assessment modality, as it is a widely available, non-radioactive, real-time and cost-effective alternative for above-mentioned techniques39. Additionally, CEUS is also suitable for use in patients with renal and hepatic failure because of the pulmonary elimination of the contrast agent48.The need for non-invasive techniques allowing early evaluation of clinical patients treated with fast-acting anti-vascular treatments is evident. To date, non-invasive techniques such as DCE-MRI, PET, and near-infrared spectroscopy have been explored to demonstrate changes in tumour perfusion and viability in both rodent tumour models and human patients49. Power Doppler US is a technique that displays the strength of the amplitude in colour, rather than speed or direction information, by using the amplitude of a Doppler shift to detect moving matter50. Noise is assigned a uniform colour that remains constant while flow is shown as a contrasting colour. As a result, PDUS has three to five times the sensitivity of conventional colour Doppler for the detection of flow51. Although the sensitivity of the technique certainly varies with both vessel calibre and flow rate, previous studies have demonstrated Doppler detection of flow through tumour vessels as small as 10–20 μm53.Vascular imaging such as US allows quantification of the number and spacing of blood vessels and to assess blood flow in a non-invasive and functionally relevant way. Therefore, US has the potential to assess the immediate efficacy of anti-vascular therapyin vivo and in situ manner44. Standardized power Doppler settings and image-processing protocols enable a precise and objective evaluation of the VI and make PDUS a convenient and immediately applicable way to determine the efficacy of anti-vascular agents in a therapeutic setting.The results of the PDUS clearly confirmed that CA4P treatment of spontaneous solid tumours in dogs resulted in an acute tumour vascular response within 10 min after start of the infusion. The statistically significant correlation between intratumoural VI and the gold standard MVD at 72 h supports the hypothesis that PDUS offers an objective and reliable assessment of the tumour vascularity at the time of US scanning in an 54 if highly excited or fearful.The susceptibility of PDUS to flash artefacts, because of its high motion sensitivity, is one of its major limitations. Patient cooperation is necessary to decrease artefacts resulting from excessive movement (e.g. panting), as prominent soft-tissue motion may preclude the use of PDUS. For human patients, this is not likely to form a problem; veterinary patients may need sedation with drugs known not to interfere with vascularisation TM consists of sulfur hexafluoride-filled microbubbels (MB’s) in a phospholipid shell48. The mean diameter of the sulfur hexafluoride-filled MB’s is 2.5 µm, resulting in a strictly intravascular trajectory of the bubbles without any interstitial component55. At low acoustic power, alternating expansion and contraction of the MB’s result in a vibration, generating harmonic echoes. The harmonic signal produced by the MB’s is greater than that of the surrounding tissue, making CEUS a very sensitive imaging technique, well suited for the evaluation of blood flow in functional vessels56.The CEUS contrast agent SonoVue57, followed by a slow clearance. Post-treatment, the time constant for contrast to reach the tumour (TTP) was increased, likely indicating a less aggressive behaviour of the tumour after CA4P treatment. Together with a significant drop in tumour blood volume (PE and WiAUC) and increase in tumour transit time (RT and mTT), it is possible that CA4P treatment induced a slower tumour progression, despite a continued growth of the tumour if no adjuvant therapy is administered58. Of particular interest was that the software for analysis of the CEUS data allowed for an easy and objective distinction between the measurements of tumour centre versus periphery, which is important in the determination of drug efficacy and prognosis. Results post-CA4P-treatment clearly demonstrated a high susceptibility of the tumour centre to necrosis formation while the perfusion of the tumour periphery was consistently poorly affected, which is in accordance with previous research59.After CA4P administration, all tumours demonstrated significant changes in the CEUS perfusion characteristics. Pre-treatment, CEUS demonstrated a rapid inflow of the MB’s into the entire tumour, a distinctive feature of malignant tumours39, the CEUS parameter maximum intensity (IMAX) was found to be closely correlated with MVD. This parameter represents the average US contrast agent concentration of the analysis region of interest (ROI) (tumour tissue) relative to that of the reference ROI  which is assumed to be 100%. Calculating the IMAX necessitates obtaining images including both tumour tissue and healthy tissue in the same image. This is perfectly feasible in rodent tumour models, with tumours reaching only several mm’s in diameter. However, in pet animal or human patients with spontaneous tumours, which can easily reach several cm’s in diameter, it is not possible to obtain images containing both tumoural and healthy tissue. Additionally, for some tumours it can be difficult to distinguish healthy tissue from tumour infiltrated tissue, based on US alone. Therefore, IMAX is not always a useful parameter in a clinical setting. Notwithstanding the fact that the CEUS parameters assessed in the current research were not significantly correlated to the MVD results, previous research demonstrated that CEUS provides an excellent non-invasive quantitative and qualitative alternative for tissue biopsies, as it has proven to be at least as sensitive a method for tumour angiogenesis monitoring as histopathology60. In this research, CEUS was not considered a reliable alternative, but rather an excellent adjunct to PDUS to assess tumour blood velocity between the different tumour areas before and after CA4P treatment. The necessity for intravenous administration and the availability of the MB’s and the dedicated software can be considered as drawbacks for the use of CEUS. However, the authors are of the opinion that the addition of CEUS to PDUS is relevant and beneficial, and the combination of these imaging modalities is an attractive and preferable alternative to sequential biopsies for the evaluation of CA4P treatment response.Although the CEUS parameters demonstrated significant reduction in perfusion in the tumour centre, the correlation between the CEUS parameters and the gold standard MVD failed to reach statistical significance. In the study carried out by Zhang and colleaguesThere are some limitations to this clinical trial. Only a small number of patients with a variety of tumour types and stages were assessed. Additionally, only dogs with superficial tumours were enrolled in this proof-of-concept study because a superficial location made it practical and acceptable to obtain a 72 h post-treatment tumour biopsy. Lastly, due to ethical regulations, only tumoural tissue was biopsied and imaged by PDUS and CEUS, while healthy tissue was not evaluated. Therefore, it cannot be confirmed that the effect of CA4P was truly limited to tumoural tissue without healthy tissue being affected at all.This research confirms that measurement of the intratumoural VI by means of PDUS is correlated highly with the conventional MVD assay and is potentially useful for clinical monitoring of CA4P treatment. Assessment of the CEUS parameters support these findings and additionally prove primary response location to be the tumour centre. Additionally, our results emphasize that dogs with spontaneous cancer offer a novel and relevant model for studies of vascular disrupting therapy.Considering the US results of this study, the clinical value of this non-invasive modality warrants further exploration and development of this technology during assessment of vascular targeting therapies.45.Dogs with histopathologically confirmed, superficial and well-defined solid tumours, suitable for US examination, were enrolled. Specifications on patient selection have been described elsewhereEuropean Communities Council Directive (86/609/EEC)/principles of laboratory animal care . Written informed consent was obtained from all patient owners before entry into the study. The use of CA4P in canine patients was approved  by the Belgian Federal Agency for Medicines and Health Products (FAMHP).Approval was obtained from the local research Ethical Committee  of the Faculty of Veterinary Medicine of Ghent University, Belgium and from the Deontological Committee of the Federal Public Service of Health, Food Chain Safety and Environment, Belgium. The study protocol adhered to the guidelines and regulations of the Pre-treatment tumour assessment consisted first of histopathological necrosis assessment and immunohistochemical MVD evaluation. A preliminary PDUS determined the imaging plane with maximum tumour perfusion and subsequently, after patient sedation, at least dual 6-mm punch biopsies were taken in this region. Further evaluation by medical imaging was then postponed for a minimum of two weeks, to preclude any influence  caused by the biopsy taking. 72 h following the CA4P-treatment and after all other assessments were completed, 6-mm punch biopsies were taken in the vicinity of the pre-treatment biopsies.45. Lyophilized CA4P  with a purity of >99% was purchased from Selleck Chemicals LLC , and stored under light exclusion at a temperature of −20 °C. For the preparation of every personalized solution, the drug was accurately weighed  and reconstituted in a sterile phosphate buffered saline (PBS)  solution. Subsequently, the required volume of CA4P solution was pipetted and transferred to sterile amber vials. Finally, PBS was added until a final volume of 10 mL was reached. All CA4P solutions were prepared in a biological safety cabinet and were administered within 30 min of preparation as an intravenous infusion at a dose of 75 mg m−2 61, over a time span of 30 min using a volumetric pump.Specifications on the preparation and administration of CA4P have been described in our previous study®62. The density of the vascular signals within the demarcated tumour was then calculated and expressed as the VI  by a researcher blinded to patient identification and tumour treatment status.To obtain the PDUS images, a linear 12-5 MHz transducer was used on a dedicated ultrasound machine , operated at a Doppler gain of 76%, low persistence, pulse repetition frequency 500 Hz, and wall filter 47 Hz. The depth and position of the focus (single focus) were adapted based on the size of the tumour. The settings were standardized and repeated at each imaging session. The plane with maximum vascular signals was found by slowly moving the probe over the tumour, both in longitudinal and transverse orientation. The colour window was reduced in size to just cover the tumour to improve the colour sensitivity and frame rate. Stable vascular images and movies were captured and stored for quantitative analyses and the scan plane was documented to enable future scanning at the identical location. During the CA4P infusion, PDUS was performed at the identical tumour location at 10, 20 and 30 min. The PDUS examinations were repeated at 24 and 72 h PI at matched locations. At a later occasion, the captured images were retrieved and the boundary of the tumour was manually delineated on the computer with the use of the software Fiji®, Bracco, Milan, Italy), consisting of lipid-stabilized MB’s with a sulphur hexafluoride gas core, was injected IV as a bolus (0.04 mL kg−1) immediately followed by a 2.5 mL flush of sterile saline 45. Images were acquired using a linear 12-5 MHz probe. Machine settings were standardized: gain optimized to start with a nearly black baseline image , dynamic range 50, mechanical index 0.09, and persistence off. The depth and position of the focus (single focus) were adapted based on the size of the tumour. During pre-treatment scanning, the scan plane was recorded for future assessment. All dogs received three injections of contrast media. Based on pre-existing knowledge, the second and third injection results were used since they resulted in more consistent enhancement than the first injection63. Between injections, the output power was adjusted to maximum with continued imaging of the tumour until background echogenicity was similar to pre-injection levels, indicating destruction of most residual contrast bubbles. In our experience, complete wash-out of the MBs was accomplished within 120 s, therefore the time frame for data collection was set at 0–120 s.As described in our previous study, to perform the CEUS, a MB contrast agent  for objective quantitative analysis. Per study, three tumour ROI  were drawn manually on the CEUS image  for two min after bolus injection. Clips were then exported to an offline computer, where they were randomized and anonymized. Subsequently, the clips were analysed using integrated dedicated specialized computer software  was applied at 1:1600 dilution for 30 min at room temperature. Subsequently, HRP was linked to the primary antibodies using Envision Link Rabbit kit K401111-2 . Sections were then rinsed, incubated with 3,3′-diaminobenzidine solution, and counterstained with Haematoxylin.The tumour tissue samples were fixed in a 10% buffered formalin solution, embedded in paraffin, and sectioned at 4 μm. Per biopsy, four sections were cut at different depth within the biopsy to acquire technical replicate sections. Two sections with a 12 μm interstice were stained with Haematoxylin and Eosin (HE) for the histopathological evaluation of necrosis. For evaluation of the MVD, two sections with a 12 μm interstice were stained with the endothelial cell marker (vWF). Deparaffinized and hydrated sections underwent pre-treatment by 15 min incubation with Proteinase K S300402-2  and subsequently by 5 min incubation with H® software. In every HE-stained section, the relative amount of necrotic tumour tissue was expressed as the ratio of necrotic tumour tissue to the total tumour area. The averages of the five ratios were then calculated for each tumour biopsy. Microvessel counts were calculated in every vWF-stained section, based on the total number of vWF-positive structures within the tumour area and expressed as the MVD (ratio of tumour blood vessels mm−2 of tumour tissue). The averages of the five MVDs were then calculated for each tumour biopsy. Only uninterrupted round vWF-positive structures containing a lumen were counted as microvessels. Larger vessels with thick muscular walls and vessels with a lumen greater than approximately eight red blood cells were excluded, as suggested by Bosari and colleagues64. All measurements were performed blinded to patient identification and tumour treatment status. To facilitate recognition of vWF-positive structures, a H-DAB filter was applied within the FiJi® software. Figure Histological assessment of all biopsies was performed with the use of a Leica light microscope . Slides were first scanned at low power (×20) to identify tumoural tissue. Five fields within tumoural tissue per section were then selected randomly as counting frame at higher power (×100). Density measures were obtained by overlaying a grid on the counting frame and quantifying the crossing points that hit the area of interest, with the use of the Fiji45. Except for the histological data, all data were analysed with nonparametric paired tests due to the non-normal distribution of the data using R (version 3.4.1). The Wilcoxon signed rank test and Friedman’s test  were used for comparing paired samples across two or more time points respectively. The Mack-Skilling’s test was used as replicated measures variant of the Friedman’s test accounting for multiple replicates per sample in the CEU data . Post hoc analysis of Friedman’s tests was performed using Wilcoxon signed rank tests and all p-values were corrected using the Holm-Bonferroni method . A two-sided p-value of ≤0.05 was considered to indicate statistical significance.The statistical analyses were performed as described previously45, using generalized linear mixed models to account for variability of individual patients and biopsies using random effects. The package lme4 (version 1.1) was used in R (version 3.4.1). Given evidence of overdispersion, negative binomial generalized linear mixed models were fitted with the number of blood vessels or the necrosis surface as an outcome, treatment as a fixed effect, along with a random intercept and random slope to allow for differences in treatment effect between individual patients and control for correlation among replicated measurements. An offset was added to control the reference area of tumour tissue assessed in each counting frame.Also the histological data, i.e. necrosis and blood vessel density data, were analysed as described in our previous studyThe Pearson correlation coefficient was calculated and evaluated for statistical significance  to examine the correlation between PDUS and the MVD, and between the individual CEUS parameters and the MVD.The study protocol adhered to the European Communities Council Directive (86/609/EEC)/principles of laboratory animal care . Approval was obtained from the local research Ethical Committee  of the Faculty of Veterinary Medicine of Ghent University, Belgium and from the Deontological Committee of the Federal Public Service of Health, Food Chain Safety and Environment, Belgium. Written informed consent was obtained from all patient owners before entry into the study. The use of CA4P in canine patients was approved  by the Belgian Federal Agency for Medicines and Health Products (FAMHP).


We report a case of Langerhans cell histiocytosis (LCH) occurring in the pelvis of a 2-year 11-month-old female with fluid–fluid level seen on MRI. Aspiration of the fluid during biopsy showed it to be blood with a few inflammatory cells and eosinophils. Tissue obtained during the biopsy confirmed the diagnosis to be LCH. While fluid–fluid levels have been infrequently encountered in skull lesions due to LCH, they have yet to be reported in lesions of the appendicular skeleton. The aim of this report is to familiarize radiologists with the fact that fluid–fluid levels can occur in LCH of the appendicular skeleton in children. We report a case of Langerhans cell histiocytosis (LCH) occurring in the pelvis of a 2-year 11-month-old female with fluid–fluid level seen on MRI. Aspiration of the fluid during biopsy showed it to be blood with a few inflammatory cells and eosinophils. Tissue obtained during the biopsy confirmed the diagnosis to be LCH. While fluid–fluid levels have been infrequently encountered in skull lesions due to LCH, they have yet to be reported in lesions of the appendicular skeleton. The aim of this report is to familiarize radiologists with the fact fluid–fluid levels can occur in LCH of the appendicular skeleton in children.A 2-year 11-month-old female presented to our hospital with left hip pain. Laboratory tests did not reveal any significant finding and the inflammatory markers were not elevated.T2 weighted images showed fluid–fluid level within the lesion (T1 weighted MRI showed the lesion to be well defined and isointense to muscle. A faint fluid–fluid level was noted within the lesion (–1), the lesion showed peripheral enhancement and an enhancing internal septa was identified within the lesion (T1 weighted images was high in signal intensity on the fat-saturated T2 weighted images, possibly owing to bony inflammation. Periosteal reaction was seen in both sequences. Another smaller lesion was noted within the right superior pubic ramus, with mild enhancement on post-contrast images. This lesion appeared solid and did not have a fluid–fluid level.Plain radiographs revealed an osteolytic lesion in the left iliac wing . The lese lesion . The none lesion . After te lesion . There wCT-guided aspiration of fluid contents followed by biopsy of the larger left iliac bone lesion was performed. Blood with a few inflammatory cells and eosinophils was aspirated. The biopsy  and immuA subsequent skeletal survey revealed another lesion in the T11 vertebral body. The patient was treated with intravenous vinblastine and oral prednisolone for 6 weeks and is currently well on regular follow-up.1 but are found in a wide range of bone lesions and are thus a non-specific finding.5 Fluid–fluid levels have been described in LCH lesions involving the calvarium but not of the appendicular skeleton.6This is the first reported case of a fluid–fluid level occurring in a case of LCH of the appendicular skeleton. The occurrence of fluid–fluid level is believed to be due to intratumoral haemorrhage.7 This case is important as it further highlights the non-specificity of fluid–fluid levels in osteolytic lesions and LCH should still be included in the differential diagnoses of these lesions in children.Fluid–fluid levels are characteristically described in aneurysmal bone cystsThe aim of this report is to familiarize radiologists with the fact that fluid–fluid levels can occur in LCH of the appendicular skeleton in children.This case also further highlights the non-specificity of fluid–fluid level in osteolytic lesions.Written informed consent was obtained from the patient’s parents for publication of this case report, including accompanying images.


A simple and robust co-flowing microfluidic device for double emulsion preparation is designed and assembled to visually study the double emulsion formation by the use of a microscope and high-speed camera. Using a visualization system, the transient processes of double emulsion formation in co-flowing stream are observed and recorded for a variety of flow rates. The effects of flow rates of each fluid on flow modes, drop sizes, and polydispersities are examined and analyzed. The results indicate that the detaching of the inner drops accelerates the detaching of the outer drops and speeds up the drop formation process of double emulsions. The manipulation of flow rates is capable to actively control the sizes of the inner and outer drops as well as the number of inner drops encapsulated. Without surface modification, the microfluidic device produces a variety of emulsions, including the single-core and multi-core O/W/O double emulsions as well as binary emulsions of single and double emulsions. The proposed co-flowing microfluidic device is highly desirable in producing double emulsions in an easy and cheap way. Monodispersed drops in emulsions ,2 are idAmong various microfluidic devices, co-flowing configuration is the most commonly used design with all fluids flowing coaxially . The emuThe formation processes of double emulsions involve two nesting interfaces that interact with each other, leading to complicated mechanisms that still lack comprehensive experimental insights. The design and optimization of microfluidic devices require full exploration of the dynamic behaviors of the emulsification processes. Therefore, a three-dimensional co-flowing microfluidic device is designed and assembled in the current investigation with the outlets of the inner fluid and middle fluid on the same cross section where the double emulsions are formed in one step. Based on this microfluidic device, the hydrodynamic behaviors of various drop formation modes are examined via a visualization system using a microscope and high-speed camera. The influence of flow rates on flow modes, drop sizes, and polydispersities are examined and analyzed.A simple and robust co-flowing microfluidic device for the production of O/W/O double emulsion is assembled at low cost without requirement for the modification of surface wettability. First, a poly(dimethylsiloxane) (PDMS) brick with fluid passages is molded as shown in w/w) solution of Poly 1788  was used as the middle fluid. Three syringe pumps  are used to inject the fluids into the devices. The visualization system is comprised of a microscope , a high-speed camera , a computer, and a light source. All experiments are conducted at constant room temperature of 22 °C.The PDMS brick is made of SYGRID 184  and cured at 85 °C for 4 h. Silicone oil  with the viscosity of 20 mPa·s and 500 mPa·s is used as the inner fluid and outer fluid, respectively, while 5%  where vi = qi/(π·R12) is the mean velocity of the inner fluid and qi is the flow rate of the inner fluid.To quantitatively describe the fluid dynamics during the formation of double emulsions, the distances from the front of the inner and outer interfaces to the outlets of the inner and middle fluid are measured as the lengths of the inner and outer drop , indicating the separation of the outer drop. As plotted in Lo turns up a discontinuous faster increment after the separation of the inner drop, attributed to enhancement of middle fluid flow caused by the detaching of the inner drop. Likewise, separation of the outer drop is also characterized by the shrinking of the neck and pinching off of the interface. Note that the outer interface squeezes the middle fluid upstream during pinching-off leading to the front of the inner interface been pushed backwards as shown in τ = 41.96–41.97) are retracted by the interfacial tension, lengths of both inner and outer drop decrease in a short period of time.As demonstrated in Lo and Li indicate that the formation process of multi-core double emulsion can be divided into several stages: growth, separation of the first inner droplet, separation of the second inner droplet, separation of the Nth inner droplet, and separation of the outer drop. The growth stage is similar as that during the formation of double emulsions with single core. However, due to the insufficient viscous force from the outer fluid which cannot overcome the interfacial tension of the outer interface, the outer drop continues growing after the detaching of the first, second, and (N − 1)th inner drops. As plotted in Under the condition of low flow rate of the outer fluid, double emulsions with multi-cores  can be pτ = 0–3.19, the faster growing outer drop reaches the critical volume while the inner drop is still growing. A neck on the outer interface develops downstream of the inner drop and the increasing of Lo is slightly speed up when a single drop of the middle fluid without inner drop is detaching. During this stage, the variation of Li fluctuates due to radial squeeze from the outer interface and the inner interface is stretched along the flow direction. After the first pinching-off, both inner and outer interfaces retract back to the tip of the capillaries and the process enters the second growth stage. The axial growth of both inner and outer drops slow down slightly while Lo is still increasing much faster than Li. It is worth noticing that the narrowest part of the outer interface is downstream of the inner drop (τ = 9.31) when the outer drop begins detaching. However, this part of the interface does not develop into a neck. The pinching-off motion of the inner interface accelerates the fluid flow upstream and the inner drop is pushed through the narrow part of the outer interface. The neck on the outer interface is then developed upstream with the detached inner drop leading to the production of a double emulsion drop.Apart from continuous production of double emulsions, alternative generation of monodispersed single and double emulsion drops is also observed when the flow rate of the middle fluid is much larger than the inner fluid. The drop formation processes are stable and robust with easy control of drop sizes, providing a novel approach for the manufacture of binary materials or dilution of the double emulsions. As shown in Ri and outer drop Ro with increasing flow rate of the outer fluid qo. The statistics of size distributions of the drops are noted as the error bars in qo slightly due to acceleration of the middle fluid caused by the momentum transfer from the outer fluid. Reduction of the number of inner drops is also observed, leading to drastic decrement on the size of the outer drop Ro. With continuous growth of viscous force, the differences in time required for the inner drops and outer drops to reach critical volumes are lessened, hence generation of monodispersed single-core double emulsions occurs spontaneously. Increasing qo results in smaller double emulsion drops, however, the variation of Ro and Ri follows the same tendency, which suggested that the increasing viscous force of the outer fluid has no obvious influence on the thickness of double emulsions produced in the one-step co-flowing device. When the flow rate of the outer fluid flow exceeds 180 mL/h, viscous force dominates over interfacial tension that single drops of middle fluid are sheared off between every other double emulsion drop. Under this flow condition, the double emulsions are still highly monodispersed while the single drops are polydispersed due to rupture of the interface repeatedly by the viscous force.qm has decisive effect on the shell thickness (Ro − Ri) of the double emulsions and weaker influence on the double emulsion size. Ultra-thin shell double emulsions (see inset in qm and the drop size can be actively controlled by qo. Also, the shell thickness can be adjusted from 5% to 50% of Ro continuously by increasing qm without causing large variation in the size of the double emulsions. A decrease in the inner drop size with qm indicates that viscous force from the middle fluid plays critical role in the detaching process of the inner drop and controlling over the shell thickness. However, further increment in qm over a threshold alters the number of inner drops and alternative formation of single-core and dual-core double emulsions can be observed. The outer drops are monodispersed while the inner drops are polydispersed due to the same reason in the formation of multi-core double emulsion. Hence, to achieve steady production of monodispersed single-core double emulsions as well maintain better control over the formation process, the flow rate of the middle fluid should be kept at the same magnitude or smaller than the inner fluid.Compared to the outer fluid, the flow rate of the middle fluid qi has no obvious influence on the drop size of the double emulsions as shown in qi, insufficient inner fluid leads to alternative formation of monodispersed single and double emulsions that each take up to 50% percent. As qi increases, continuous formation of single core double emulsions can be observed in a wide range that the inner drop size increases slightly with qi while the outer drop size remains almost constant. Besides, the shell thickness can only be adjusted in a narrower range by the inner fluid compared to the middle fluid. However, after the increment of qi over a threshold, alternative formation of single-core and dual-core double emulsions can be observed with high polydispersity, indicating that low flow rate of the inner fluid is required to obtain steady production of monodispersed double emulsions.Similar to the middle fluid, variation of the flow rate of the inner fluid In this study, a cheap and robust co-flowing microfluidic device is proposed in this paper to generate double emulsions in one step without requirement of surface modification. The growth, deformation, and break-up of the drop interfaces are visualized through high-speed camera to determine the liquid-liquid interaction between the outer and inner drops, in an effort to elucidate the fluid dynamic behaviors during the drop formation processes. The influence of the flow rates of three-phase fluids on flow mode are examined and discussed. The results indicate that the one-step co-flowing microfluidic device is capable of producing the single-core and multi-core O/W/O double emulsions as well as the binary emulsions of single and double emulsions mixture. The detaching of the inner drops accelerates the detaching of the outer drops and hence contributes to drop formation of double emulsions. The manipulation of the flow rates of the outer fluid actively controls the drop formation modes, the number of the inner drops, the drop sizes, as well as the drop size distribution. The drop formation modes are insensitive to the middle and inner fluid flow rates. However, these flow rates significantly affect the shell thickness of the double emulsions.


In the article titled “Circulating Endocannabinoids and Insulin Resistance in Patients with Obstructive Sleep Apnea” , a grantThis study was supported by a grant from the Health Bureau of Gansu Province (no. GSWSKY-2014-28) and a grant from the Natural Science and Technology Fund of Gansu province (no. 1506RJZA254).


The aim of this study was to evaluate the pre-operative performance of an automated multiplex PCR (mPCR) system in patients with suspected periprosthetic joint infection (PJI).Under sterile conditions, synovial fluid samples from patients with a suspected PJI were collected pre-operatively. One hundred eighty microliter of the aspirate was used for analysis in the mPCR. The remaining joint fluid was sent for microbiological analysis. PJI was diagnosed by using the Musculoskeletal Infection Society (MSIS) criteria. Total percentage agreement and Cohen’s kappa coefficient were calculated to measure overall agreement.Cutibacterium spp. were detected five times by mPCR, it could only be cultured once. A higher detection rate of CoNS by mPCR (n = 7) compared to conventional culture (n = 5) was also demonstrated. In comparison to synovial fluid culture, the mPCR missed Staphylococcus aureus five times.Overall, 90 patients with a suspected PJI were included. Using MSIS criteria, 38 (42%) patients were classified as septic. Total percent agreement between mPCR and synovial fluid culture was 86% with a Cohen’s kappa of 0.68. The mPCR and synovial fluid culture showed sensitivities of 71% and 84%, respectively. Combined evaluation provided an even higher sensitivity of 92%. While With a moderate agreement between synovial fluid mPCR and culture, the mPCR system could be a useful adjunct in diagnosing a PJI pre-operatively. Due to faster availability of results and a higher detection rate of low-virulent microorganisms, it can complement conventional culture. The pre-operative detection of the causative microorganism(s) and antimicrobial susceptibility is essential for ideal surgical and antibiotic treatment of periprosthetic joint infection (PJI). An accurate and prompt evaluation is crucial for treatment success. Currently, pre-operative-collected synovial fluid cultures represent the method of choice for diagnosing an infection and planning the optimal revision surgery (one-stage vs. two-stage). Results from conventional microbiological culture-based analyses should become available within one to 14 days , coagulase-negative Staphylococci [n = 1], Cutibacterium acnes + coagulase-negative Staphylococci , coagulase-negative Staphylococci [n = 1]). Out of these, two were classified aseptic, although definitive histology indicated a low-grade infection (according to Krenn classification type III), and the serum CRP-level was elevated. It is particularly noteworthy that these patients were under antimicrobial treatment. In other three mPCR-positive patients, the MSIS criteria were positive, but no microorganism was isolated in the synovial fluid culture.In five patients, the mPCR system was able to identify pathogens, while the synovial fluid culture was not able to do so .Although an infection was present (according to the MSIS criteria) in eight cases, no microorganism was identified by mPCR, while the synovial fluid culture was positive . In one synovial fluid sample with suspected PJI, the detected pathogens did not match. The multiplex PCR system detected Klebsiella pneumoniae, and in the conventional culture of the synovial fluid, Staphylococcus aureus was isolated. Staphylococcus aureus was also cultured in the tissue and sonication fluid, and in the blood cultures, Klebsiella pneumoniae was isolated.Three synovial fluid samples showed negative results in mPCR and SF culture, although an infection was diagnosed (Cutibacterium acnes was detected by the mPCR in addition to Staphylococcus aureus (in one patient) and Cutibacterium avidum (in the other patient). However, the conventional culture only isolated Staphylococcus aureus (in the first patient) and Cutibacterium avidum (in the second patient).In two synovial fluid samples, Cutibacterium avidum/granulosum, a resistance to clindamycin was detected by conventional resistance analysis. Due to a lack of resistance gene markers, the mPCR was not able to identify this resistance. Nevertheless, in this bacterium, only the resistance gene marker [erm X] is described in the literature  was detected only by mPCR. However, macrolides or lincosamides are not clinically relevant.In one concordant Pseudomonas aeruginosa, a resistance to ciprofloxacin was detected by conventional resistance analysis. Due to a lack of resistance gene markers, the mPCR was not able to identify this resistance.In one Staphylococcus aureus was identified after one day, Cutibacterium avidum needed 14 days to be isolated.After a mean time period of 2.58 days , the microorganism was assessed by conventional culture in patients with concordant microorganisms. The antibiogram of the conventional culture was available for the physician after an average of 3.83  days. While The pre-operative diagnosis of PJI remains challenging. While serum CRP and ESR may not be accurate as diagnostic tools in the pre-operative diagnosis of PJI (and identification of infection persistence), particularly to identify low-grade PJI , 11, theStaphylococcus aureus when it came to diagnosing a PJI, possibly because of the prevailing high threshold value. However, Portillo et al. demonstrated better detectability of Staphylococcus aureus , ) are included in this device. A limitation of detectable resistance markers by the multiplex PCR system caused a lack in identification of four antibiotic resistances detected by conventional culture . In two concordant microorganisms, only mPCR identified clinical relevant resistance gene markers /aph(2″)]), while the synovial fluid culture was not able to do so. However, due to the small number of detected resistance markers, a recommendation is not possible.In 14 concordant microorganisms, resistance or a resistance marker was determined either in the multiplex PCR system or in the SF culture. A positive and negative conformity was illustrated five and eight times, respectively. However, six of the resistance markers identified by conventional culture  were not detected in the mPCR system, even though the resistance marker genes ([A limitation of the mPCR system is the lack of detectable microorganisms. However, in our study, only one of these undetectable pathogens in the mPCR was isolated in the conventional culture. Hence, it seems that the most common PJI-related microorganisms can be detected by this novel mPCR device.Staphylococcus aureus. Nevertheless, with moderate agreement compared to the conventional culture and high sensitivity of combined evaluation, this device could be a useful adjunct in pre-operative diagnosis of PJI, especially in low-grade infections.The mPCR system provides an advantage in identification of low-virulent microorganisms and has short turnaround time. In comparison with conventional culture, mPCR was inferior for detection of