NECC2), in adipocytes. Studies in 3T3‐L1 cells showed that NECC2 expression increased during adipogenesis. Furthermore, NECC2 co‐immunoprecipitated with caveolin‐1 (CAV1) and exhibited a distribution pattern similar to that of the components of adipocyte caveolae, CAV1, Cavin1, the insulin receptor and cortical actin. Interestingly, NECC2 overexpression enhanced insulin‐activated Akt phosphorylation, whereas NECC2 downregulation impaired insulin‐induced phosphorylation of Akt and ERK2. Finally, an up‐regulation of NECC2 in subcutaneous and omental adipose tissue was found in association with human obesity and insulin resistance. This effect was also observed in 3T3‐L1 adipocytes exposed to hyperglycaemia/hyperinsulinemia. Overall, the present study identifies NECC2 as a component of adipocyte caveolae that is regulated in response to obesity and associated metabolic complications, and supports the contribution of this protein as a molecular scaffold modulating insulin signal transduction at these membrane microdomains.Adipocyte dysfunction in obesity is commonly associated with impaired insulin signalling in adipocytes and insulin resistance. Insulin signalling has been associated with caveolae, which are coated by large complexes of caveolin and cavin proteins, along with proteins with membrane‐binding and remodelling properties. Here, we analysed the regulation and function of a component of caveolae involved in growth factor signalling in neuroendocrine cells, neuroendocrine long coiled‐coil protein‐2 ( The first recognizable abnormality detected in individuals destined to develop T2D is insulin resistance, in which insulin action is impaired in skeletal muscle, liver and adipose tissue.Caveolae are plasma membrane microdomains enriched in cholesterol and sphingolipids that act as signalling platforms containing a variety of signalling receptors and enzymes.Long alpha‐helical coiled‐coil proteins represent highly versatile molecules that have been proposed to act as molecular scaffolds and/or tethers that stabilize and organize membrane systems.NECC2 expression in human omental and subcutaneous adipose tissue increased in obesity and, in particular, in relation to insulin resistance. Furthermore, in vitro induction of insulin resistance by chronic exposure of 3T3‐L1 adipocytes to high concentrations of glucose and insulin also increased NECC2 content. Taken together, our data indicate that NECC2 is a component of adipocyte caveolae that is regulated in response to obesity and associated metabolic complications, and support a role for this protein as a molecular scaffold modulating insulin signal transduction at these membrane microdomains.The aim of the present study was to investigate the regulation and function of NECC2 in adipocytes and to establish its relationship to obesity and insulin sensitivity. Confocal microscopy studies showed that NECC2 immunosignal colocalized with that of CAV1, Cavin1, β‐actin and IR at the cell surface of 3T3‐L1 adipocytes. We also show that overexpression of NECC2, which co‐immunoprecipitated with CAV1, enhanced insulin‐activated Akt phosphorylation, whereas NECC2 silencing impaired IR‐dependent activation of extracellular‐regulated kinase 2 (ERK2) and Akt. Moreover, we observed that 22.1A polyclonal rabbit antiserum against rat NECC2 (residues 2‐17), anti‐NECC2, was produced and affinity‐purified as described.2.23T3‐L1 cells  were differentiated into adipocytes.At the end of the experiments, cells were harvested for RNA and/or protein determination or processed for confocal microscopy.2.3Samples of omental and subcutaneous adipose tissue were obtained from the abdominal region of 45 Caucasian individuals  undergoing diverse laparoscopic surgery procedures after ethics committee approval was obtained at the Clínica Universidad de Navarra . The study was conducted according to the principles of the Declaration of Helsinki. All participants provided written informed consent.2) were sub‐classified into three groups  following the criteria of the Expert Committee on the Diagnosis and Classification of Diabetes.Patients underwent a clinical assessment including medical history, physical examination and body composition analysis Table . Obese sTissue samples were immediately frozen in liquid nitrogen and stored at −80°C until use.2.4® Reagent) following the manufacturer′s instructions.NECC2 gene, and of 18s ribosomal RNA (rRNA) as a housekeeping gene, were measured by real‐time PCR using an iCycler™ Real‐Time PCR System . Primers are listed in Table Total RNA from 3T3‐L1 cells was extracted using the TRIzol method , incubated with PBS containing 0.3% w/v saponin and 1% w/v BSA (1 hours at RT), and then exposed  to rabbit anti‐NECC2 antibody,2.6Necc2 and CFP‐Cav1 as described previously.A standard protocol was used for co‐immunoprecipitation of NECC2 and CAV1 in HEK‐293 AD cells transfected with c‐Myc‐2.7Post‐nuclear supernatant (PNS), cytosolic (S2) and crude membrane (P2) fractions of 3T3‐L1 adipocytes at baseline and after 30 minutes of insulin treatment (100 nmol/L) were obtained by subcellular fractionation.Caveolin‐enriched membrane fractions were isolated from 3T3‐L1 adipocytes using a detergent‐free method and sucrose gradient fractionation analysis as previously described.2.8Protein extracts were obtained from cells lysed in buffer containing 50 mmol/L Tris‐HCl (pH 7.40), 150 mmol/L NaCl, 1% v/v Triton‐X‐100, 1 mmol/L EDTA and 1 μg/mL anti‐protease cocktail. 3T3‐L1 adipocytes from overexpression and silencing studies were lysed in SDS‐DTT.Protein extracts were separated by SDS‐PAGE. Antibodies against NECC2, CAV1, IR, Adiponectin, Cavin1, Perilipin1, B‐actin, A‐tubulin, Akt, pAkt, ERK1/2, p44/42 MAPK, c‐Myc and GFP were dispensed overnight (4°C) and peroxidase‐conjugated secondary antibodies were administered for 1 hour (RT). The immunoreaction was visualized using ECL plus . B‐actin, A‐tubulin and Ponceau S2.9Necc2 construct Necc2 silencing (5′‐GGAGGAGATAAGATTTAAA‐3′) was cloned using the BgIII and HindIII sites in front of the H1‐RNA promoter of the pEGFP‐RNAi plasmid as described earlier.Necc2‐shRNA plasmid (NECC2 shRNA) were kept in culture for 72 hours before performing the experiments.3T3‐L1 adipocytes were electroporated  as previously described.Necc2, cultured for 48 hours and then immunostained for cavin1. For Cav1 silencing studies, 3T3‐L1 adipocytes were transfected with Lipofetamine RNAiMAX (Invitrogen) and Cav1 siRNA  and cultured for 72 hours before NECC2 immunostaining.For colocalization analysis of NECC2 and cavin1, cells were transfected with Lipofectamine 2000  and the expression vector coding for GFP‐2.10t test and paired‐samples t test were used where appropriate. A Bonferroni correction was applied for multiple testing. A post‐hoc statistical analysis using the Tukey or Games‐Howell′s test was used to identify significant differences between groups. The contrast statistic used when the sphericity assumption was not satisfied was Huynh‐Feldt. Values were considered significant at P < 0.05.Statistical analysis was carried out using SPSS statistical software, version 19.0 for WINDOWS . The normal distribution of variables was assessed using the Kolmogorov‐Smirnov test and log‐transformed if appropriate. Repeated measures ANOVA (RM‐ANOVA), One‐Way ANOVA, Independent‐Samples 33.1Necc2 mRNA content in 3T3‐L1 cells at 0, 3, 6, 10 and 12 days of differentiation by RT‐PCR. Necc2 expression increased throughout adipogenesis, reaching a peak at day 10 (P = 0.041)  Figure a.Necc2 shRNA transfected cells   s Figure b. NECC2 ) Figure B. Immuno) Figure C. NECC2 A Figure c.3.2vs 0.0913 ± 0.1805 (n = 10), P = 0.006) , P = 0.039), without modifying NECC2 distribution at the plasma membrane , P = 0.153)   increased NECC2 accumulation at the adipocyte surface   Figure F.3.4Necc2, which enhanced NECC2 content by 59% . However, though Akt remained phosphorylated in both experimental groups after 15 minutes of insulin stimulation, pAkt/total Akt ratio in GFP‐Necc2 expressing cells was twice that observed in insulin‐treated, mock‐transfected cells at 15 minutes (P = 0.007)  Figure A. On theNecc2, which decreased NECC2 protein content by 44%    Figure d.Neither NECC2 overexpression nor silencing modified CAV1 or IR content in 3T3‐L1 cells Figure .3.5NECC2 gene is expressed in both fat depots, and that transcript levels of this protein were significantly higher in obese than in lean subjects irrespective of the fat depot and sex of the individual (data not shown). When the mRNA data from the three groups of obese individuals were analysed separately, we observed that both obese women and men with T2D exhibited significantly higher NECC2 mRNA levels in omental adipose tissue than lean subjects (P < 0.05)     Figure A. Furthe) Figure A. In sub) Figure B. Moreov) Figure B.in vitro studies including exposure of cells to fatty acids to induce adipocyte hypertrophy without  these levels reached statistical significance as compared to lean subjects. Notably, these changes parallel those previously reported for CAV1 in human adipose tissue.Based on our findings on NECC2 in 3T3‐L1 adipocytes and given the close relationship between human obesity and insulin resistance/T2D,in vitro models of adipocyte hypertrophy and/or insulin resistance.Finally, in order to get insights into the mechanisms regulating NECC2 expression in adipose tissue, we assessed the expression of this protein in In conclusion, our data provide the first demonstration of the association of a long coiled‐coil protein, NECC2, to adipocyte caveolae and identify this protein as a potential regulator of the insulin/IR system in adipocytes. In addition, they suggest that NECC2 up‐regulation in human adipose tissue may contribute, at least in part, to the development of insulin resistance in obesity.The authors confirm that there are no conflicts of interest. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Varicella-zoster virus (VZV), an exclusively human herpesvirus, causes chickenpox and establishes a latent infection in ganglia, reactivating decades later to produce zoster and associated neurological complications. An understanding of VZV neurotropism in humans has long been hampered by the lack of an adequate animal model. For example, experimental inoculation of VZV in small animals including guinea pigs and cotton rats results in the infection of ganglia but not a rash. The severe combined immune deficient human (SCID-hu) model allows the study of VZV neurotropism for human neural sub-populations. Simian varicella virus (SVV) infection of rhesus macaques (RM) closely resembles both human primary VZV infection and reactivation, with analyses at early times after infection providing valuable information about the extent of viral replication and the host immune responses. Indeed, a critical role for CD4 T-cell immunity during acute SVV infection as well as reactivation has emerged based on studies using RM. Herein we discuss the results of efforts from different groups to establish an animal model of VZV neurotropism. Varicella-zoster virus (VZV), a member of the human alphaherpesvirus family, causes chickenpox (varicella) mostly in children and establishes a latent infection in cranial, dorsal root and autonomic ganglia. Latent VZV can reactivate decades later to produce shingles (zoster). VZV-specific cell-mediated immunity (CMI) declines with age, resulting in zoster and associated neurological complications that are also seen in immunocompromised organ transplant recipients and in patients with cancer or AIDS. The actual mechanism of reduction in VZV-specific CMI and associated virus reactivation remains unclear. VZV produces chickenpox and shingles only in humans, underscoring the need for an animal model to study VZV neuropathogenesis. Attempts by multiple groups to establish VZV infection in guinea pigs and mice by experimental inoculation have resulted in seroconversion without clinical symptoms ,3,4,5,6.There is a strong belief, reflected in the introductions to many papers, that VZV grows only in human cells. Although humans are its only natural host and VZV was first cultured in cells of human origin , early sIt took another decade for investigators to explore the use of guinea pigs as in vivo models of VZV infection. This exploration included ocular infections, such as keratitis, and latency in the trigeminal ganglion ,16. In aSeveral investigators used what were then newly developed molecular diagnostic techniques  to test the usefulness of guinea pigs as models for the study of VZV pathogenesis, clinical disease and antiviral therapy ,3,4,19. Gershon and colleagues ,27 origiAfter a successful infection of isolated guinea pig neurons with VZV in vitro and the demonstration that VZV is a natural ENS pathogen, experiments were carried out to reevaluate in vivo VZV infection and latency in guinea pigs. A new two-step approach was used, involving confirmation that VZV established latency in the DRG, CNG and the ENS of guinea pigs after viremia induced by infusion of VZV-infected lymphocytes, followed by the administration of tacrolimus to reactivate the virus, a step based on the successful use of tacrolimus-induced immunosuppression to reactivate simian varicella virus in monkeys . VZV wasViremia inevitably occurs during varicella . T-cellsORF63, is also expressed in cotton rat ganglia, while another transcript which has rarely been detected during latency in humans, ORF40, is usually not expressed in ganglia of latently infected cotton rats [The cotton rat has been used in models of numerous virus infections, including respiratory syncytial virus, influenza virus, parainfluenza virus, measles virus, hantavirus, adenovirus and herpes simplex virus. Infection of cotton rat fibroblasts with VZV results in productive infection (unpublished observations). Inoculation of cotton rats intramuscularly along the side of the thoracic and lumbar spine with cell-associated virus results in a latent virus infection, with VZV DNA in the DRG. A viral transcript that is expressed during latency in human ganglia, ton rats . It is iton rats  in ganglORF1, ORF2, ORF10, ORF13, ORF17, ORF21, ORF 32, ORF47, ORF 57, ORF61 and ORF66, have been shown to be dispensable for latent infection in cotton rats based on the infection of animals with VZV with deletions or stop codons in the genes [ORF21, which is essential for growth in cell culture, was dispensable for the establishment of latency [ORF17 is severely impaired for replication in cell culture at 37 °C, the mutant virus established latency at levels similar to those of wild-type virus in cotton rats, which have a core temperature of 39 °C [ORF61 is severely impaired in the cell culture, the virus establishes latency at levels similar to those of the wild-type virus [The role of many VZV genes in latent infection has been studied in the cotton rat. Several virus genes, he genes ,52,53,54of 39 °C . Third, ORF4, ORF29 and ORF63, shows impaired latency and lower levels of viral DNA in DRG of cotton rats compared with animals infected with wild-type virus [ORF4 and ORF29 are essential for growth in cell culture and can only be grown in cells expressing the proteins. In each of these experiments, VZV was passaged once in non-complementing cells, and animals were inoculated with non-complementing cells containing the virus. ORF63 is the most abundant transcript in latently infected human ganglia [ORF63 had levels of VZV DNA in their ganglia similar to those in animals inoculated with VZV expressing ORF63 at three days after infection; however, at six weeks, both the percentage of latently infected animals and the viral load in the ganglia were reduced in the animals infected with VZV deleted for ORF63 [ORF63 revealed a strong correlation between mutants impaired for replication in cell culture and those impaired for the establishment of latency [VZV with deletions or stop codons in other genes, pe virus ,56,57,58 ganglia , and itsor ORF63 . Use of ORF62 and ORF63 and rearrangements in the 3’ portion of the genome was found to replicate to wild-type titers in the cell culture, induce high titers of neutralizing antibody in guinea pigs and was significantly impaired for latency in cotton rats. Thus, the establishment of latency with the mutant virus did not strictly correlate with virus replication in vitro. This VZV mutant could represent a safer vaccine, since it might be less likely to reactivate from latency.The cotton rat model was used in developing a VZV mutant that was impaired for latency, but not for replication, in vitro . VZV witORF63) is also expressed in cotton rat ganglia, while a transcript rarely expressed during latency in humans (ORF40) is also usually not expressed in latently infected cotton rats. Ganglia can be removed immediately after death to reduce the risk of postmortem virus reactivation, and the level of latent VZV can easily be quantified in ganglia. However, like all small animal models of VZV, infection of cotton rats does not result in acute disease (varicella) or virus reactivation (zoster), and unlike human ganglia, explanted ganglia from cotton rats do not undergo viral reactivation. In addition, compared with other small animal models, cotton rats are difficult to work with since they are very aggressive animals, immunologic reagents are much more limited for these animals than for mice, and fewer suppliers sell these animals; accordingly, this model has not been used for different studies, including innate adaptive immunity of VZV, by researchers other than those who originally reported its use.Overall, the cotton rat model has several features that make it an attractive model to study VZV. Animals can be reliably infected with the virus, and a transcript expressed during latency in human ganglia  tissue xenografts in mice with severe combined immunodeficiency, i.e., SCID-hu mice, has facilitated in vivo investigations of the molecular mechanisms of VZV pathogenesis ,62. VZV 3) was surgically implanted under the left renal capsule of a sedated six-week-old male scid/scid homozygous C.B.17 mouse  is characterized by extensive cytopathic changes, the fusion of neurons with their surrounding satellite cells to form polykaryons and the production of infectious virus in both neurons and satellite glial cells (SGCs) ,66. The VZV and HSV-1 infection of DRG xenografts differ significantly, in that HSV-1 infects neurons only; the capacity of VZV to infect SGCs and fuse neurons and SGCs is unique . Of noteAnalyses of VZV determinants of neurovirulence have focused on strain comparison  and the use of VZV recombinant viruses generated by overlapping cosmid or bacterial artificial chromosome (BAC) systems. Importantly, infection of DRG xenografts using live attenuated varicella vaccine virus demonstrated that vaccine Oka (vOka) exhibits the same pattern of acute replication followed by viral DNA persistence as two clinical isolates, parent Oka virus and VZV-S . The expIn summary, the SCID-hu DRG model has enabled substantial progress in the understanding VZV virulence factors and neurotropism. That VZV exhibits tropism for both neurons and SGCs and can cause fusion of these unique cell types distinguishes VZV from HSV neuropathogenesis, and helps to explain the often prolonged recovery from zoster and the occurrence of post-herpetic neuralgia. While this review is restricted to neurotropism, it should be noted that SCID-hu model is useful for studies of innate immune response. Although many questions remain, further advances are expected to provide better insight into the clinical manifestations of varicella and herpes zoster.VZV encodes 70 unique ORFs. In a genome-wide mutagenesis study, each of the VZV ORFs was individually deleted using VZV BAC technology . That stTo further test ORF7 neurotropism in more physiologically relevant systems, the SCID-hu DRG xenograft mouse model was used . A humanCercopithecine herpesvirus 9 within the Varicellovirus genus of alphaherpesviruses.Simian herpesviruses were identified as the causative agent responsible for epizootic outbreaks of monkey chickenpox in Old World monkeys . Virus i2–104 plaque-forming units per ml) compared to many other herpesviruses. Such is also the case for VZV grown in vitro [In the laboratory, SVV is grown in cell cultures of simian origin, usually African green  or rhesus (LM-MK2) monkey kidney cells . Infectein vitro . SVV andSVV virions have typical herpesvirus morphology containing at least 30 protein species ranging in size from 16 to >200 kD, including at least six envelope glycoproteins (46–115 kD) . SVV andAlthough SVV and VZV are two different viruses, their genomes are similar in size, structure, genetic content ,87,88 anORF62 encodes a major transactivator protein (IE62), which is expressed early in infection and induces transcription of SVV IE and early genes [ORF61 and 63 also encode transcriptional activators [ORF8), ribonucleotide reductase (ORF19), thymidine kinase (ORF36) and uracil DNA glycosylase (ORF59) [Like other herpesviruses, SVV gene expression during viral replication is understood to be coordinately regulated into immediate early (IE), early and late phases. SVV ly genes . SVV IE tivators . SVV ear (ORF59) ,95. The  (ORF59) ,96,97,98ORF63 and ORFs41 and 49, which encode structural proteins, are abundantly expressed during an acute disease. In contrast, SVV gene expression is restricted during viral latency. An SVV latency-associated transcript (LAT), identical to VLT [ORF61 transactivator gene. Although SVV LAT was discovered first, it is not as well characterized as VLT. The simian varicella model provides a useful approach to further explore the role of VLT in viral latency.During acute varicella infection of NHP, most of the SVV genome is transcribedd, as confirmed by the transcriptome analysis of bronchial alveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMCs) . SVV IE l to VLT ,100. Sim–, ORF61–, UDG– and dUTPase [Escherichia coli and facilitating the generation of SVV mutants and recombinant viruses [SVV infection of non-human primates (NHP) provides the only experimental animal model that closely simulates human varicella and studies involving innate and adaptive immunity. The study of SVV molecular biology and pathogenesis is facilitated by the ability to generate gene-specific viral mutants. Initially, an SVV cosmid recombination system for generating SVV mutants was used , in whic dUTPase ,101,102. dUTPase . More re viruses ,105,106. viruses .Despite being a different herpesvirus infecting a different host, SVV is pathogenic in NHP causing an acute respiratory infection with varicella (chickenpox) and later establishment of latency in sensory and enteric neurons, similar to disease produced by VZV infection in humans. Multiple species of NHP are susceptible to natural SVV infection ,108,109.Experimental respiratory inoculations of SVV in AGM results in an infection of epithelial, myeloid and lymphoid cells. A Virus then enters the bloodstream infecting PBMCs to cause a viremia, with further hematogenous dissemination to the skin, lymph nodes, spleen and other organs ,119. PeaThe first signs of clinical disease, seen frequently in AGMs and sometimes in macaques, are a fever and enlarged lymph nodes at 6–14 dpi ; Traina-At 7–14 dpi, a robust adaptive immune response develops, characterized by the presence of both binding and neutralizing antibodies and the detection of antigen-specific CD4 and CD8 central and effector memory T-cells in both BAL and PBMC samples ,128,129.In RM, a virus is cleared from the peripheral organs within 7 dpi, with establishment of latency ,138. In Accumulating evidence points to the important role of T-cells in SVV dissemination, establishment and maintenance of latency. First, it was shown that T-cells in the BAL are susceptible to and can support SVV viral replication in vivo . MoreoveA significant complication of aging is the reactivation of latent VZV, manifesting as zoster, post-herpetic neuralgia (PHN), pneumonitis, blindness, stroke, myocardial infarction and other multi-organ diseases. VZV latency is controlled largely by cell-mediated immunity and reactivation is due to its decline, disruption or loss . In humaThe NHP model of varicella reactivation has been successfully developed in SVV-infected cynomolgus and RM and AGMs ,127,142 In another study, more monkeys developed zoster when treated with tacrolimus alone rather than with both tacrolimus and irradiation . As mentSVV reactivation was observed in infected RM treated with Trio-XTP . Zoster Similar to observations in cynomolgus macaques, numbers of circulating PBMC were significantly reduced in RM within a week after the start of the Trio-XTP treatment . Flow cyAssessment of cytokine and chemokine levels in serum after immunosuppression and during SVV reactivation showed a strong increase in the pro-inflammatory mediators MCP-1, eotaxin, IL-6, IL-8, MIF, RANTES and HGF, but lower levels of the anti-inflammatory mediator IL-1Ra in the circulation . The infSubclinical reactivation of latent SVV in RM was demonstrated using thymectomy along with either CD4 or CD8 depletion . FollowiAfter primary infection, VZV establishes a life-long latent infection in neurons of the cranial nerve, dorsal root and autonomic ganglia, and reactivates later to produce several neurological disorders. It has been a challenge to study VZV neurotropism using an animal model since the virus causes diseases only in humans. In this review, we have described most of the animal models that have been adopted to study VZV neurotropism. The advantages and the disadvantages of each of the models are listed in


Take rates were high for human CRC cells (>73%) and for CCA cells (90%). We have demonstrated that this method reliably induces CRCLM and CCAs, in which tumour volume can be monitored throughout using HF-US and/or BLI. This provides a promising experimental tool for future testing of cancer therapeutics in an orthotopic model.Mouse models of human diseases are an essential part of the translational pipeline. Orthotopic tumour mouse models are increasingly being used in cancer research due to their increased clinical relevance over subcutaneous xenograft models, particularly in relation to metastatic disease. In this study, we have developed orthotopic colorectal cancer liver metastases (CRCLM) and primary cholangiocarcinoma (CCA) models in BALB/c nude mice using minimally invasive ultrasound-guided intrahepatic injection. Due to its minimally invasive nature, the method reduced risk from surgical complications whilst being fast and easy to perform and resulted in measurable tumour volumes 1 to 3 weeks post-injection. Tumour volumes were monitored  The most common site of metastasis is the liver, amounting to 70% of all cases2. Cholangiocarcinoma (CCA) although relatively rare, has a five-year survival of approximately 5%3. Both are characterised as being difficult to treat with poor long-term survival of patients. Therefore, there is a pressing need for more reliable models of these diseases for pre-clinical evaluation of anti-cancer therapies. Currently, orthotopic models recapitulating these human cancers are time-consuming to establish with poor or unreliable tumour formation.Metastatic cancer is the leading cause of death for patients diagnosed with colorectal cancer (CRC) with a five-year survival of stage-IV disease at diagnosis just 7–8%5. Mouse models of CRC and/or liver metastases are either transplantable or genetically modified, with the latter generally taking a long time to establish and rarely metastasise7. Transplantable models are much faster to generate but involve direct surgical implantation of cancer cells or tissues into the caecum or spleen, or direct injection into the portal vein11. The most commonly exploited models of colorectal cancer liver metastases (CRCLM) are injection site-dependant, either by surgical inoculation of the spleen or caecum involving implantation or subserosal injection13. Intrasplenic injection results in the rapid formation of liver tumours by drainage into the hepatic portal vein via the splenic vein but often involves the removal of the spleen; a vital immune organ. Caecal injections are technically more challenging with a higher risk of post-surgical complications, but with the advantage of allowing the study of invasion from the primary site. However, this results in the slow (up to four months) and unreliable formation of metastases in the liver in addition to lung, lymph nodes and peritoneal tumours10.In CRC, cancer cells from the primary tumour disseminate via the haematogenous route to secondary sites in the liver15. The process of generating these models is generally long and expensive. A syngeneic rat model of intrahepatic CCA using a rat-derived CCA cell line surgically implanted into the left hepatic duct with/without surgical ligation of the bile duct, reported 100% success rates16. Although this model has the advantage of producing bile duct obstruction (which is a common feature of CCA), the development of the model involved invasive surgery and does not utilise human-derived cancer cells.Pre-clinical orthotopic models of CCA generally involve chemotoxic agents or gene knock-out techniquesin vivo19. Tumours grown subcutaneously do not accurately represent the natural tumour microenvironment which contains fibroblasts, immune cells and an extracellular matrix which have all been shown to be influencing factors for tumour establishment and growth20. Subcutaneous models, in general, have been poor predictors of drug efficacy in humans and therefore the need for more predictive models of human cancers21. Hence, the generation of in vivo models of advanced disease in an orthotopic setting are necessary for more effective translation of novel therapies in order to improve treatment outcomes. Immune compromised or competent mice can be used depending on the cell lines used (patient-derived and/or human xenografts or syngeneic grafts) for testing of cytotoxic or immunotherapies, respectively.The tumour microenvironment is thought to play an important role in influencing the growth, response to treatment and tumour immune responses To this end, we have developed a minimally-invasive method to create orthotopic liver tumours that recapitulate cancers in the liver. Ultrasound-guided percutaneous injections were used to develop a pre-clinical orthotopic model of CRCLM, which was minimally-invasive, fast and easy to perform and was amenable to monitoring tumour growth regularly and economically via HF-US. The human colorectal adenocarcinoma cell line, SW620, that was first derived from a lymph node metastasis was used with or without Matrigel in BALB/c nude mice to assess the feasibility of generating CRCLM. TFK-1, an extra-hepatic CCA cell line transfected to express luciferase (TFKLuc2B2), was used for the generation of an orthotopic CCA model which had the advantage of longitudinal tumour growth monitoring using BLI as well as HF-US imaging.An orthotopic human CRCLM mouse model was developed using ten 5-week-old female BALB/c nude mice. Mice were randomised into two groups of five and anaesthetised with 5% (v/v) isoflurane gas in medical air at a flow rate of 2 L/min via an induction chamber, then positioned supine on the ultrasound platform with a nose cone for and maintained at 3% (v/v). Ultrasound gel was applied liberally to the abdomen and mice were imaged in B mode (2D) using a 40 MHz (RM-704) transducer with the VisualSonics Vevo 770 system, to determine and visualise the largest liver lobe Fig. . A 1 ml Post-injection, anaesthesia was ceased and mice were placed in a warm recovery chamber and observed until fully recovered then returned to their original housing. The whole procedure from induction of anaesthesia to recovery took less than five minutes. Animals’ heart and respiration rates were monitored during the entire procedure through electrode pads on the ultrasound platform using the Vevo’s Physiological Controller Unit. Post-operative analgesics were not necessary with such a minimally-invasive technique. One mouse from the Matrigel group did not recover post-procedure for unknown reasons.Due to the internal location of the tumours, tumour volume was monitored by HF-US imaging. The condition of each animal was monitored by daily visual inspection for clinical signs of pain or distress such as abnormal behaviours, piloerection, reduced mobility, body posture or isolation from cage mates. The Mouse Grimace Scale was also used to assess each animal for signs of pain. All indicators suggested that the animals were in minimal discomfort, if any, after this technique. Animals were weighed weekly and if they lost >10% of pre-experimental body mass, would be monitored daily. If weight loss reached 20%, animals would be humanely euthanised, however, in this case, all animals remained at a healthy weight throughout.in vivo HF-US imaging, the orthotopic xenograft take rate was 100% using Matrigel  and 20% without. One of the tumours without the use of Matrigel grew lower down in the liver and was out of the imaging region and upon dissection take rate was recalculated to 40%. Tumour doubling times for tumours with or without Matrigel were 3.9 ± 1.2 days compared to 3.4, respectively, with no statistically significant differences between groups, suggesting Matrigel did not influence tumour growth rate in this model. End-point liver mass was used to determine any significant differences between livers with orthotopic tumours and those with no tumours . Tumour volume was calculated by outlining the tumour area of multiple 2D sections at regular intervals by-hand throughout the image scan, which generated a 3D reconstruction and tumour volume. Animals were euthanised 35 days post-inoculation. Figure urs Fig. . Althougurs Fig. . Body maurs Fig. .Figure 2By histology, tumours were observed in 4/4 100%) of livers injected with cells in Matrigel and 2/5 (40%) without Matrigel . Weekly HF-US imaging was used for longitudinal monitoring of tumour growth . Figure With the success and ease of this method for generation of CRCLM, the same technique was implemented to produce an orthotopic model of primary CCA. Ten 6 to 10-week-old female BALB/c nude mice were injected as described above with a 50 µl bolus of 5 million TFKLuc2B2 cells in PBS using HF-US (Matrigel not used). The gallbladder was used as a landmark for ultrasound-guided percutaneous injection into the liver as it was the closest relevant structure to the bile duct which could be located using ultrasound Fig. . HF-US iIn addition to HF-US imaging, bioluminescence imaging (BLI) was also performed up to twice weekly Fig. , demonst24. This model is therefore, an excellent example of the National Centre for the Replacement, Refinement and Reduction of Animals in Research principles involving refinement of technique and development of techniques which minimise the ‘pain, suffering, distress or lasting harm’, directly improving welfare of animals in research25. This model represents a refinement of current technique with fewer animals required than with traditional orthotopic models of CRC, as tumour take rates were high and subsequent growth could be monitored longitudinally and non-invasively.To the best of our knowledge, this is the first known demonstration of non-surgical, orthotopic models of human CRCLM and primary CCA using ultrasound-guided intrahepatic injection. This method was minimally invasive thereby reducing risk from surgical complications, whilst fast and easy to perform resulting in measurable tumour volumes 1–3 weeks later. No loss in body mass or changes to normal behaviour were noted at any point during the experiments, with the exception of one CCA mouse at day-16. The use of a 29-gauge needle to inject cells resulted in no visible wound, an advantage over surgical inoculation which would require stitches, resulting in less inflammation and minimal healing time with this method. It has been widely suggested that pain and resulting stress can impact experimental outcomes resulting in poor reliability and reproducibility of experiments involving animals27. Due to the small volumes that could be injected into the intrahepatic space, reduced cell numbers could be used (compared to subcutaneous tumour models) and the Matrigel may have provided a more favourable environment for growth. The Matrigel may also have held the cells in place by the formation of a plug (Matrigel solidifies rapidly above 10°C) therefore preventing leakage of cells from the injection site, as seen in the group without Matrigel. Recently, a group has successfully demonstrated surgical intrahepatic inoculation of hepatocellular carcinoma cells with 100% take rates, however, 50% of these also showed abdominal and pelvic tumours from leakage during intrahepatic injection or potentially a cell line specific result28. The non-surgical transplantation of CRC cells into liver has not been done before, but orthotopic pancreatic and bladder cancer models have been successfully developed using ultrasound-guided injection, demonstrating the diversity of models which can be created using minimally invasive methods30.Tumour burden consisted of obvious, large tumours at/near the injection site, with minimal dissemination into the abdominal cavity, a distinct advantage for interventional studies. The necessity of Matrigel may be cell line dependent, it was not necessary for tumour growth and was not used for the CCA cell line TFKLuc2B2, but was however shown to improve SW620 orthotopic rates from 40% to 73–100%. Matrigel is comprised of extracellular matrix proteins, collagen IV and growth factors which can be used to improve cell engraftment in difficult to grow cell lines or primary patient-derived cancer cellsin vivo. Both are relatively cheap, quick and safe imaging modalities as demonstrated here. Without the use of imaging, the outcome may not be known until end-point dissection and histological analysis, which would be time-consuming and require many more mice to determine appropriate end-points. As shown here, end-point liver mass can also be used to quantitate the degree of CRCLM tumour growth, as livers with tumours increased in mass significantly compared to those without. Other imaging modalities such as BLI (as shown here), magnetic resonance imaging, micro computed tomography or fluorescence33 may also be used for in vivo tumour volume assessment but may not be as economical, rapid or as accurate as HF-US for measurement of tumour volumes in vivo34.HF-US and BLI were used to identify the location of tumours and to monitor the progress of tumour growth Orthotopic tumours grow differently to the commonly used subcutaneous models and therefore the importance of using a model which takes into consideration the cancers natural microenvironment is vital for faster and more reliable clinical translation. The formation of large single tumours could reflect late-stage CRC, where large tumours in the liver would require de-bulking  via adjuvant chemotherapy or radiation, prior to surgical resection. This model provides a promising experimental tool for future testing of anti-cancer agents for the management of metastatic CRC or primary CCA with the possibility of primary liver tumours such as hepatocellular carcinoma, hepatoblastoma or liver metastases from other cancers. The use of syngeneic cell lines in immunocompetent mice or patient-derived cells in immunodeficient mice should also be possible using this method and requires further evaluation.In summary, this method has successfully demonstrated the development of a minimally-invasive model of CRCLM and CCA. The relatively quick and reliable formation of tumours allows for ease of monitoring of tumour volume via HF-US or BLI, and therefore a means of testing efficacy of novel therapeutics quickly and easily. This model provides a promising experimental tool for future testing of anti-cancer agents in an orthotopic setting.35) were transfected with luciferase for bioluminescence imaging, clones were selected and one was termed TFKLuc2B2. Cells were regularly screened for mycoplasma and were short random repeat (STR) DNA profile authenticated. Cells were maintained in vented T150 tissue culture flasks  using Roswell Park Memorial Institute medium 1640  supplemented with 10% (v/v) foetal calf serum (FCS)  at 37°C in a 5% CO2 atmosphere. Cells were passaged at 70–80% confluence by washing with sterile phosphate buffered saline (PBS) pH 7.4  before being harvested by trypsinisation. Culture medium was added back into the flask to block the action of trypsin before cells were collected and centrifuged at 400 g for 5 minutes using an Eppendorf™ 5810 R centrifuge . Cells were re-suspended in PBS and the total cell count was obtained manually using a Neubauer haemocytometer, and the concentration adjusted using PBS. SW620 cells were further adjusted to 5 × 105 cells/ml using ice-cold Matrigel  or PBS immediately prior to injection. TFKLuc2B2 cells were adjusted to 1 × 108 cells/ml using PBS prior to injection.The SW620 cell line was originally obtained from the American Type Culture Collection (ATCC). Human extrahepatic CCA cells TFK-1  Act 1986.BALB/c nude mice were originally obtained from Charles River Laboratories  and maintained in-house. All mice were maintained under high health status conditions and were specific pathogen free status. Animals were housed in individually ventilated cages with access to food and water 36, and imaged in B mode. Vevo 770 version 3 software  was used for post-acquisition 3D reconstructions and used to calculate tumour volumes37. A video showing HF-US imaging of a CRCLM tumour is included in the Online Resource HF-US was performed weekly in the CRCLM model and at 3 weeks for the CCA model to measure tumour volume. Using a Vevo 770 HF-US system  a 40 MHz (RM-704) transducer  was held above the animal in supine positionin vivo BLI of CCA tumours, 150 mg/kg of D-Luciferin  was administered intraperitoneally five minutes prior to imaging using an IVIS Spectrum . Auto-exposure settings were used with a maximum exposure time of one minute. Living image software version 4.4  was used to analyse bioluminescence signals. Standardised regions of interest were used to calculate average radiance (photons/second/cm2/steradian) across imaging sessions.For Immediately after sacrifice by cervical dislocation, livers were collected and washed in ice-cold PBS, weighed and fixed by immersion in 4% (w/v) paraformaldehyde. Tissues were blocked in paraffin wax, 5 µm sections cut and stained with Haematoxylin and Eosin  for visual examination. Images were obtained using an Eclipse E1000 light microscope and NIS-Elements BR software .2 boxes were placed randomly throughout the tumour section using RandomSpot software version 6.0238 and the number of CD31 positive vessel were counted manually. Microvessel density was calculated as the number of CD31 positive vessels per mm2.CD31-positive blood vessels were stained following the manufacturer’s instructions. In brief, sections were deparaffinised and heat-mediated antigen retrieval in 10 mM citrate buffer (pH6) was performed for 10 minutes. Endogenous peroxidases were blocked with 0.3% v/v hydrogen peroxide followed by blocking of endogenous avidin/biotin . Sections were then incubated for 1-hour with the primary antibody . After washing, a biotinylated anti-rat secondary antibody was applied for 30 minutes . ABC/HRP solutions  were then applied and visualised with 3,3′-Diaminobenzidine . Slides were scanned using an Aperio slide scanner  at × 20 magnification. Ten 0.25 mm©2017 GraphPad Software, Inc., CA, USA). The type of analysis used in each experiment and statistical significance are shown in figure legends.All statistical analyses were performed using GraphPad Prism 7 (Supplementary InformationOnline Resource 1Online Resource 2Online Resource 4


Tumour angiogenesis is modulated on both an epigenetic and protein level and has potential implications for immune cell responses. However, the importance of related angiogenic biomarkers in cholangiocarcinoma (CCA) is unknown. This study assessed human CCA samples for the expression of angiogenesis-associated microRNAs, angiopoietins (Angs) and monocytes expressing the Ang-receptor, TIE2, with regards to prognostic significance after liver resection.Angiogenic miRNAs were analysed in frozen samples of intrahepatic CCA  and hilar CCA . Ang-1 and Ang-2, as well as TIE2-expressing monocytes (TEMs), were detected in paraffin-embedded iCC sections (n = 88). MiRNA expression and the abundance of TEMs and Angs were correlated with clinicopathological characteristics and survival.MiR-126 was downregulated in 76.7% of all CCA samples, with high relative expression associated with smaller tumours and reduced lymph node metastasis. High Ang-1 expression was associated with less lymphangiosis carcinomatosa and better histological grading . The absence of TEMs in iCC correlated with elevated CA19-9 levels. High relative miR-126 and low miR-128 levels were associated with improved survival in iCC and HC, respectively . High miR-126, low miR-128 and TEMs were independent prognostic factors for recurrence-free and overall survival .These results suggest that angiogenic miRNAs, Angs and TEMs are of prognostic value in CCA. In addition to the possible functional links between angiogenic miRNA expression profiles, Angs and immune-cell responses by TEMs, these data have clinical implications as novel diagnostic tools. MicroRNAs (miRNAs) are a large family of endogenous small noncoding RNAs (19–24 nucleotides) that play a crucial regulatory role by binding to the 3′ untranslated regions of target mRNAs and blocking translation or initiating mRNA degradation . MoreoveRecent research revealed that mature vessels express high levels of miR-126, which promotes the remodelling and stabilisation effects of angiopoietin-1 (Ang-1) . AnotherAng family members and VEGF are potent growth factors and important modulators of tumour-related angiogenesis; however, they could also represent functional antagonists . VEGF isTie2-expressing monocytes (TEMs) were recently shown to exert profound proangiogenic activities and constitute preeminent immunological compounds that were distinct from tumour-associated macrophages (TAMs) –24. The Angiogenic miRNAs, Angs and TEMs were suggested to have clinical significance in human malignancies where the interplay between tumour immunology and angiogenesis plays a central role in tumour progression . TEMs exThe relative expression levels of several miRNAs thought to regulate vascular growth factors involved in Ang–TIE2 signalling were initially assessed in CCA samples , 32–36. MiR-126 was proposed as a potent tumour suppressor that regulates Ang-1 signalling . The relIn HC, high miR-126 relative expression was associated with a smaller tumour mass (largest tumour diameter < 50 mm)  of the 46 patients in the invasive frontAng1-low group revealed a G1 tumour status (p = 0.038).In iCC, the high density of Ang-1 in tumour tissue was associated with reduced lymphangiosis carcinomatosa. In the tumourAng1-high group, 27 (67.5%) of the 40 patients had no presence of lymphangiosis carcinomatosa. In the tumourAng1-low group, 22 (45.8%) of the 48 patients had no lymphangiosis carcinomatosa (p = 0.042). Furthermore, the high abundance of Ang-1 in the tumour-infiltrating front correlated with a better histological grading. A well-differentiated (G1) tumour was diagnosed in 22 (84.6%) of the 26 patients in the invasive frontSimilarly to Ang-1, Ang-2 was also uniformly dense in all tumour areas, including the tumour-infiltrating front; however, Ang-2 distribution was not significantly associated with clinicopathological characteristics in iCC.TEM-positive group) and tumour-infiltrating front (invasive frontTEM-positive group) in 56 patients, while the rest of the tumour specimens  displayed a complete absence of infiltrating TEMs. No invading TEMs were observed in adjacent healthy liver tissue.In addition to angiogenic miRNAs and the angiopoietin axis, this study also investigated the impact of TEMs in CCA. In iCC, TEMs displayed a uniform density in the tumour, tumour-infiltrating front and perivascular areas. The same expression pattern was previously described for HC Figure  31]. In. In31]. TEM-positive group, 43 (79.6%) of the 54 patients had normal CA19-9 serum levels, whereas in the invasive frontTEM-negative group, only 15 (57.7%) of the 26 patients had normal values (p = 0.040).The absence of TEMs in the tumour-infiltrating front was associated with pathological CA19-9 levels. In the invasive frontThe TEM density was not significantly associated with other clinicopathological markers or angiopoietin expression in iCC; however, a trend was observed between reduced local and overall tumour recurrence and mitigated formation of fibrosis in tumours with a high TEM density (data not shown).In iCC, survival was significantly decreased in patients with high relative miR-128 expression in the tumour in comparison to those with low miR-128 expression  Figure . The 1-,P = 0.047; Table This study also analysed whether miRNAs, Angs and TEMs, in addition to other clinicopathological parameters, could predict the outcome after resection for CCA. Clinicopathological parameters reported previously in the same cohort of patients included histological differentiation, T status, overall and local tumour recurrence, multiple tumour nodules, tumour size, perineural sheet infiltration and lymph node involvement . Using mThis study analysed the tissue density of infiltrating TEMs and Angs and the expression of related angiogenic miRNAs in tumour samples from patients who underwent resection for CCA. The main findings were five-fold: (1) a high Ang-1 density was associated with reduced lymphangiosis carcinomatosa and improved histological grading in iCC; (2) angiogenic TEMs and miRNAs were differentially expressed in iCC and HC; (3) associated with established tumour markers, tumour growth and lymph node involvement; (4) served as independent prognosticators and (5) impacted on survival rates.In the current work we show that high miR-126 and low miR-128 expression levels were associated with beneficial effects in CCA. Moreover, in accordance with previously published results in HC, the results showed that Ang-1 and TEMs correlated with improved tumour profiles and, together with angiogenic miRNAs, affected patient outcomes in CCA [Little information is currently available regarding the impact of angiogenesis in human CCA. Angiogenic miRNAs that regulate potent (lymph) angiogenic factors were shown to impact tumour progression in various models of human disease. Experimental data suggest a potential role of VEGF and angiopoietin-related pathways in CCA progression . In addiRecent data suggest that miRNAs play important roles in liver biology and diseases, although the clinical impact of miRNAs in CCA remains to be further elucidated. Downregulation of miR-126 induces carcinogenesis, and miR-126 acts as a tumour suppressor and is also crucially involved in Ang-1 signalling and vessel maturation , 40–44. Based on previous findings linking the angiopoietin axis to TEMs in HC, this study explored the impact of angiogenic miRNAs that influence Ang-1-related pathways on CCA progression. A potential mechanism was hypothesised that linked miR-126 to the activation of Ang-1-dependent mechanisms, which mediated enhanced infiltration of TEMs, which in turn exerted a beneficial effect on CCA progression Figure . In thisGiven the growing interest in the miRNA-dependent modulation of (lymph) angiogenic signalling, the importance of other angiogenic miRNAs in CCA progression was investigated. Recent results are inconsistent; however, they do suggest that miR-128 is involved in malignant diseases, either as a tumour suppressor or oncogene , 46–54. This is the first work to document the expression of miR-128 in CCA. Moreover, it also shows that the upregulation of intratumoural miR-128 expression is a negative prognostic factor for patient survival following surgery Table . These dA negative prognostic value of the angiopoietin axis and TEMs was documented in highly vascularised malignancies; however, there is also a growing body of evidence that suggests that TEMs can have a positive impact. Research into HCC and pancreatic adenocarcinoma showed a negative influence of TEMs and Angs , 57–59. The M1 or M2 polarisation state of TAMs differentially affects tumour progression and patient prognosis, therefore TIE2 expression on TEMs may be only the tip of the iceberg. In addition, further discrimination of distinct subpopulations within TEMs may deliver new insights regarding novel functions and differential influences on disease progression. It should be noted that results published to date that have revealed a negative prognostic value of TEMs have been performed in highly vascularised tumours where tumour progression relies on prominent tumour microvasculature , 63. TheThere is a scarce amount of data available with respect to the clinical significance of the angiopoietin axis and TEMs in CCA. Previously, the significance of TEMs was established with respect to their circulating blood population . Howeverin vitro co-culturing of TEMs and tumour-derived factors or tumour cells to measure and verify angiogenic responses will help to mechanistically elucidate the proposed effects. In summary, angiogenic miRNAs, the related Ang-1 axis and corresponding TEMs were associated with beneficial tumour profiles and improved outcomes in human CCA. However, further research is required to investigate the molecular mechanisms linking TEMs to miRNAs and related tumour angiogenesis.The descriptive nature of the results is a limitation of the current work. Hence functional tests  at the Department of Surgery, Charité–Universitätsmedizin Berlin were included in the study. CCA was confirmed histopathologically and was classified according to the Union for International Cancer Control. The study was approved by the Ethics Committee of Charité–Universitätsmedizin Berlin (ID: 111 EA1/318/15).Clinicopathological characteristics of the study population were reported previously . BrieflyAll protocols used for immunohistochemistry, histology, cellular infiltrate quantification and angiopoietin density were described in detail previously , 64, 65.Briefly, TEMs were defined by their coexpression of CD14 and TIE2. The TEM density in the whole tumour area and in the tumour-infiltrating front was semiquantitatively scored as 1 = negative or 2 = positive. For statistical analysis, a score of 1 was categorised as the absence of abundance (or negative), while a score of 2 was categorised as the presence of abundance (or positive).TEM-positive group, n = 55, and tumourTEM-negative group, n = 33) or by the ‘positive’ or ‘negative’ abundance of TEMs in the tumour-infiltrating front . The tumour-infiltrating front was identified in 87 of the 88 tumour samples.Patients with iCC were divided into groups either by the ‘positive’ or ‘negative’ abundance of TEMs in the tumour , while scores of 2 and 3 were categorised as high abundance (or positive).Ang1-low group, n = 48; tumourAng2-low group, n = 66; tumourAng1-high group, n = 40 or tumourAng2-high group, n = 22) or by the ‘low’ or ‘high’ abundance of Angs in the tumour-infiltrating front . The tumour-infiltrating front was identified in 72 of the 88 tumour samples stained for Ang-1 or Ang-2.Patients with iCC were divided into groups either by the ‘low’ or ‘high’ abundance of Angs in the tumour  and purified as described in the user manual but with some modifications . Purified RNA was reverse transcribed, and qPCR was performed . RNU6 was used as an internal control in miRNA studies. The relative expression was calculated using the comparative ΔΔCT method, and the values were expressed as 2−ΔCT [Samples of CCA and matched adjacent noncancerous liver tissue were collected after liver resection. Samples were snap frozen, and the relative expression of angiogenic miRNAs (hsa-miR-107 (MS00031255), which regulates VEGF-D signalling; hsa-miR-126 MS00003430), which regulates Ang-1 signalling; hsa-miR-128 (MS00008582), which regulates VEGF-C signalling and hsa-miR-145 (MS00003528), which regulates Ang-2 signalling) was assessed by quantitative reverse transcriptase PCR , 32–34. 30, whichas 2−ΔCT .t-test or non-parametric Mann-Whitney U test was used. A Two-Way Analysis of Variance (Two-Way ANOVA) was performed to test whether the two independent variables, entity’ (HC versus iCC) and ‘tissue sample’  affect the expression of the selected miRNAs. Two-Way ANOVA was followed by Sidak's multiple comparison test for a pairwise comparison. Survival analysis, univariate analysis and Kaplan–Meier curves were performed and generated using SPSS. Comparison of categorical and continuous variables was performed using the Chi-squared test and the Wilcoxon test, respectively. Survival data were compared using the log-rank test. Variables that significantly influenced survival in the univariate analysis were entered into a Cox regression analysis. When p ≤ 0.05 the differences were considered statistically significant.Statistical analysis was performed using GraphPad Prism  and SPSS software program (version 23). Data were described as means +/− standard deviations (SD) or standard error of mean . D'Agostino & Pearson omnibus normality test was used to test for normal distribution. For comparison of two groups unpaired


Mycobacterium tuberculosis periprosthetic joint infection (TBPJI) is a rare complication of hip/knee joint arthroplasty. The outcomes of hip/knee TBPJI treatment are still unreported. The objective of this study was to investigate the outcomes of hip/knee TBPJI following treatment with two-stage exchange arthroplasty.From 2003 to 2013, 11 patients with TBPJI (six hips and five knees) were treated with two-stage exchange arthroplasty at our institution. We collected and analyzed variables including demographic data, comorbidities, microbiological data, duration of symptoms, and types of antibiotic used in bone cement.vs. 40%).At the most recent follow-up, the success rate of two-stage exchange arthroplasty was 63.3% (7 of 11). All five knee treatments resulted in infection eradication and successful prosthesis reimplantation. However, only two hip TBPJI treatments resulted in successful outcomes; two patients died and two experienced chronic infection. Overall, secondary bacterial infections were common in patients with TBPJI . Streptomycin in bone cement increased the success rate (83.33% M. tuberculosis culture is recommended when treating TBPJI in areas of high tuberculosis prevalence.More than one third of the patients treated with two-stage exchange arthroplasty for TBPJI showed infection relapse or uncontrolled infection. Streptomycin-loaded interim cement spacers appeared to help ensure successful treatment. Routine  Staphylococcus species, which constitute 50–60% of all isolates). If we excluded death that was not related to treatment and patients who refused further management, the success rate increased to 77.8% (7/9) even after repeated surgical debridement. If streptomycin was impregnated in the cement spacer, the success rate increased to 83.3% (5/6). Routine


CO and lactate were also measured.In adult recipients (ASA III to IV) scheduled for liver transplantation, intraoperative fluid management was guided by pulse pressure variations (PPV). PPV of ≥15% (Fluid Responding Status-FRS) indicated fluid resuscitation with 250 ml albumin 5% boluses repeated as required to restore PPV to < 15% (Fluid non-Responding Status-FnRS). Simultaneous blood samples from central venous and pulmonary artery catheters (PAC) were sent to calculate central venous to arterial CO2 gaps between the two states (FRS and FnRS) was poor with AUC of ROC of 0.698 and 0.570 respectively. Central CO2 gap was significantly higher in FRS than FnRS (P = 0.016), with no difference in the pulmonary CO2 gap between both states. The central and Pulmonary CO2 gaps are weakly correlated to PPV . There was no correlation between both CO2 gaps and both CO and lactate.Sixty seven data points were recorded (20 FRS and 47 FnRS). The discriminative ability of central and pulmonary CO2 gaps cannot be used as valid tools to predict fluid responsiveness or to guide fluid management during liver transplantation. CO2 gaps also do not correlate well with the changes in PPV or CO.Central and the Pulmonary COClinicaltrials.gov Identifier: NCT03123172. Registered on 31-march-2017. End-stage liver disease (ESLD) patients undergoing orthotopic liver transplantation can be prone to severe hemodynamic and metabolic changes. In the dissection phase; bleeding and hypovolemia are frequent , while i2 gradient and central venous oxygen saturation  relationship during high-risk surgery  and is affected by cardiac output and global CO2 production, as well as the complex relationship between PCO2 and CO2 content [2 does not exceed 6 mmHg. Elevated [Pulm(P-a) CO2] gradient has been observed in all types of circulatory failure   used to guide intraoperative fluid management. If pulse pressure variation (PPV) was more than 15%, the patient was considered as a fluid responder and received a 250-ml boluses of 5% albumin to maintain ≤15% PPV Arterial, central venous and pulmonary artery blood samples were collected and analyzed . We calculated the central venous to arterial CO2 gap [C(v-a) CO2] and the pulmonary mixed venous to arterial CO2 gap [Pulm(P-a) CO2] at two time periods, 30 min after the start of the pre-anhepatic dissection phase and 30 min after the reperfusion of the transplanted graft. No data was recorded during the an-hepatic phase or during partial or complete obstruction of the IVC by either clamping or surgical manipulation.The study was designed to include 20 adult (> 18 years) ASA III to IV physical status patients with an end-stage liver disease (ESLD) scheduled for orthotopic liver transplantation. Patients were excluded if they were less than 18 years old or suffering from chronic respiratory disease. Induction of anesthesia was performed using propofol, fentanyl, and atracurium and maintained with sevoflurane adjusted to achieve an expired minimal alveolar concentration (MAC) between 1 and 2% in a mixture of air/oxygen, fentanyl infusion (1–2 μg/kg/h), and atracurium infusion (0.5 mg/kg/h). Patients were mechanically ventilated  with a 6–8 ml/kg tidal volume and respiratory rate adjusted to maintain the ETCOA transfusion trigger of 7 g/dL guided the need for blood transfusion while. Fresh frozen plasma and platelets were transfused if the INR reached > 1.5 and the count was < 50,000/μl respectively guided by thromboelastography and according to the severity of bleeding.2 and pulmonary CO2 gaps were recorded apart from during the anhepatic phase and IVC obstruction as described earlier. Cardiac output (CO), lactate, central venous oxygen saturation (ScvO2) and PPV were all recorded throughout the procedures.Patient characteristics; age, weight, MELD Score, child score and associated HCC were recorded. Intraoperatively central CO2 gaps to predict fluid responsiveness appreciated by PPV. Area Under the Curve (AUC) for Receiver Operating Characteristic (ROC) was used to calculate the discriminative ability of both CO2 gaps to distinguish between FRS and FnRS with calculation of a cutoff value for either CO2 gaps should it be existing.Primarily, the current study aimed to investigate the ability of CO, a comparison between central and pulmonary CO2 gaps in both fluid states (FRS and FnRS), the correlation of the CO2 gaps to the hemodynamic and metabolic parameters , the correlation between hemodynamic and metabolic parameters (CO and lactate) and fluid responsiveness (FRS and FnRS) were also studied.Secondarily2 gap to be3.8 (1.7). Assuming a mean difference of 30% between responding and non-responding and by using G power software  with a power of 0.8 and 0.05 alpha error sample size was calculated to be 20 patients.The sample size was calculated after obtaining preliminary data of seven fluid non-responding status data points, which revealed a mean (SD) of the central CO2 gaps, cardiac output and lactate level are presented as mean (SD). Mann-Whitney test was performed for comparison of cardiac output and the Central and the Pulmonary CO2 gaps. The Receiver Operating Characteristic (ROC) curves were constructed, and the area under the curve (AUC) calculated to compare the performance of the central CO2 gap and the pulmonary CO2 gap in predicting fluid responsiveness. MedCalc version 12.1.4.0  generated values with the highest sensitivity and specificity (Youden index). Comparison of the AUC of the ROC curves used a Hanley-McNeil test. Correlations between either central CO2 gap and pulmonary CO2 gap and each of CO, lactate and PPV were done using Pearson moment correlation equation. A P value of less than 0.05 was considered statistically significant. All but ROC curves statistical calculations were done using SPSS  statistical program.Central and pulmonary COTwenty patients  were enrolled in the study. Their mean (SD) age was 53.1(7.6) years, mean (SD) weight 79.2 (11.5) kg, and mean (SD) height 170.1 (7.2) cm. Thirteen patients had ESLD following hepatitis C, two patients had a hepatocellular carcinoma (HCC), and five patients had combined hepatitis C and HCC. Median (range) of MELD score was 17 (13–29). Fourteen patients had a child-class C, and six patients had a child-class B and fifteen patients presented with ascites. There were 67 data points recorded (20 FRS points and 47 FnRS points).2 gap, pulmonary CO2 gap, lactate, ScvO2, and CO are presented in Table 2 gap was significantly higher in fluid-responder compared to the fluid non-responders (P = 0.016). Lactate level, ScvO2, pulmonary CO2 and CO were comparable between both FRS and FnRS.Mean values of central CO2 gap and PPV    or between pulmonary CO2 gap and CO (r = 0.22) with P = 0.076. Also, there was no correlation between either central or pulmonary CO2 gap and the lactate level(r) = 0.071 and 0.202 respectively.There was no correlation between central CO2 gaps valid indicators of fluid responsiveness in liver transplant patients? And is there a difference between the central and the pulmonary CO2 gaps in this setting? Second, do central and pulmonary CO2 gaps correlate with other hemodynamic and metabolic parameters such as CO, PPV and lactate? Third, are there any differences between fluid responding and fluid non-responding states in the hemodynamic and metabolic parameters?The target of the current study was to answer three questions; first, are the central and the pulmonary CO2 gap was significantly higher in FRS than in FnRS during the pre- and post anhepatic phase of liver transplantation surgery, however the ability of the central CO2 gap to predict fluid responsiveness was weak (AUC = 0.698) and the cutoff gap value to predict fluid responsiveness was 3.6 mmHg. On the other hand, the pulmonary CO2 gap was comparable between FRS and FnRS. (2) Both central and pulmonary CO2 gaps were comparable  and both showed significant correlation . Possibly this contradiction between the two findings is the result of the presence of intrapulmonary shunt [2 gap in cases of septic shock, both gaps cannot be used alone as valid indicators of fluid responsiveness despite the central CO2 gap in our patients being higher in fluid responder, but the diagnostic validity of which remained weak. Based on our findings, veno-arterial CO2 gap cannot be relied upon as a tool to predict fluid responsiveness in these patients with complex hemodynamic and pathophysiological changes. Additionally, both CO2 gaps  are approximate and the central CO2 gap can replace the pulmonary [For the first question, there were two main findings; (1) central COry shunt  in our pry shunt . The simulmonary –22., both CO2 gaps were only correlated with PPV but not with cardiac output or lactate level. PPV is a validated monitor for prediction of fluid responsiveness in major abdominal surgeries [2 gaps with PPV, despite being significant, was weak. This supports our finding that the CO2 gaps cannot be used alone as a valid predictors of fluid responsiveness in liver transplant patients.Answering the second questionurgeries  however,2 gap in our patients supports the disputed validity of CO2 gaps as sole monitor of fluid responsiveness. Mekontso et al. [2 gap and lactate level during hypoxic metabolic states with decreased oxygen consumption. Mekontso et al. used the ratio, rather than the absolute value, of CO2 gap to arterio-venous oxygen difference to relate to lactate levels.Lactate level reflects both tissue anaerobic metabolism and the ability of the liver to metabolize it, with both conditions present in liver transplant patients during different phases of the transplant procedure . Lactate level is a validated parameter to monitor adequate fluid resuscitation and the absent correlation between lactate and the COo et al.  confirme2 production (VCO2), changes in cardiac output result in large changes in pulmonaryCO2 gap at low cardiac output values, whereas changes in cardiac output will not result in significant changes in pulmonary CO2 gap at the high values of cardiac output [2 gaps and the CO in our patients known to have a high CO as part of the pathophysiology of liver cirrhosis.For a constant total COc output , 24 This, pulmonary CO2 gap and CO. These findings support the verdict not to rely only on CO2 gaps alone as valid indicators of fluid responsiveness.Moving forward to the third question, FRS and FnRS patients were comparable regarding their lactate level2 gaps correlated with PPV. Cuschieri et al. [2 gaps in their studies of critically ill patients and on septic patients. In the current study, there was no correlation between central and pulmonary CO2 gaps with cardiac output., many studies [2 gap in low cardiac output states due to venous flow stasis which decreased with increased cardiac output. Cuschieri et al. [2 gap and the pulmonary CO2 gap with cardiac index. Troskot et al. [2 gradient could predict fatal outcomes in non-ventilated patients only. Also, Mallat et al. [2 gap and cardiac index using PICCO technology at time 0 (start of the study) and at time 6 (6 h after resuscitation) and found a correlation between CO2 gap and CI at T0  and at T6 (r = − 0.54 P < 0.0001). Also, the changes in CI between T0 and T6 were also correlated with changes in CO2 gap .In our study, both central and pulmonary COi et al.  and Van i et al.  showed s studies , 25, 27 t et al.  concludet et al.  in their2 gap did not correlate with cardiac output presumably due to the hyperdynamic state of the hepatic patient which preserves systemic blood flow even in states of tissue hypo-perfusion. Mecher et al. [2 gap; high gap group > 6mmhg and normal gap group < 6 mmHg. They found normal gap group to have a high cardiac index (3 ± 0.2) despite circulatory failure. In this group; the gap did not change after fluid resuscitation (pre-fluid gap 4 ± 0 vs. post fluid 4 ± 1 mmHg) with an increase in cardiac index. While in the other group cardiac index was lower (2.3 ± 0.2) and gap decreased after resuscitation.In our study, the central COr et al.  studied 2 gap or pulmonary CO2 gap and the lactate level. This was consistent with the study of Vallee et al. [2 gap with cut off value of 6 mmHg, low gap (< 6 mmHg), and high gap (> 6 mmHg). Patients’ resuscitation resulted in significantly larger clearance of lactate in low gap group than high gap group. There was also no correlation between CvCO2 gap and lactate level at time of inclusion T0  and poor correlation at six hours T6  and twelve hours T12 .In our results, there was no correlation between either central COe et al.  in which2 gap and cardiac index at baseline  but not between pulmonary CO2 gap and lactate at baseline (p = 0.58). Also, Mecher et al. [2 gap and lactate after fluid resuscitation in all patients with severe sepsis and systemic hypo-perfusion involved in the study.In agreement with our results, Monnet et al.  found thr et al.  showed n2 gap between fluid responding and non-responding states, the validity of CO2 gap is poor which makes its use to guide fluid resuscitation in liver transplant recipient is questionable.The present study had several limitations. First, This is a single center experience. Second, we avoided periods of marked hemodynamic instability caused by manipulation of the liver and downward retraction of the inferior vena cava which may intermittently obstruct venous return and causing hemodynamically significant changes in preload. Such changes in the preload are typically transient and may not reflect the actual volume status of the patient. Finally, we did not compare the CO2 gaps recorded during the pre-anhepatic phase to the CO2 gaps recorded during the neo-hepatic phase as the two periods represent different hemodynamic and pathophysiologic situations with the presence of a cirrhotic liver in the former and a potentially healthy graft in the latter. A future study can check this aspect.fCO2 gap was found to be complementary tool for early resuscitation of patients with circulatory failure . In the 2 gap and pulmonary CO2 gaps could not be used to predict fluid responsiveness or to guide adequate fluid management during living related liver transplantation. Both CO2 gaps could be used interchangeably, and both did not correlate well with changes in cardiac output or lactate level. These results suggest that CO2 gap may not be a good hemodynamic endpoint of resuscitation of patients undergoing living related liver transplant.Both central CO


Immune checkpoint inhibitors such as Nivolumab work by preventing the inactivation of host T-cells by tumour cells, thereby allowing the T-cells to attack the tumour cells, which results in tumour tissue necrosis. We describe a 78-year-old woman with metastatic lung adenocarcinoma treated with Nivolumab after disease progression following first-line chemotherapy. Computed tomography (CT) after 3 cycles showed a smaller left lower lobe (LLL) primary and stable right lower lobe (RLL) metastatic lesion. CT after 9 cycles showed a reduced RLL mass and an increase in LLL primary. However, CT after 15 cycles showed that the RLL mass had further reduced in size but the LLL mass was significantly larger. The biopsy of the LLL lesion showed necrotic areas and reactive inflammatory changes, without residual malignancy. A repeat CT after further 4 cycles confirmed tumour regression in both the primary and the metastatic lesions. There was a prior reported case of pseudoprogression in a non-small-cell lung cancer patient who had 7 cycles of Nivolumab, and it was diagnosed during a further line of chemotherapy. Here, we report a patient with pseudoprogression during treatment with Nivolumab and at a much later time, after 15 cycles. Nivolumab works as a checkpoint inhibitor by binding to the T-cell programmed death- (PD-) 1 receptors and therefore preventing the tumour cell PD-ligand 1 (PD-L1) from binding to them and inactivating the T-cells. The use of this therapy is now applied to several malignancies such as melanoma, non-small-cell lung cancer (NSCLC), and urological malignancies, with more studies ongoing for other types of cancers. This recent advancement with immune checkpoint inhibitors has therefore posed its own challenges in the assessment of response to treatment. There have been several reports of pseudoprogression on scheduled CT imaging during the first few weeks of immunotherapy treatment in melanoma and NSCLC. Here, we report the second case of delayed pseudoprogression with Nivolumab in the treatment of NSCLC with the first reported case of a pseudoprogression which occurred after 7 cycles of Nivolumab and a further line of chemotherapy [A 78-year-old woman was diagnosed with stage IV adenocarcinoma of the left lung in November 2015 after presenting with a history of haemoptysis. Her only medical history was hypercholesterolaemia. She underwent a bronchoscopy and biopsy of a lesion in the LLL, which confirmed TTF-1-positive adenocarcinoma of the lung. Her tumour status was epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase rearrangement negative. Her initial CT at diagnosis showed a large LLL tumour measuring 5.3 × 7.9 × 6.3 cm with volume loss, satellite nodules, and surrounding interstitial changes. There was a severe encasement and narrowing of the pulmonary vessels, pleura infiltration with discrete pleural nodularity in the left upper lobe, and a small effusion. Bilateral pulmonary metastases were seen with a large nodule in the RLL measuring 2.2 × 2.9 cm. There were also enlarged necrotic appearing lymph nodes in the left hilar and subaortic region, which measured 12 mm.She was initially commenced on palliative chemotherapy with carboplatin and pemetrexed. After 3 cycles of chemotherapy, her restaging CT showed an increase in size of the nodular lesion of RLL measuring 3.8 × 3.5 cm with LLL measuring 5.3 × 3.5 × 5.9 cm and subaortic node of 9 mm . She wasA restaging scan after 9 cycles of Nivolumab in September 2016 showed some reduction in the RLL mass measuring 3.1 × 2.8 cm, an increase in LLL lesion 4.3 × 3.9 cm . A furthA further repeat CT scan was performed after further 4 cycles in March 2017 which confirmed tumour regression with the LLL mass measuring 6.5 cm × 5.3 cm. The mass in the RLL also showed a reduction in size measuring 2.1 × 2.0 cm in maximum axial diameter .The treatment with immunotherapy is dependent upon the ability of the immune system to recognise malignant from normal cells and this, in part, relies on the antigenicity of the tumour cells , 3. Tumoγ) from tumour-infiltrating lymphocytes (TIL) are thought to lead to the upregulation of PD-L1 [Another method of evasion by tumour cells is by decreasing their immunogenicity , 5. Thisof PD-L1 , 8.Tumour microenvironment also plays a crucial role in the escape of tumour cells from elimination by the immune cells. Some tumours recruit immunosuppressive leukocytes to generate an immunosuppressive microenvironment, and the elimination of these immunosuppressive cell populations has been found to restore T-cell infiltration into tumour tissues and their ability to mediate antitumour activity .The mechanism underlying pseudoprogression is, however, less clear. The phenomenon of apparent increase in tumour burden could be explained by either transient immune cell infiltration, extensive inflammation, vascular permeability, and oedema or continued tumour growth preceding a delayed effect of immune cells . Hence, Pseudoprogression in immunotherapy-treated solid tumours can be either early or delayed. The assessment by current gold standard RECIST criteria can be misleading and irRECIST is a better way to assess the CT imaging of these patients. The importance of clinical assessment could not be overstated, and in cases which pseudoprogression is suspected, a biopsy should be performed. The development of liquid biopsy has certainly offered the prospect of a less invasive way of diagnosing these cases of pseudoprogression.


We analysed the effects of a leucine-rich diet on the signalling pathway of protein synthesis in muscle during a tumour growth time-course.The exact signalling mechanism of the mTOR complex remains a subject of constant debate, even with some evidence that amino acids participate in the same pathway as used for insulin signalling during protein synthesis. Therefore, this work conducted further study of the actions of amino acids, especially leucine, in vivoWistar rats were distributed into groups based on Walker-256 tumour implant and subjected to a leucine-rich diet and euthanised at three different time points following tumour development . We assessed the mTOR pathway key-proteins in gastrocnemius muscle, such as RAG-A-GTPase, ERK/MAP4K3, PKB/Akt, mTOR, p70S6K1, Jnk, IRS-1, STAT3, and STAT6 comparing among the experimental groups. Serum WF  and muscle protein synthesis and degradation were assessed.The tumour-bearing group had increased serum WF content, and the skeletal-muscle showed a reduction in IRS-1 and RAG activation, increased PKB/Akt and Erk/MAP4K3 on the 21st day, and maintenance of p70S6K1, associated with increases in muscle STAT-3 and STAT-6 levels in these tumour-bearing rats.Meanwhile, the leucine-rich diet modulated key steps of the mTOR pathway by triggering the increased activation of RAG and mTOR and maintaining JNK, STAT-3 and STAT-6 levels in muscle, leading to an increased muscle protein synthesis, reducing the degradation during tumour evolution in a host, minimising the cancer-induced damages in the cachectic state. Branched-chain amino acids, especially leucine, play a prominent role in the regulation of protein metabolism. Both in vivo and in vitro studies corroborate the fact that a diet supplemented with leucine stimulates protein synthesis, particularly in skeletal muscle –4, and mCellular signalling in the setting of protein synthesis involves the regulation of protein turnover in skeletal muscle, a complex process that generally involves an interaction between gene transcription and protein translation and degradation. The stimulus and signal of this process each come from outside the cell; the primary stimulating agents are anabolic hormones, such as insulin, growth factors, glucose and nutrients, such as amino acids, especially leucine –10.The mammalian protein kinase target of rapamycin, mTOR, plays a central role in the development of skeletal muscle stimulation through growth by nutrition. mTOR acts as a sort of strategic centre from which various cellular processes are regulated , 12. Thi, the primary purpose of our research. Therefore, the primary purposes of our research include investigating the time-course of tumour growth under a leucine-rich diet and the effects of this diet on the activation of upstream and down-stream proteins involved in protein synthesis in the gastrocnemius muscle of Walker-tumour-bearing rats.Leucine is the primary amino acid that activates protein synthesis in skeletal muscle , 9. SomeThe semi-purified isocaloric diets were a normal-protein diet (C), containing 18% protein , and leun = 72 animals; 90-days-old, weighing 180–200 g) were obtained from the Centre/UNICAMP animal facilities , and received food and water ad libitum under light-dark cycles (12/12 h each) and constant temperature (22 + 2 °C) and humidity (50–60%). The animals were distributed into 12 groups based on whether they were implanted with Walker-256 tumour cells , their periods of tumour development analysis , and whether they received leucine-rich diet supplementation. Each group contained a minimum of six animals as follows: 1. Control rats  subjected to a normoproteic diet and euthanised on the 7th, 14th and 21st days of the experiment; 2. Leucine rats  subjected to leucine-rich diet and euthanised on the 7th, 14th and 21st days of the experiment; 3. Tumour-bearing rats  subjected to a normoproteic diet, implanted with Walker 256 carcinosarcoma cells and euthanised on the 7th, 14th and 21st days following tumour development; 4. Leucine tumour-bearing rats  subjected to leucine-rich diet, implanted with Walker 256 carcinosarcoma cells and euthanised on the 7th, 14th and 21st days after tumour development.Females Wistar rats  according to the manufacturer’s instructions and expressed as pM, using a standard provided by the manufacturer with a range of 1 to 500 pM, and fluorescent flow cytometry with Luminex equipment .panTyr, p-Akt/PKBThr308, p-ERK/MAPK Thr185/Tyr187, p-mTORSer2448, p-70S6KThr412, p-STAT3Ser727, p-STAT6Tyr641, p-Jnk Thr/Tyr185, as provided by the manufacturer, and the Millipore cell signalling kits tested for each antibody, using the IP Western assay; the HeLa cell lysate was used as a positive control (provided by the Milliplex map kit detection by Millipore).The gastrocnemius muscle samples were homogenised in a buffer sample  according to the manufacturer’s instructions, and analyses of the phosphorylated signalling proteins IRS-1, Stat-3, Stat-6, Jnk, ERK/MAPK, Akt-PKB, p70S6K and mTOR were completed using multiplex kits , according to the manufacturer’s instructions. The fluorescence of coupling beads with capture antibodies specific for each protein of interest was measured using the Luminex®200™ system . The analyses were carried out using xPonent® 3.1 Software  and the Luminex®200™ system. The protein phosphorylation are as follows: p-IRS-1Serum samples were also resolved to analyse the proteolysis-induced factor-like Walker Factor (WF) as previously described by Yano and colleagues . The priThe gastrocnemius muscles were homogenised with homogenisation buffer , centrifuged, and divided into aliquots for a total protein analysis . We also3; 3.4 mM KCl; 1 mM CaCl2; 1 mM MgSO4 and 1 mM KH2PO4, pH 7.4) supplemented with 5.5 mM glucose and 0.01% (w/v) albumin. The muscles were pre-incubated for 30 min at 37 °C with continuous gassing (95% O2–5% CO2). After this, new KHB buffer supplemented with 5 μCi/ml of L-(3H) phenylalanine  was added, and the incubation continued for further 2 h. At the end of this period, the muscles were homogenised in trichloroacetic acid , centrifuged at 10.000×g and measured the total protein -Leu/mg/h) when compared to W group (W = 14.00 ± 1.31 nmol [3H]-Leu/mg/h)  was not significantly different but showed an increased value (WL = 18.77 ± 2.57 nmol [14C]-KIC /mg/h).KIC incorporation indicates protein synthesis, stimulated by indirect effect of leucine, which could be affected by tumour growth, as we noted here where the W group had a decrease in muscle KIC incorporation Fig. , but wasIn order to analyse the tumour wasting effect, we analysed the muscle protein degradation by tyrosine released which was increased in both tumour-bearing groups, W and WL, compared to C and L groups Fig. b. These The mTOR complex integrates various signalling pathways that are modulated by different stimuli, including insulin, hypoxia, energy stress and nutrients ; the lasThe present work contributed to deepening further the results obtained in previous studies . TherefoBriefly, the Walker-256 tumour as an experimental model of cachexia shows that its development increases the muscle protein degradation by increasing the proteolysis-induction factor-like – Walker-Factor - (WF) levels, but also decreases the muscle protein synthesis by reducing the insulin level, which was associated with lower activation of IRS-1 in W group, especially after the 14th and 21st day after implant. In parallel, we verified increases in the levels of phosphorylated Akt-PKB and ERK/MAPK only at 21 days of tumour growth. Although, these high levels of Akt did not lead to activation of mTOR via in muscle of these tumour-bearing hosts, which clearly showed that the mTOR pathway was compromised likely starting by the 14th day of tumour development. On the other hand, one of the downstream components of mTORC1, phospho-STAT3 had some variable profile with an expressive rise in the muscle of these tumour-bearing groups on day 21. Indeed, STAT3 mediates the expression of a variety of genes in response to cellular stimuli and therefore plays a key role in many cellular processes, including cell apoptosis and inflammatory processes , 28. STAIn order to investigate some of the benefits of leucine-rich diet over the damage effects of tumour development on muscle protein, here we analysed the profile of these same key proteins of mTOR signalling pathway, including phosphorylated proteins, from up-stream and down-stream steps, which provided some interesting point how the leucine-rich diet could modulate the tumour-induced damages in this experimental model of cachexia.In fact, the leucine-rich diet clearly demonstrated that could ensure maintenance of serum insulin levels throughout the course of tumour development, improving the muscle protein synthesis. Indeed, as presented here, Petruzzelli et al.  observedIndeed, the analysis of the concentration of phosphor-mTOR in the gastrocnemius muscle revealed an increase in this concentration in the tumour-bearing group that received a leucine-rich diet between the 14th and the 21st day of the experiment compared with the control group and also with the non-supplemented tumour-bearing group. Interestingly, leucine has been described by some researchers as an inhibitor of protein degradation and a stimulator of protein synthesis in cachectic hosts , 38. In Considering the activation of downstream mTOR pathway, the maintenance of STAT3 phosphorylation occurred in WL group showed the leucine influence, since the group without tumour cells or bearing a tumour had constant levels of phospho-STAT3 over the three study days compared with the C group. Indeed, STAT3 mediates the expression of a variety of genes in response to cellular stimuli, in this case being modulated by leucine effects. We suggest that the STAT3 could be likely activated in various pathways characterised by the activity of cytokines and growth factors, including interleukins IL-6, as previously noted in your study , but undAfter analyse those time-course experiments we choose the 21st day as a point of more pronounced effect of a tumour and leucine-rich diet on animals’ body. Therefore, radioactive experiments were performed only on the 21st day of tumour development.Leucine stimulates muscle protein synthesis and modules the activity of various proteins involved in the control of mRNA translation . LeucineThese finds clearly show the minimisation of tumour-induced damage effects when these tumour-bearing rats were subjected to leucine-rich diet. Leucine supplementation appeared to reduce the effects of stress in the host body, as well as effects of the inflammatory processes generated by the presence of the tumour cells improving the host’s responses and maintaining lean body mass Fig. .This work demonstrates for the first time a time-course study with action of leucine anticipating and enhancing the parameters analysed in the setting of tumour growth; this involves the stimulation of protein synthesis, which results in reduced cachexia and occurs primarily in the experiment that achieves the best values on the 21st day following the start of the experiment. These results are consistent with those observed in previous studies performed by our research group. It was also noted that leucine stimulates various points of the mTOR pathway, including different anabolic hormones via, such as insulin. Further study is necessary in order to completely understand the effects of leucine on protein synthesis and protein degradation at the molecular level.


Since the advent of immunohistochemistry for the diagnosis of stromal tumours, the incidence of leiomyosarcomas has significantly decreased. Nowadays, gastric leiomyosarcoma is an exceptionally rare tumour. We report the second case in the English literature of gastric leiomyosarcoma revealed with massive bleeding and hemodynamic instability and diagnostic pitfalls that we encountered.A 63-year-old woman, with 2 years’ history of dizziness and weakness probably related to an anaemic syndrome, presented to the emergency room with hematemesis, melena and hemodynamic instability.On examination, she had conjunctival pallor with reduced general condition, blood pressure of 90/45 mmHg and a pulse between 110 and 120 beats per minute. On digital rectal examination, she had melena. Laboratory blood tests revealed a haemoglobin level at 38 g/L.The patient was admitted to the intensive care department. After initial resuscitation, transfusion and intravenous Omeprazole continuous infusion, her condition was stabilized. She underwent upper gastrointestinal endoscopy showing a tumour of the cardia, protruding in the lumen with mucosal ulceration and clots in the stomach. Biopsies were taken. Histological examination showed interlacing bundles of spindle cells, ill-defined cell borders, elongated hyperchromatic nuclei with marked pleomorphism and paranuclear vacuolization. Immunohistochemistry showed positivity for Vimentine, a strong and diffuse immunoreactivity for smooth muscle actin (SMA). Immunoreactivities for KIT and DOG1 were doubtful.Computed tomography scan revealed a seven-cm tumour of the cardia, without adenopathy or liver metastasis.The patient underwent laparotomy. A total gastrectomy was performed without lymphadenectomy. Post-operative course was uneventful.Histological examination of the tumour specimen found the same features as preoperative biopsies with negative margins. We solicited a second opinion of an expert in a reference centre for sarcomas in France, who confirmed the diagnosis of a high grade gastric leiomyosarcoma.Gastric leiomyosarcoma is a rare tumour. Diagnosis is based on histological examination with immunohistochemistry, which could be sometimes confusing like in our case. The validation of a pathological expert is recommended. Gastrointestinal stromal tumours (GISTs) were considered to be of smooth muscle origin. They were misdiagnosed as leiomyomas and leiomyosarcomas. Since the advent of immunohistochemistry for the diagnosis of stromal tumours, the incidence of leiomyosarcomas has significantly decreased. Nowadays, gastric leiomyosarcoma is an exceptionally rare tumour . DiscoveWe present the case of a gastric leiomyosarcoma revealed by a massive upper gastrointestinal bleeding and diagnostic pitfalls that we encountered.A 63-year-old woman, with 2 years’ history of dizziness and weakness probably related to an anaemic syndrome, presented to the emergency room with hematemesis, melena and hemodynamic instability. There was no history of chronic liver disease, dyspepsia, ulcer disease, nonsteroidal anti-inflammatory drugs or aspirin use.On examination, she had conjunctival pallor with reduced general condition, blood pressure of 90/45 mmHg and a pulse between 110 and 120 beats per minute. On digital rectal examination, she had melena. There were no abdominal wall varices, no hepatomegaly, and no palpable mass or adenopathy.Laboratory blood tests revealed a haemoglobin level at 38 g/l with haematocrit at 13.4%. The mean corpuscular volume was in the normal range.The patient was admitted to the intensive care department. After initial resuscitation, transfusion and intravenous Omeprazole continuous infusion, her condition was stabilized. She underwent upper gastrointestinal endoscopy showing a tumour of the cardia, protruding in the lumen with mucosal ulceration and clots in the stomach Fig. . BiopsieComputed tomography (CT) scan revealed a seven-cm tumour of the cardia, without adenopathy or liver metastasis Fig. .Fig. 2CTAfter multidisciplinary meeting, we suspected the diagnosis of stromal tumour of the cardia with high risk of re-bleeding and we decided to perform a total gastrectomy.The patient underwent laparotomy. There was a nine-cm tumour of the cardia and the fundus, and no sign of peritoneal seeding or liver metastasis. A total gastrectomy was performed without lymphadenectomy Fig. . Post-opHistological examination of the tumour specimen found the same features as preoperative biopsies with negative margins Fig. . We soliBefore the late 90’s, GISTs were misdiagnosed as leiomyomas and leiomyosarcomas . AdvanceFirst, gastric leiomyosarcomas are generally insidious since they have a predominant extraluminal component . It may Second, arising between the muscularis propria and muscularis mucosa layers, diagnosis of leiomyosarcomas relies on histological examination of deep biopsies. Conventional endoscopy usually yields superficial and normal mucosa. Endoscopic ultrasonography, on the other hand, has been proved to be of great sensitivity, up to 97% , in the Third, histological examination was not evident. In the first and second pathology reports, based respectively on endoscopic biopsies and resection specimen, we had doubts about the positivity of KIT and DOG1 immunoreactivities. These two markers present the basis for the diagnosis of GISTs. Miettinen et al. declared that sensitivities of DOG1 and KIT were nearly identical: 94.4% and 94.7% , 9. MoreFinally, the localization of the tumour in the cardia is exceptional and presents a challenge for the surgeon especially if adjacent structures, such as the aorta, are invaded. In fact, surgery is the only curative option for leiomyosarcomas. The type of surgery depends on the size and localization of the tumour . It rangIn conclusion, gastric leiomyosarcoma is a rare tumour. Diagnosis is based on histological examination with immunohistochemistry, which could be sometimes confusing like in our case. The validation of a pathological expert is recommended. Treatment depends on surgery with a very little place reserved for chemotherapy and radiotherapy in advanced cases , 18. Pro


The Funding section is incorrect. The correct funding information is: This study was funded by Health Research Board, Ireland grant HPF-2016-1671 to SP.