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1.  Sprouty 1 predicts prognosis in human epithelial ovarian cancer 
American Journal of Cancer Research  2015;5(4):1531-1541.
Sprouty proteins are evolutionary-conserved modulators of receptor tyrosine kinase (RTK) signaling. We have previously reported inverse correlation of the Sprouty 1 (Spry1) protein expression with ovarian cancer cell proliferation, migration, invasion and survival. In the present study, the expression status of Spry1 protein and its clinical relevance in patients with epithelial ovarian cancer were explored. Matched tumor and normal tissue samples from 100 patients with epithelial ovarian cancer were immunohistochemically stained for Spry1. Expression of ERK, p-ERK, Ki67, FGF-2, VEGF and IL-6 and their correlation with Spry1 were also evaluated. In addition, correlation between Spry1 and clinicopathological characteristics and predictive significance of Spry1 for overall survival (OS) and disease-free survival (DFS) were analysed. Our data indicated that Spry1 was significantly downregulated in tumor tissues (p=0.004). Spry1 showed significant inverse correlation with p-ERK/ERK (p=0.045), Ki67 (p=0.010), disease stage (p=0.029), tumor grade (p=0.037), recurrence (p=0.001) and lymphovascular invasion (p=0.042). It was revealed that Spry1 low-expressing patients had significantly poorer OS (p=0.010) and DFS (p=0.012) than those with high expression of Spry1. Multivariate analysis showed that high Spry1 (p=0.030), low stage (p=0.048) and no residual tumor (p=0.007) were independent prognostic factors for a better OS, among which high Spry1 (p=0.035) and low stage (p=0.035) remained as independent predictors of DFS, too. We also found that the expression of Spry1 significantly correlates with the expression of Spry2 (p<0.001), but not that of Spry4. In conclusion, we report for the first time to our knowledge that Spry1 protein is downregulated in human epithelial ovarian cancer. Spry1 expression significantly impacts tumor behavior and shows predictive value as an independent prognostic factor for survival and recurrence.
PMCID: PMC4473329  PMID: 26101716
Disease free survival; epithelial ovarian cancer; overall survival; prognostic biomarker; Sprouty 1
2.  Minocycline attenuates hypoxia-inducible factor-1α expression correlated with modulation of p53 and AKT/mTOR/p70S6K/4E-BP1 pathway in ovarian cancer: in vitro and in vivo studies 
Hypoxia-inducible factor (HIF)-1α is the key cellular survival protein under hypoxia, and is associated with tumor progression and angiogenesis. We have recently shown the inhibitory effects of minocycline on ovarian tumor growth correlated with attenuation of vascular endothelial growth factor (VEGF) and herein report a companion laboratory study to test if these effects were the result of HIF-1α inhibition. In vitro, human ovarian carcinoma cell lines (A2780, OVCAR-3 and SKOV-3) were utilized to examine the effect of minocycline on HIF-1 and its upstream pathway components to elucidate the underlying mechanism of action of minocycline. Mice harboring OVCAR-3 xenografts were treated with minocycline to assess the in vivo efficacy of minocycline in the context of HIF-1. Minocycline negatively regulated HIF-1α protein levels in a concentration-dependent manner and induced its degradation by a mechanism that is independent of prolyl-hydroxylation. The inhibition of HIF-1α was found to be associated with up-regulation of endogenous p53, a tumor suppressor with confirmed role in HIF-1α degradation. Further studies demonstrated that the effect of minocycline was not restricted to proteasomal degradation and that it also caused down-regulation of HIF-1α translation by suppressing the AKT/mTOR/p70S6K/4E-BP1 signaling pathway. Minocycline treatment of mice bearing established ovarian tumors, led to suppression of HIF-1α accompanied by up-regulation of p53 protein levels and inactivation of AKT/mTOR/p70S6K/4E-BP1 pathway. These data reveal the therapeutic potential of minocycline in ovarian cancer as an agent that targets the pro-oncogenic factor HIF-1α through multiple mechanisms.
PMCID: PMC4396050  PMID: 25973298
Minocycline; HIF-1; p53; AKT; mTOR; VEGF; ovarian cancer
3.  Anticancer properties of novel aminoacetonitrile derivative monepantel (ADD 1566) in pre-clinical models of human ovarian cancer 
Monepantel (MPL) is a new anthelmintic agent approved for the treatment of nematode infections in farm animals. As a nematicide, it acts through a nematode-specific nicotinic receptor subtype which explains its exceptional safety in rodents and mammals. In the present study, we evaluated its potential as an anticancer agent. In vitro treatment of epithelial ovarian cancer cells with MPL resulted in reduced cell viability, inhibition of cell proliferation and suppression of colony formation. Proliferation of human ovarian surface epithelial cells and other non-malignant cells were however minimally affected. MPL-induced inhibition was found to be independent of the acetylcholine nicotinic receptor (nAChR) indicating that, its target in cancer cells is probably different from that in nematodes. Analysis of MPL treated cells by flow cytometry revealed G1 phase cell cycle arrest. Accordingly, MPL treated cells expressed reduced levels of cyclins D1 and A whereas cyclin E2 expression was enhanced. Consistent with a G1 phase arrest, cellular levels of cyclin dependent kinases (CDKs) 2 and 4 were lower, whereas expression of CDK inhibitor p27kip was increased. In cells expressing the wild-type p53, MPL treatment led to increased p53 expression. In line with these results, MPL suppressed cellular thymidine incorporation thus impairing DNA synthesis and inducing cleavage of poly (ADP-ribose) polymerase (PARP-1). Combined these pre-clinical findings reveal for the first time the anticancer potential of monepantel.
PMCID: PMC4163619  PMID: 25232496
Monepantel; ovarian cancer; cell cycle; cyclins; PARP-1
4.  Monepantel induces autophagy in human ovarian cancer cells through disruption of the mTOR/p70S6K signalling pathway 
We have recently shown that the novel anthelmintic drug monepantel (MPL) inhibits growth, proliferation and colony formation, arrests the cell cycle and induces cleavage of PARP-1 in ovarian cancer cell lines. Here we report on the mechanism behind the anticancer properties of MPL. The cytotoxic effect of MPL on ovarian cancer cells (OVCAR-3 and A2780) was investigated employing a panel of tests used for the detection of apoptosis and autophagy. Apoptosis and autophagy were defined by caspase activity, DNA-laddering, Annexin-V and acridine orange (AO) staining. Autophagy markers such as LC3B, SQSTM1/p62 and mammalian target of rapamycin (mTOR) pathway related proteins were assessed by western blotting and ELISA techniques. MPL did not activate caspases 3 or 8, nor did it alter the percentage of Annexin V positive stained cells. Failure to cause DNA laddering and the inability of z-VAD-fmk to block the MPL antiproliferative effects led to the ruling out of apoptosis as the mechanism behind MPL-induced cell death. On the other hand, accumulation of acidic vacuoles with distinct chromatin morphology and an increase in punctuate localization of green fluorescent protein-LC3B, and MPL-induced changes in the expression of SQSTM1/p62 were all indicative of MPL-induced autophagy. Consistent with this, we found inhibition of mTOR phosphorylation leading to suppression of the mTOR/p70S6K signalling pathway. Our findings provide the first evidence to show that MPL triggers autophagy through the deactivation of mTOR/p70S6K signalling pathway.
PMCID: PMC4163620  PMID: 25232497
Monepantel; ovarian cancer; apoptosis; PARP; autophagy; mTOR; p70S6K
5.  Effects of sex hormones on survival of peritoneal mesothelioma 
Previous studies have suggested the presence of steroid receptors as a favourable prognostic factor in peritoneal mesothelioma (PM). This study aims to investigate possible hormonal effects on survival of PM.
This is a retrospective study of prospectively collected data of 52 consecutive patients with PM who underwent cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) by the same surgical team at St George Hospital in Sydney, Australia, between April 1996 and April 2013. Females were arbitrarily divided into assumed premenopausal (<51 years old; n = 15) and assumed postmenopausal (≥51 years old, n = 9). In each gender group, patients were furthered divided into three age groups (<40, 40–60, >60). A significant statistical difference was defined as p < 0.05.
Females with epithelial mesothelioma had a significantly higher survival than males (p = 0.023). They also had a better overall median survival (>60 months) than males (43 months), although this difference was not statistically significant (p = 0.098). Survival of postmenopausal females became similar to males after excluding benign cystic mesothelioma.
The better survival in premenopausal females could probably be explained by higher levels of oestradiol and progesterone. Also, our data suggests that higher rates of benign cystic mesothelioma in females was not the key reason for the better survival in female patients, further supporting the hypothesis of hormonal links with survival of PM. Therapeutic effects of sex steroid hormones on PM may be a valuable area to explore.
PMCID: PMC4491202  PMID: 26111523
Peritoneal mesothelioma; Hormone; Female; Survival
6.  Impact of treatment modality and number of lesions on recurrence and survival outcomes after treatment of colorectal cancer liver metastases 
Ablative strategies have been used to treat and facilitate hepatic resection (HR) in patients with otherwise unresectable colorectal liver metastases (CLM). We evaluated the efficacy of HR, concomitant HR and ablation and isolated ablation on recurrence and survival outcomes after treatment of CLM in patients with 1-4 and ≥5 lesions, respectively.
A retrospective review of a prospectively collected hepatobiliary surgery database was performed on patients who underwent treatment for isolated CLM between 1990 and 2010. Pre-operative and treatment characteristics were compared between patients who underwent HR, concomitant HR and ablation and ablation alone. The impact of treatment modality on survival and recurrence outcomes was determined.
A total of 701 patients met inclusion criteria; 550 patients (78%) had 1-4 lesions and 151 patients (22%) had ≥5 lesions. Overall median survival for the entire cohort was 35 months with 5- and 10-year survival of 33% and 20%, respectively. Overall median and 5-year recurrence-free survival (RFS) was 13 months and 21%, respectively. For patients with 1-4 lesions, median survival was 37 months with 5-year survival of 36%. Stratified by procedure type, 5-year survival was 41% in patients who underwent HR, 35% in patients who underwent concomitant HR and ablation and 13% in patients who underwent ablation alone (P<0.001). For patients with ≥5 lesions, median survival was 28 months with 5-year survival of 23% without difference between treatment groups (P=0.078).
HR appears to be the most effective strategy for patients with 1-4 lesions. When ≥5 lesions are present, ablative strategies are useful in facilitating HR in otherwise unresectable patients.
PMCID: PMC3904025  PMID: 24490042
Colorectal cancer; hepatectomy; liver metastases; resection; survival; ablation
7.  Defective removal of ribonucleotides from DNA promotes systemic autoimmunity 
Genome integrity is continuously challenged by the DNA damage that arises during normal cell metabolism. Biallelic mutations in the genes encoding the genome surveillance enzyme ribonuclease H2 (RNase H2) cause Aicardi-Goutières syndrome (AGS), a pediatric disorder that shares features with the autoimmune disease systemic lupus erythematosus (SLE). Here we determined that heterozygous parents of AGS patients exhibit an intermediate autoimmune phenotype and demonstrated a genetic association between rare RNASEH2 sequence variants and SLE. Evaluation of patient cells revealed that SLE- and AGS-associated mutations impair RNase H2 function and result in accumulation of ribonucleotides in genomic DNA. The ensuing chronic low level of DNA damage triggered a DNA damage response characterized by constitutive p53 phosphorylation and senescence. Patient fibroblasts exhibited constitutive upregulation of IFN-stimulated genes and an enhanced type I IFN response to the immunostimulatory nucleic acid polyinosinic:polycytidylic acid and UV light irradiation, linking RNase H2 deficiency to potentiation of innate immune signaling. Moreover, UV-induced cyclobutane pyrimidine dimer formation was markedly enhanced in ribonucleotide-containing DNA, providing a mechanism for photosensitivity in RNase H2–associated SLE. Collectively, our findings implicate RNase H2 in the pathogenesis of SLE and suggest a role of DNA damage–associated pathways in the initiation of autoimmunity.
PMCID: PMC4382239  PMID: 25500883
8.  Albumin nanoparticles increase the anticancer efficacy of albendazole in ovarian cancer xenograft model 
The poor prognosis of patients with drug resistant ovarian cancer and the lack of targeted therapy have raised the need for alternative treatments. Albendazole (ABZ) is an anti-parasite compound capable of impairing microtubule formation. We hypothesized that ABZ could be repurposed as a potential anti-angiogenic drug due to its potent inhibition of vascular endothelial growth factor (VEGF) in ovarian cancer with ascites. However, the poor aqueous solubility of ABZ limits its potential for cancer therapy. In this study, we have assembled ABZ with bovine serum albumin into nanoparticles with a size range of 7–10 nm (BSA-ABZ) and 200–250 nm (Nab-ABZ). We further examined the anticancer effects of ABZ carrying nanoparticles in ovarian cancer cells, in both in vitro and in vivo models.
Drug release studies demonstrated that about 93% of ABZ was released from BSA-ABZ 10 nm in comparison to 83% from Nab-ABZ 200 nm at pH 7.4 in 8 days. In vitro cell proliferation studies showed that the BSA-ABZ 10 nm exhibited the highest killing efficacy of ovarian cancer cells with surprisingly least toxicity to healthy ovarian epithelial cells. Confocal microscopy and fluorescence activated cell sorting analysis (FACS) revealed more efficient internalization of the BSA-ABZ 10 nm by cancer cells. For in vivo studies, we examined the tumor growth, ascites formation and the expression of VEGF and secreted protein acidic and rich in cysteine (SPARC) in tumor samples and only VEGF in plasma samples. The BSA-ABZ 10 nm reduced the tumor burden significantly (p < 0.02) at a much lower drug dose (10 μg/ml) compare to free drug. Both formulations were capable of suppressing the ascites volume significantly (p < 0.05) and reducing the number of ascites cells. The expression of VEGF and SPARC was also reduced, which indicates the underlying therapeutic mechanism of the ABZ.
Our data suggest that the BSA-ABZ may hold promise for the treatment and control of progression of ovarian cancer with ascites. However further studies are required to examine the efficacy of both the formulations in aggressive models of recurrent ovarian cancer with respect to particle size and dosing parameters.
Electronic supplementary material
The online version of this article (doi:10.1186/s12951-015-0082-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4409778  PMID: 25890381
Angiogenesis; Albendazole; BSA-ABZ 10 nm; Nab-ABZ 200 nm; VEGF; Ascites; Ovarian cancer
9.  Leukotriene B4-mediated sterile inflammation favors susceptibility to sepsis in murine type 1 diabetes 
Science signaling  2015;8(361):ra10.
The chronic systemic inflammation in type I diabetes mellitus (T1DM), which is driven by signaling through the interleukin-1 (IL-1) 1 receptor (IL1R) and the adaptor protein myeloid differentiation factor 88 (MyD88), may be associated with the enhanced susceptibility of diabetics to systemic bacterial infection (sepsis). We hypothesized that low insulin concentrations trigger the enzyme 5-lipoxygenase (5-LO) to produce the lipid mediator leukotriene B4 (LTB4), serving as a trigger of systemic inflammation and increased susceptibility to polymicrobial sepsis in T1DM. In support of this hypothesis, we found that the abundance of MyD88 and its direct transcriptional regulator, STAT-1 were higher in peritoneal macrophages from two mouse models of T1DM compared to nondiabetic mice. Expression of Alox5, synthesis of LTB4, and concentrations of the proinflammatory cytokine IL-1β were also increased in peritoneal macrophages and serum from T1DM mice. Insulin treatment restored LTB4 concentrations and Myd88 and Stat1 expression in T1DM mice. T1DM mice lacking Alox5 or treated with a 5-LO inhibitor showed reduced Myd88 and Il1b mRNA expression and increased IL-1 receptor antagonist concentration. The transcription factor cJun drove LTB4-dependent transcription of Stat1 in macrophages from T1DM mice. Compared to wild-type or untreated diabetic mice, T1DM mice lacking 5-LO or treated with a 5-LO inhibitor survived polymicrobial sepsis and showed reduced production of proinflammatory cytokines and decreased bacterial counts, suggesting that high LTB4 concentrations contribute to enhanced susceptibility to sepsis in T1DM. These results uncover a role for LTB4 in promoting sterile inflammation in diabetes and enhanced susceptibility to sepsis in T1DM.
PMCID: PMC4356178  PMID: 25628460
eicosanoid; SIRS; sepsis; diabetes; leukotriene; 5-LO inhibitor; translational
10.  Human Sprouty1 Suppresses Urokinase Receptor-Stimulated Cell Migration and Invasion 
ISRN biochemistry  2013;2013:598251.
The urokinase-type plasminogen activator receptor (uPAR) has been implicated in several processes in tumor progression including cell migration and invasion in addition to initiation of signal transduction. Since uPAR lacks a transmembrane domain, it uses the interaction with other proteins to modulate intracellular signal transduction. We have previously identified hSpry1 as a partner protein of uPAR, suggesting a physiological role for hSpry1 in the regulation of uPAR signal transduction. In this study, hSpry1 was found to colocalize with uPAR upon stimulation with epidermal growth factor (EGF), urokinase (uPA), or its amino terminal fragment (uPA-ATF), implicating a physiological role of hSpry1 in regulation of uPAR signalling pathway. Moreover, hSpry1 was able to inhibit uPAR-stimulated cell migration in HEK293/uPAR, breast carcinoma, and colorectal carcinoma cells. In addition, hSpry1 was found to inhibit uPAR-stimulated cell invasion in breast carcinoma and osteosarcoma cell lines. Increasing our understanding of how hSpry1 negatively regulates uPAR-stimulated cellular functions may determine a distinctive role for hSpry1 in tumour suppression.
PMCID: PMC4393002  PMID: 25937961
11.  Peritonectomy HIPEC—contemporary results, indications 
PMCID: PMC3752351  PMID: 23997522
12.  Does the expression of BCL2 have prognostic significance in malignant peritoneal mesothelioma? 
Background Malignant peritoneal mesothelioma (MPM) is a rare neoplasm of the peritoneal membrane that is causally related to asbestos exposure. Survival after treatment is poor. Current therapy involving hyperthermic intraperitoneal chemotherapy has improved survival in selective patients. In the past, several prognostic factors have been identified in MPM patients and this has prompted the development of new therapies and patient management. Since BCL2, an antiapoptotic oncoprotein, is a favourable prognostic factor in breast cancer, we investigated to determine the significance of BCL2 in MPM. Materials and Methods Forty two archival patient tumour sections embedded in paraffin blocks were sectioned and subjected to immunohistochemistry to detect BCL2. The staining intensity and abundance was classified using standard procedures and classified into two groups (0-4 = low & 5-8 = high expression). The distribution of BCL2 groups was examined in the different clinicopathological categories to determine prognosis using Kaplan–Meier survival analysis. Results: Univariate analysis revealed that in almost all clinicopathological categories, high BCL2 expression predisposed patients to a favourable prognosis. Independent of BCL2 expression, univariate analysis also showed that male gender, sarcomatoid histology, high PCI and age at diagnosis ≥ 60 years were associated poor prognosis. Multivariate analysis indicated that for all tumours, males and females, high BCL2 expression was associated with good prognosis. Further, independent of BCL2, age ≥ 60 years is an unfavourable prognostic factor. Conclusion: Expression of BCL2 may serve to distinguish prognosis within the individual clinicopathological categories. BCL2 is also an independent variable in all tumours, males and females, with high expression being associated with good prognosis.
PMCID: PMC3696537  PMID: 23841030
BCL2; prognosis; peritoneal mesothelioma; survival
13.  An MHC Class II Dependent Activation Loop Between Adipose Tissue Macrophages and CD4+ T cells Controls Obesity-Induced Inflammation 
Cell reports  2014;9(2):605-617.
An adaptive immune response triggered by obesity is characterized by the activation of adipose tissue CD4+ T cells by unclear mechanisms. We have examined if interactions between adipose tissue macrophages (ATMs) and CD4+ T cells contribute to adipose tissue metainflammation. Intravital microscopy identifies dynamic antigen dependent interactions between ATMs and T cells in visceral fat. Mice deficient in major histocompatibility complex class II (MHCII) showed protection from diet-induced obesity. Deletion of MHCII expression in macrophages led to an adipose tissue specific decrease in the effector/memory CD4+ T cells, attenuation of CD11c+ ATM accumulation, and improvement in glucose intolerance by increasing adipose tissue insulin sensitivity. Ablation experiments demonstrated that the maintenance of proliferating conventional T cells is dependent on signals from CD11c+ ATMs in obese mice. These studies demonstrate the importance of MHC Class II restricted signals from ATMs that regulate adipose tissue T cell maturation and metainflammation.
PMCID: PMC4252867  PMID: 25310975
14.  Peritonectomy and hyperthermic intraperitoneal chemotherapy as treatment for desmoplastic small round cell tumour 
The St George Hospital specialises in peritonectomy and hyperthermic intraperitoneal chemotherapy (HIPEC) for treatment of intra-abdominal malignancies. Despite performing around 800 peritonectomy and HIPEC procedures, we have rarely encountered desmoplastic small round cell tumours (DSRCT). We present our experiences with DSRCT, and propose peritonectomy and HIPEC as a treatment option for DSRCT.
Presentation of case
This is a case series of 3 cases. The first case was a 26-year-old male who presented with appendicitis which we diagnosed as DSRCT and treated with peritonectomy and HIPEC. The second case was a 14-year-old male referred to our centre for peritonectomy and HIPEC after initial presentation with a pelvic mass and treatment with chemotherapy. The third case was a 21-year-old male referred to our centre for peritonectomy and HIPEC for recurrent DSRCT after previously being treated with neoadjuvant chemotherapy and surgery without HIPEC.
DSRCT is a rare, almost exclusively intra-abdominal malignancy, which predominantly affects young males. Survival prognosis remains poor in DSRCT despite conventional treatment with surgery, chemotherapy and radiotherapy; however, HIPEC has offered promising survival results. Our recurrences with peritonectomy and HIPEC at 6 months and 15 months are comparable with the literature of 8.85 months.
In our experience, patients with DSRCT who present with nodal involvement or recurrent disease tend to recur early despite treatment with peritonectomy and HIPEC. Longer term follow up of our patients and future studies involving HIPEC in DSRCT would be useful in assessing long-term clinical outcomes and survival.
PMCID: PMC4336428  PMID: 25598401
Desmoplastic small round cell tumour (DSRCT); Hyperthermic intraperitoneal chemotherapy (HIPEC); Peritonectomy
15.  Effectiveness of early and aggressive administration of fresh frozen plasma to reduce massive blood transfusion during cytoreductive surgery 
Cytoreductive surgery (CRS) combined with perioperative intraperitoneal chemotherapy (PIC) has been consistently associated with high volume blood loss and red blood cell (RBC) transfusion. This study evaluates the effectiveness of the introduction of a novel protocol to reduce blood loss and subsequent intra-operative transfusion in patients with high volume disease.
One hundred and thirty-one consecutive patients with high volume disease (peritoneal cancer index ≥16) who underwent CRS and PIC were evaluated. Group I consisted of the sixty patients (46%) treated before June 2006. Group II consistent of the seventy-one (54%) patients treated after June 2006 under the new protocol. The clinical and treatment-related data of patients in the two groups were compared.
Group II was associated with reduced intra-operative red blood cell transfusion (P<0.001), reduced cryoprecipitate transfusion (P=0.020), reduced platelet transfusion (P<0.001), reduced fresh frozen plasma transfusion (P=0.024), reduced operation length (P<0.001), reduced crystalloid administration (P<0.001) and reduced colloid administration (P<0.001). Group II was also associated with increased transfusion of FFP in the first half of the surgical intervention relative to the second half [FFP1st:FFP2nd ratio >1 (P<0.001)] and increased transfusion of RBC in the first half of the surgical intervention relative to the second half [RBC1st:RBC2nd ratio ≥1 (P=0.016)].
Early administration of fresh frozen plasma combined with restrictive fluid resuscitation may reduce overall intra-operative transfusion of RBC and other blood components.
PMCID: PMC3562619  PMID: 23449950
Peritonectomy; cytoreductive surgery; perioperative intraperitoneal chemotherapy; morbidity; mortality; blood transfusion
16.  Flow Cytometry Analyses of Adipose Tissue Macrophages 
Methods in enzymology  2014;537:297-314.
Within adipose tissue, multiple leukocyte interactions contribute to metabolic homeostasis in health as well as to the pathogenesis of insulin resistance with obesity. Adipose tissue macrophages (ATMs) are the predominant leukocyte population in fat and contribute to obesity-induced inflammation. Characterization of ATMs and other leukocytes in the stromal vascular fraction from fat has benefited from the use of flow cytometry and flow-assisted cell sorting techniques. These methods permit the immunophenotyping, quantification, and purification of these unique cell populations from multiple adipose tissue depots in rodents and humans. Proper isolation, quantification, and characterization of ATM phenotypes are critical for understanding their role in adipose tissue function and obesity-induced metabolic diseases. Here, we present the flow cytometry protocols for phenotyping ATMs in lean and obese mice employed by our laboratory.
PMCID: PMC4244227  PMID: 24480353
17.  Hepatocytes isolated from neoplastic liver-immunomagnetic purging as a new source for transplantation 
AIM: To investigate whether hepatocytes isolated from macroscopically normal liver during hepatic resection for neoplasia could provide a novel source of healthy hepatocytes, including the development of reliable protocols for malignant cells removal from the hepatocyte preparation.
METHODS: Hepatocytes were procured from resected liver of 18 patients with liver tumors using optimised digestion and cell-enrichment protocols. Suspensions of various known quantities of the HT-29 tumor cell line and patient hepatocytes were treated or not with Ep-CAM-antibody-coated immunomagnetic beads in order to investigate the efficacy of tumor-purging by immunomagnetic depletion, using a semi-quantitative RT-PCR method developed to detect tumor cells. Immunomagnetic bead-treated or bead-untreated tumor cell-hepatocyte suspensions were transplanted intra-peritoneally in Balb/C nude mice to assess the rates of tumor development.
RESULTS: Mean viable hepatocyte yield was 9.3 x 106 cells per gram of digested liver with mean viability of 70.5%. Immunomagnetic depletion removed tumor cells to below the RT-PCR detection-threshold of 1 tumor cell in 106 hepatocytes, representing a maximum tumor purging efficacy of greater than 400 000-fold. Transplanted, immunomagnetic bead-purged tumor cell-hepatocyte suspensions did not form peritoneal tumors in Balb/C nude mice. Co-transplantation of hepatocytes with tumor cells did not increase tumorigenesis of the tumor cells.
CONCLUSION: Immunomagnetic depletion appears to be an effective method of purging contaminating tumor cells to below threshold for likely tumorigenesis. Along with improved techniques for isolation of large numbers of viable hepatocytes, normal liver resected for neoplasia has potential as another clinically useful source of hepatocytes for transplantation.
PMCID: PMC2742930  PMID: 18763285
Hepatocyte transplantation; Immunomagnetic purging; Isolation of hepatocytes
18.  Streptozotocin is equally diabetogenic whether administered to fed or fasted mice 
Laboratory animals  2013;47(4):257-265.
Streptozotocin (STZ) is a selective pancreatic β cell toxin used to generate experimental hyperglycemia in rodent models. Several laboratory animal protocols suggest that STZ be administered to fasted rodents to minimize competition between STZ and glucose for low affinity GLUT2 transporters on β cells. However, whether the diabetogenic effects of multiple low dose (MLD)-STZ administration are enhanced by fasting has not been addressed. Given that repeated bouts of fasting can cause undue metabolic stress in mice, we compared the efficacy of MLD-STZ injections (50 mg/kg body weight daily for 5 days) to induce experimental hyperglycemia in both NOD/SCID/γchainnull and C57BL/6J mice that were either ad libitum fed (STZ-Fed) or that had been fasted for 6 h (STZ-Fasted) prior to the time of STZ administration. Both STZ-Fed and STZ-Fasted mice had significantly worse glucose tolerance than vehicle-treated control mice 10 days after initiation of the MLD-STZ regimen. In C57BL/6J mice, fasting glucose levels, serum insulin levels, β cell mass, and glucose disposal during intraperitoneal glucose tolerance tests (IPGTTs) were indistinguishable between STZ-Fed and STZ-Fasted mice 20 days after MLD-STZ. The glucose intolerant phenotypes persisted for 20 weeks thereafter, irrespective of whether C57BL/6J mice were fed or fasted at the time of STZ injections. However, STZ-Fasted C57BL/6J mice experienced significant weight loss during the repeated bouts of fasting/re-feeding that were required to complete the MLD-STZ protocol. In summary, induction of experimental hyperglycemia can be achieved using the MLD-STZ protocol without repeated bouts of fasting, which have the potential to cause metabolic stress in laboratory mice.
PMCID: PMC3773005  PMID: 23760565
streptozotocin; fasting; diabetes; animal protocol refinement
19.  Approach to Rectal Cancer Surgery 
Rectal cancer is a distinct subset of colorectal cancer where specialized disease-specific management of the primary tumor is required. There have been significant developments in rectal cancer surgery at all stages of disease in particular the introduction of local excision strategies for preinvasive and early cancers, standardized total mesorectal excision for resectable cancers incorporating preoperative short- or long-course chemoradiation to the multimodality sequencing of treatment. Laparoscopic surgery is also increasingly being adopted as the standard rectal cancer surgery approach following expertise of colorectal surgeons in minimally invasive surgery gained from laparoscopic colon resections. In locally advanced and metastatic disease, combining chemoradiation with radical surgery may achieve total eradication of disease and disease control in the pelvis. Evidence for resection of metastases to the liver and lung have been extensively reported in the literature. The role of cytoreductive surgery and hyperthermic intraperitoneal chemotherapy for peritoneal metastases is showing promise in achieving locoregional control of peritoneal dissemination. This paper summarizes the recent developments in approaches to rectal cancer surgery at all these time points of the disease natural history.
PMCID: PMC3389703  PMID: 22792451
20.  Adipose Tissue Macrophages Function As Antigen-Presenting Cells and Regulate Adipose Tissue CD4+ T Cells in Mice 
Diabetes  2013;62(8):2762-2772.
The proinflammatory activation of leukocytes in adipose tissue contributes to metabolic disease. How crosstalk between immune cells initiates and sustains adipose tissue inflammation remains an unresolved question. We have examined the hypothesis that adipose tissue macrophages (ATMs) interact with and regulate the function of T cells. Dietary obesity was shown to activate the proliferation of effector memory CD4+ T cells in adipose tissue. Our studies further demonstrate that ATMs are functional antigen-presenting cells that promote the proliferation of interferon-γ–producing CD4+ T cells in adipose tissue. ATMs from lean and obese visceral fat process and present major histocompatibility complex (MHC) class II–restricted antigens. ATMs were sufficient to promote proliferation and interferon-γ production from antigen-specific CD4+ T cells in vitro and in vivo. Diet-induced obesity increased the expression of MHC II and T-cell costimulatory molecules on ATMs in visceral fat, which correlated with an induction of T-cell proliferation in that depot. Collectively, these data indicate that ATMs provide a functional link between the innate and adaptive immune systems within visceral fat in mice.
PMCID: PMC3717880  PMID: 23493569
21.  Sex-specific selection for MHC variability in Alpine chamois 
In mammals, males typically have shorter lives than females. This difference is thought to be due to behavioural traits which enhance competitive abilities, and hence male reproductive success, but impair survival. Furthermore, in many species males usually show higher parasite burden than females. Consequently, the intensity of selection for genetic factors which reduce susceptibility to pathogens may differ between sexes. High variability at the major histocompatibility complex (MHC) genes is believed to be advantageous for detecting and combating the range of infectious agents present in the environment. Increased heterozygosity at these immune genes is expected to be important for individual longevity. However, whether males in natural populations benefit more from MHC heterozygosity than females has rarely been investigated. We investigated this question in a long-term study of free-living Alpine chamois (Rupicapra rupicapra), a polygynous mountain ungulate.
Here we show that male chamois survive significantly (P = 0.022) longer if heterozygous at the MHC class II DRB locus, whereas females do not. Improved survival of males was not a result of heterozygote advantage per se, as background heterozygosity (estimated across twelve microsatellite loci) did not change significantly with age. Furthermore, reproductively active males depleted their body fat reserves earlier than females leading to significantly impaired survival rates in this sex (P < 0.008). This sex-difference was even more pronounced in areas affected by scabies, a severe parasitosis, as reproductively active males were less likely to survive than females. However, we did not find evidence for a survival advantage associated with specific MHC alleles in areas affected by scabies.
Increased MHC class II DRB heterozygosity with age in males, suggests that MHC heterozygous males survive longer than homozygotes. Reproductively active males appear to be less likely to survive than females most likely because of the energetic challenge of the winter rut, accompanied by earlier depletion of their body fat stores, and a generally higher parasite burden. This scenario renders the MHC-mediated immune response more important for males than for females, which implies a relatively stronger selection pressure on MHC genes in males than in females.
PMCID: PMC3340304  PMID: 22335968
MHC; Sex-specific selection; Heterozygosity advantage; Alpine chamois
22.  CX3CR1 Deficiency Does Not Influence Trafficking of Adipose Tissue Macrophages in Mice With Diet-Induced Obesity 
Obesity (Silver Spring, Md.)  2012;20(6):1189-1199.
Adipose tissue macrophages (ATMs) accumulate in fat during obesity and resemble foam cells in atherosclerotic lesions, suggesting that common mechanisms underlie both inflammatory conditions. CX3CR1 and its ligand fractalkine/CX3CL1 contribute to macrophage recruitment and inflammation in atherosclerosis, but their role in obesity-induced adipose tissue inflammation is unknown. Therefore, we tested the hypothesis that CX3CR1 regulates ATM trafficking to epididymal fat and contributes to the development of adipose tissue inflammation during diet-induced obesity. Cx3cl1 and Cx3cr1 expression was induced specifically in epididymal fat from mice fed a high-fat diet (HFD). CX3CR1 was detected on multiple myeloid cells within epididymal fat from obese mice. To test the requirement of CX3CR1 for ATM trafficking and obesity-induced inflammation, Cx3cr1+/GFP and Cx3cr1GFP/GFP mice were fed a HFD. Ly-6cLow monocytes were reduced in lean Cx3cr1GFP/GFP mice; however, HFD-induced monocytosis was comparable between strains. Total ATM content, the ratio of type 1 (CD11c+) to type 2 (CD206+) ATMs, expression of inflammatory markers, and T-cell content were similar in epididymal fat from obese Cx3cr1+/GFP and Cx3cr1GFP/GFP mice. Cx3cr1 deficiency did not prevent the development of obesity-induced insulin resistance or hepatic steatosis. In summary, our data indicate that CX3CR1 is not required for the recruitment or retention of ATMs in epididymal adipose tissue of mice with HFD-induced obesity even though CX3CR1 promotes foam cell formation. This highlights an important point of divergence between the mechanisms regulating monocyte trafficking to fat with obesity and those that contribute to foam cell formation in atherogenesis.
PMCID: PMC4006981  PMID: 22252034
23.  Correction: Neuropeptide Y Is Produced by Adipose Tissue Macrophages and Regulates Obesity-Induced Inflammation 
PLoS ONE  2013;8(9):10.1371/annotation/c2432ace-1dd7-4b02-8980-e4e6c085beba.
PMCID: PMC3812369
24.  Ki67-BCL2 index in prognosis of malignant peritoneal mesothelioma 
Background: malignant peritoneal mesothelioma (MPM) is a rare peritoneal mesothelial neoplasm. Ki67 and BCL2 are established prognostic markers in several cancers. High Ki67 expression indicates tumour progression, whilst similar expression of BCL2 retards tumour replication. Traditionally, prognosis in MPM is gauged with a single biomarker assessed separately in a dichotomous manner. Here, we examine prognosis with dual biomarkers incorporated in a model to predict survival. Materials and methods: Forty two MPM archival patient tumours were screened for Ki67 and BCL2 by immunohistochemistry and evaluated using standard methods. Ki67 and BCL2 expression was incorporated into a prognostic model to develop Ki67-BCL2 index. Using this index, three hazard groups were identified (high, medium and low risk). Kaplan-Meier survival analysis was performed to assess the significance of these hazard groups in the various clinicopathological categories. Results: In all clinicopathological categories, high risk group showed poor prognosis compared to low risk group (p = < 0.001). Compared to medium risk, high risk group carried poor prognosis in all tumours, females, epitheloid tumours, peritoneal cancer index (PCI) < 20, ≥ 20, age at diagnosis (AAD) < 60, and ≥ 60 years. Independent of the Ki67-BCL2 index, male, sarcomatoid, PCI ≥ 20 and AAD ≥ 60 were poor prognostic factors. High risk group was an independent poor prognostic factor in all tumours, males, females and age < 60 years. The distribution of high risk: low risk group in male and female was 3: 2 and 2: 3, respectively, indicating a gender difference. Comparing hazard ratios generated by Ki67-BCL2 index to that of either Ki67 or BCL2, as a single prognostic biomarker, there was a reduction of HR values. Conclusion: Ki67-BCL2 index seems to suggest a more sensitive method of predicting prognosis. However, the current model needs further evaluation in an independent large cohort sample.
PMCID: PMC3744020  PMID: 23977450
Ki67; BCl2; prognosis; malignant peritoneal mesothelima
25.  Carbohydrate antigen 19-9 (CA 19-9) is an independent prognostic indicator in pseudomyxoma peritonei post cytoreductive surgery and perioperative intraperitoneal chemotherapy 
Pseudomyxoma peritonei (PMP) is characteristically divided into two histopathological subtypes; disseminated peritoneal adenomucinosis (DPAM) and peritoneal mucinous carcinomatosis (PMCA). The latter is associated with a worse prognosis. However, even within the DPAM group, there is a considerable variation in outcome. In this study we investigate the role of baseline serum tumor markers CA 19-9, CEA and CA-125 in further stratifying survival.
Over 16 years, 218 patients with PMP were treated with cytoreductive surgery (CRS) and perioperative intraperitoneal chemotherapy (PIC) at our institution. A CA-125 level of >35 U/L, CA 19-9 of >40 U/mL and CEA of >3 ng/mL were considered positive or elevated outside the laboratory reference range. The impact of clinicopathologic and treatment-related variables on overall survival (OS) was analyzed with the Kaplan Meier method. Survival curves were compared using the log-rank test. Variables deemed significant by univariate analyses were entered into multivariate analysis using the Cox proportional hazards model.
Within the DPAM group, the 5-year survival of patients who were CA 19-9 positive versus those with normal values were 58% and 90% respectively (P<0.001). Other variables found to negatively impact on OS in univariate analyses were completeness of cytoreduction (CC) score 2/3 (P<0.001), peritoneal cancer index (PCI) >25 (P<0.001) and male gender (P=0.017). In the Cox regression model, only CA 19-9 positivity was found to be an independent prognostic factor for OS (P=0.034). In addition to marker positivity, the absolute level of CA 19-9 was also prognostically significant. In patients with CA 19-9>1,000 U/mL, the 5-year survival was 23%, in contrast to 90% in patients with CA 19-9<100 U/mL (P<0.001).
In the PMCA cohort, only CC-score was found to be associated with OS (P<0.001).
Our study provides relevant prognostic information for the DPAM subtype in staging and prioritizing surgery; as even in apparently indolent disease, some patients have poorer survival. CA 19-9 elevation may also be useful in identifying patients who would potentially benefit from adjuvant therapy and/or closer post-operative surveillance.
The potential role of CA 19-9 in mediating tumor cell adhesion and disease progression in PMP should be further investigated to deepen our understanding of the disease’s inherent biological behavior. If a true relationship exists, CA 19-9 may be a conceivable target for immunotherapy.
PMCID: PMC3635178  PMID: 23730513
Carbohydrate antigen 19-9 (CA 19-9); pseudomyxoma peritonei (PMP); disseminated peritoneal adenomucinosis (DPAM); peritoneal mucinous carcinomatosis (PMCA)

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