Long-Term Changes to the Microbiome, Blood Lipid Profiles and IL-6 in Female and Male Swedish Patients in Response to Bariatric Roux-en-Y Gastric Bypass.

Lipid metabolism dysregulation is a critical factor contributing to obesity. To counteract obesity-associated disorders, bariatric surgery is implemented as a very effective method. However, surgery such as Roux-en-Y gastric bypass (RYGB) is irreversible, resulting in life-long changes to the digestive tract. The aim of the present study was to elucidate changes in the fecal microbiota before and after RYGB in relation to blood lipid profiles and proinflammatory IL-6. Here, we studied the long-term effects, up to six years after the RYGB procedure, on 15 patients' gut microbiomes and their post-surgery well-being, emphasizing the biological sex of the patients. The results showed improved health among the patients after surgery, which coincided with weight loss and improved lipid metabolism. Health changes were associated with decreased inflammation and significant alterations in the gut microbiome after surgery that differed between females and males. The Actinobacteriota phylum decreased in females and increased in males. Overall increases in the genera Prevotella, Paraprevotella, Gemella, Streptococcus, and Veillonella_A, and decreases in Bacteroides_H, Anaerostipes, Lachnoclostridium_B, Hydrogeniiclostridium, Lawsonibacter, Paludicola, and Rothia were observed. In conclusion, our findings indicate that there were long-term changes in the gut microbiota after RYGB, and shifts in the microbial taxa appeared to differ depending on sex, which should be investigated further in a larger cohort.

Acute appendicitis: A block-randomized study on active observation with or without antibiotic treatment.

BACKGROUND: Antibiotic treatment of unselected patients with acute appendicitis is safe and effective. However, it is unknown to what extent early provision of antibiotic treatment may represent overtreatment due to spontaneous healing of appendix inflammation. The aim of the present study was to evaluate the role of antibiotic treatment versus active in-hospital observation on spontaneous regression of acute appendicitis. METHOD: Patients who sought acute medical care at Sahlgrenska University Hospital were block-randomized according to age (18-60 years) and systemic inflammation (C-reactive protein <60 mg/L, white blood cell <13,000/µL), in combination with clinical and abdominal characteristics of acute appendicitis. Study patients received antibiotic treatment and active observation, while control patients were allocated to classic active "wait and see observation" for either disease regression or the need for surgical exploration. According to our standard surgical care, certified surgeons in charge decided whether and when appendectomy was necessary. In total, 1,019 patients were screened for eligibility; 203 patients met inclusion criteria, 126 were accepted to participate, 29 declined, and 48 were missed for inclusion. RESULTS: The antibiotic group (n = 69) and the control group (n = 57) were comparable at inclusion. Appendectomy at first hospital stay was 28% and 53% for study and control patients (χ2, P < .004). Life table analysis indicated a time-dependent difference in the need for appendectomy during follow-up (P < .03). Antibiotics prevented surgical exploration and appendectomy by 72% to 50% compared to 47% to 37% in the control group across the time course follow-ups between 5 and 1,200 days. CONCLUSION: Early antibiotic treatment is superior to traditional "wait and see observation" to avoid surgical exploration and appendectomy.

Parathyroid hormone related protein (PTHrP) in patients with pancreatic carcinoma and overt signs of disease progression and host tissue wasting.

BACKGROUND: Cancer-cachexia is a complex syndrome secondary to physiological mechanisms related to classical hormone and immune alterations, where contributions of neuro-endocrine involvement have been less evaluated. Therefore, the aim of our study was to explore relationships between PTHrP and whole body metabolism in patients with progressive pancreatic carcinoma; relevant to "fat tissue browning". METHODS: Patient serum samples and clinical information were retrieved from earlier translational projects (1995-2005), at Sahlgrenska University Hospital in Gothenburg. Blood PTHrP levels were determined at Harvard medical School (2014). Patient data included: medical history, clinical laboratory tests, food diaries, resting metabolic expenditure, body composition, exercise capacity, Health-Related Quality of Life (SF-36) and mental disorders (HAD-scales). RESULTS: Serum PTHrP was detectable in 17 % of all samples without significance to tumor stage. PTHrP-negativity at inclusion remained during follow-up. Mean PTHrP concentration was 262±274 pg/ml, without sex difference and elevation over time. PTHrP-positive and negative patients experienced similar body weight loss (%) at inclusion, with a trend to deviate at follow ups (16.8±8.2% vs. 13.1±8.2%, p<0.06), where PTHrP concentrations showed correlations to weight loss, handgrip strength and Karnofsky performance, without difference in exercise capacity. PTHrP-positivity was related to increased whole body fat oxidation (p<0.006-0.01) and reduced carbohydrate oxidation (p<0.01-0.03), independently of peripheral lipolysis. Metabolic alterations in PTHrP-positive patients were related to reduced Health Related Quality of life (SF: p<0.08, MH: p<0.02), and increased anxiety and depression (HAD 1-7: p<0.004; HAD 8-14: p<0.008). CONCLUSION: Serum PTHrP positivity in patients with pancreatic carcinoma was related to altered whole body oxidative metabolism; perhaps induced by "browning" of fat cells?

Initiation of muscle protein synthesis was unrelated to simultaneously upregulated local production of IGF-1 by amino acids in non-proliferating L6 muscle cells.

BACKGROUND: IGF-1 is considered an important regulator of muscle protein synthesis. However, its role in stimulation of muscle protein synthesis by amino acids (AA) is not clear, despite pronounced alterations in IGF-1 mRNA expression and signaling in muscle tissues by feeding. This study evaluates the role of locally produced IGF-1 and IGF-1 signaling when skeletal muscle protein synthesis is activated by increased amino acid availability in confluent, non-proliferating cells. METHODS: L6 skeletal muscle cells were subjected to amino acid starvation (24 h, 0.14 mM) followed by 18 h amino acid refeeding in Low AA (0.28 mM) or High AA concentrations (9 mM). Protein synthesis rates were estimated by L-[U-14C]-phenylalanine incorporation into cellular proteins. IGF-1 and IGF-1 receptor mRNA expression were quantified by real time PCR. SiRNA knockdown, antibodies and chemical inhibitors were used to attenuate muscle IGF-1 production and signaling. RESULTS: High AA concentrations (9mM) increased IGF-1 mRNA expression (+ 30%, p<0.05) and increased L-[U-14C]-phenylalanine incorporation compared to Low AA in confluent, non-proliferating muscle cells. Blocking IGF-1 signaling by chemical inhibitors reduced IGF-1 mRNA upregulation (~50%, p< 0.01), without decrease of protein synthesis. SiRNA knockdown of IGF-1 reduced protein synthesis, mainly explained by reduced cell proliferation. High AA or IGF-1 inhibitors did not change IGF-1 receptor mRNA expressions. CONCLUSION: Amino acids increased IGF-1 mRNA expression and stimulated muscle protein synthesis. However, simultaneous upregulation of IGF-1 mRNA did not relate to increased protein synthesis by amino acids. The results indicate that increased IGF-1 mRNA expression is rather a covariate to amino acid initiation of protein synthesis in non-proliferating muscle cells; effects that may be related to unrecognized metabolic activities, such as transport of amino acids.

Reduced tumor growth in EP2 knockout mice is related to signaling pathways favoring an increased local anti­tumor immunity in the tumor stroma.

Inflammatory signaling through prostaglandin E2 receptor subtype 2 (EP2) is associated with malignant tumor growth in both experimental models and cancer patients. Thus, the absence of EP2 receptors in host tissues appears to reduce tumor growth and systemic inflammation by inducing major alterations in gene expression levels across tumor tissue compartments. However, it is not yet well­established how signaling pathways in tumor tissue relate to simultaneous signaling alterations in the surrounding tumor­stroma, at conditions of reduced disease progression due to decreased host inflammation. In the present study, wild­type tumor cells, producing high levels of prostaglandin E2 (MCG 101 cells, EP2+/+), were inoculated into EP2 knockout (EP2­/­) and EP2 wild­type (EP2+/+) mice. Solid tumors were dissected into tumor­ and tumor­stroma tissue compartments for RNA expression microarray screening, followed by metabolic pathway analyses. Immunohistochemistry was used to confirm adequate dissections of tissue compartments, and to assess cell proliferation (Ki­67), prostaglandin enzymes (cyclooxygenase 2) and immunity biomarkers (CD4 and CD8) at the protein level. Microarray analyses revealed statistically significant alterations in gene expression in the tumor­stroma compartment, while significantly less pathway alterations occurred in the tumor tissue compartment. The host knockout of EP2 receptors led to a significant downregulation of cell cycle regulatory factors in the tumor­stroma compartment, while interferon γ­related pathways, chemokine signaling pathways and anti­tumor chemokines [chemokine (C­X­C motif) ligand 9 and 10] were upregulated in the tumor compartment. Thus, such gene alterations were likely related to reduced tumor growth in EP2­deficient hosts. On the whole, pathway analyses of both tumor­ and tumor­stroma compartments suggested that absence of host EP2 receptor signaling reduces 'remodeling' of tumor microenvironments and increase local immunity, probably by decreased productions of stimulating growth factors, perhaps similar to well­recognized physiological observations in wound healing.

Determinants of change in resting energy expenditure in patients with stage III/IV colorectal cancer.

BACKGROUND & AIMS: Resting energy expenditure (REE) is variable in cancer and might be influenced by changes in tumor burden, systemic inflammation, and body composition. The objective of this study was to assess REE change and the predictors of such in patients with stage III or IV colorectal cancer. METHODS: REE was measured via indirect calorimetry and fat mass and fat-free mass (FFM) were assessed using dual X-ray absorptiometry as part of a unique analysis of two studies. C-reactive protein (CRP) was measured as an inflammatory marker. Linear regression was used to assess the determinants of REE at baseline and REE change, with days between baseline and follow-up measures included as a covariate. RESULTS: One-hundred and nine patients were included at baseline (59.6% male; 67 ± 12 years; body mass index 24.1 ± 4.3 kg/m2); 49 had follow-up data (61.2% male; 65 ± 12 years; body mass index 25.4 ± 4.3 kg/m2), with median follow-up of 119 days (interquartile range: 113-127 days). At baseline, age, FFM, and CRP explained 68.9% of the variability in REE. A wide variability in REE change over time was observed, ranging from -156 to 370 kcal/day, or -13.0 to 15.7%/100 days. CRP change (1.7 ± 0.4 mg/L, p < 0.001) and stage (81.3 ± 38.7, p = 0.042) predicted REE change in multivariate analysis, controlling for age, FFM change, and days between visits (R2: 0.417 ± 88.2, p < 0.001). CONCLUSIONS: Age, FFM, and CRP predicted REE at a single time point. REE change was highly variable and explained by inflammation and stage. Future research should investigate the validity and feasibility of incorporating these measures into energy needs recommendations.

LPS immune challenge reduces arcuate nucleus TSHR and CART mRNA and elevates plasma CART peptides.

BACKGROUND: The aim was to examine the impact of lipopolysaccharide-induced systemic inflammation on expression of mRNA for cocaine- and amphetamine-regulated transcript (CART) and the thyrotropin receptor (TSHR) and its ligands in CNS areas of relevance for feeding controls and metabolism. Lipopolysaccharide effects on plasma levels of TSH and CART peptides were also examined. METHODS: Lipopolysaccharide (150-200 µg/mouse) was injected in C57BL/6J mice and tissue and plasma samples taken after 24 h. To establish if plasma increase in CART peptide levels were prostanoid dependent, indomethacin was given via the drinking water beginning 48 h prior to LPS. We evaluated mRNA expression for CART, TSHR, TSHß, and thyrostimulin in brain and pituitary extracts. Plasma levels of TSH, CARTp, and serum amyloid P component were analyzed by ELISA. RESULTS: Lipopolysaccharide suppressed TSHR mRNA expression in the arcuate nucleus and the pituitary. CART mRNA expression was reduced in the arcuate nucleus but elevated in the pituitary of mice treated with Lipopolysaccharide, whereas plasma TSH remained unchanged. Plasma CART peptide concentration increased after LPS treatment in a prostanoid-independent manner, and CART peptide levels correlated positively to degree of inflammation. CONCLUSIONS: Our findings suggest that central and peripheral CART is affected by acute inflammation. Considering the role of the arcuate nucleus in feeding controls, our data highlight TSHR and CART as putative neuroendocrine signaling components that respond to inflammation, perhaps to maintain weight and metabolic homeostasis during states of disease.

Estrogen biosynthesis in cultured skeletal muscle cells (L6) induced by amino acids.

BACKGROUND: Previous investigations have indicated upregulation of gene expression in cellular pathways related to the biosynthesis of steroids in response to amino acids (AA) in skeletal muscle cells. This suggests AA as modulators of de novo synthesis of sex steroids for muscle growth and improved functional capacity. The aim of the present study was to investigate if increased availability of amino acids induced biosynthesis of sex steroids in skeletal muscles. METHODS: Confluent L6 muscle cells were cultured in media with various AA concentrations (0.3 or 9 mM AA or 2.1 mM branched-chain (BCAA) only), following pre-culture in serum-free medium. Sex steroids were quantified by gas chromatography-tandem mass spectrometry (GC-MS/MS). Mevalonate (diphospho-) decarboxylase enzyme (MVD) was quantified by Western blot. RESULTS: The experiments confirmed that estradiol and estrone increased in both L6 cell lysates and in conditioned media at the end of experiments on confluent cells, while progesterone or androgenic steroids were not detected in either cell lysates or culture media. Estradiol (+ 31 ± 3%) and estrone (+ 18 ± 4%) increased significantly in cells cultured at 9 mM AA (p < 0.001 vs. 0.3 mM AA, n = 10). Similarly, MVD protein increased at 9 mM AA (p < 0.001 vs. 0.3 mM AA, n = 17). An addition of BCAA alone to media increased MVD-protein levels to the same extent as all AA (p < 0.01 vs. 0.3 mM AA, n = 3). CONCLUSION: Female sex steroids and MVD enzyme production increased significantly in response to amino acid availability. The results indicate a role of amino acids as modulators of local muscle estrogen synthesis in muscle cells from rats at feeding.

EGFR, but not COX-2, protein in resected pancreatic ductal adenocarcinoma is associated with poor survival.

The effects of EGFR and COX-2 protein overexpression on clinical outcomes in pancreatic ductal adenocarcinoma (PDAC) patients remains unclear. Therefore, the aim of the present study was to evaluate the protein expression of epithelial growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) in tumor cells in surgically resected PDAC, in comparison with clinicopathological characteristics and clinical outcomes. Immunohistochemical staining of formalin-fixed paraffin-embedded tissue derived from surgically resected tumors was performed. Tissue slides were evaluated for membrane wild-type EGFR and cytoplasmic COX-2 staining using a histoscore system. Statistical associations between EGFR and COX-2 staining and clinicopathological characteristics were examined to predict survival. In a cohort of 32 resected PDAC patients, high EGFR protein expression in tumor cells was significantly associated with shorter median overall survival (7.9 vs. 39.2 months, P=0.0038). The corresponding hazard ratio (HR) for patients with high EGFR protein expression in tumor cells was 3.12 [95% confidence interval (CI): 1.39-7.00, P=0.006]. COX-2 protein expression was not associated with survival (22.6 vs. 24.5 months P=0.60; HR 1.22 95% CI: 0.59-2.51, P=0.60). Following multivariate Cox regression analysis, high EGFR protein expression in tumor cells (P=0.043) remained as significant independent prognostic factor for survival. In conclusion, high wild-type EGFR protein expression, but not COX-2 protein expression, in tumor cells is a prognostic factor for reduced overall survival following pancreatic tumor resection, supporting a role for EGFR in identifying resected patients that may benefit from EGFR-targeted therapy.

Overnight Steady-State Infusions of Parenteral Nutrition on Myosin Heavy Chain Transcripts in Rectus Abdominis Muscle Related to Amino Acid Transporters, Insulin-like Growth Factor 1, and Blood Amino Acids in Patients Aimed at Major Surgery.

BACKGROUND: Evaluation of improvements by nutrition support to severely ill patients requires sensitive methods to demonstrate activation of protein synthesis in various tissues from groups with a limited number of patients to be statistically efficient. This study examines effects of standard parenteral nutrition (PN) on abdominal muscle transcripts of amino acid (AA) transporters, myosin heavy chains (MHCs), and the insulin-like growth factor 1 and its receptor (IGF-1/IGF-1R) in patients aimed at major surgery. METHODS: Twenty-two randomized patients received steady-state PN (0.16 gN/kg/d, 30 kcal/kg/d) or saline infusions for 12 hours before operation. Blood samples and muscle biopsies were obtained at operation start. Muscle messenger RNA (mRNA) levels of AA transporters (solute carrier family members SNAT2, LAT1, LAT3, LAT4, TAUT, PAT1, CD98), IGF-1, IGF-1R, MHC isoforms (MHC1, MHC2A, MHC2X), and LAT3 protein were quantified and related to concentrations of AA, IGF-1, insulin, and metabolic substrates in blood. RESULTS: Muscle mRNA LAT3, LAT4, IGF-1R, and MHC2A increased by PN infusion, with correlations to specific AA transporters and MHC isoforms (P < .01-.05). TAUT and LAT3 correlated to slow (MHC1) and fast (MHC2A, MHC2X) isoforms (P < .001-.02). Muscle IGF-1 mRNA correlated to plasma essential AAs, whereas IGF-1R mRNA was related to LAT3, MHC2A, and serum IGF-1 (P < .001-.03). CONCLUSIONS: The results confirm that short-term preoperative PN activates transcription of AA transporters and myosin isoforms. Thus, combinations of methods on gene transcription and translation of muscle proteins can be applied to define efficient combinations of nutrition and hormones to catabolic patients in preoperative and postoperative settings.

Fractional uptake of circulating tumor cells into liver-lung compartments during curative resection of periampullary cancer.

Circulating tumor cells (CTCs) are able to predict outcome in patients with breast, colon and prostate cancer and appear to be promising biomarkers of pancreatic carcinoma. The aim of the present study was to demonstrate a statistically significant portal-arterial difference of CTCs during curative resection of periampullary cancer. A commercially available instrument (IsofluxR) was used to quantify blood content of CTC in 10 patients with periampullary cancer according to preoperative diagnostics. Portal and arterial blood samples (~8 ml each) were simultaneously collected intra-operatively following surgical dissection prior to division of the pancreas for tumor removal. Quantitative CTC analyses were performed according to standardized protocols for immune-magnetic enrichment of CTC. Flow cytometry was applied for qualitative evaluations of various CTC markers in 7 patients. There was a statistically significant difference in the number of CTCs collected in the portal blood [58±14 cells per 100 ml; mean ± standard error (SE)] vs. arterial blood [24±7 cells per 100 ml (SE), P<0.025]. A fractional uptake of ≥40% across liver and lung compartments of assumed malignant CTC was estimated to correspond to the appearance of ~410 tumor cells per minute during pancreatic resections based on estimated hepatic blood flow, measured tumor cell mass and tumor cell proliferation activity. Complications in the collection of portal blood were not observed. A significant uptake across liver or lung compartments of potentially malignant tumor CTCs from periampullary carcinoma may represent a model to capture, define and characterize cell clones with metastatic potential in liver and lung tissues following surgical resection.

Host knockout of E-prostanoid 2 receptors reduces tumor growth and causes major alterations of gene expression in prostaglandin E2-producing tumors.

Prostaglandin E2 (PGE2) is elevated in a variety of malignant tumors and has been shown to affect several hallmarks of cancer. Accordingly, the PGE2 receptor, E-prostanoid 2 (EP2), has been reported to be associated with patient survival and reduced tumor growth in EP2-knockout mice. Thus, the aim of the present study was to screen for major gene expression alterations in tumor tissue growing in EP2-knockout mice. EP2-knockout mice were bred and implanted with EP2 receptor-expressing and PGE2-producing epithelial-like tumors. Tumor tissue and plasma were collected and used for analyses with gene expression microarrays and multiplex enzyme-linked immunosorbent assays. Tumor growth, acute phase reactions/systemic inflammation and the expression of interleukin-6 were reduced in EP2-knockout tumor-bearing mice. Several hundreds of genes displayed major changes of expression in the tumor tissue when grown in EP2-knockout mice. Such gene alterations involved several different cellular functions, including stemness, migration and cell signaling. Besides gene expression, several long non-coding RNAs were downregulated in the tumors from the EP2-knockout mice. Overall, PGE2 signaling via host EP2 receptors affected a large number of different genes involved in tumor progression based on signaling between host stroma and tumor cells, which caused reduced tumor growth.

Preoperative overnight parenteral nutrition (TPN) improves skeletal muscle protein metabolism indicated by microarray algorithm analyses in a randomized trial.

Loss of muscle mass is associated with increased risk of morbidity and mortality in hospitalized patients. Uncertainties of treatment efficiency by short-term artificial nutrition remain, specifically improvement of protein balance in skeletal muscles. In this study, algorithmic microarray analysis was applied to map cellular changes related to muscle protein metabolism in human skeletal muscle tissue during provision of overnight preoperative total parenteral nutrition (TPN). Twenty-two patients (11/group) scheduled for upper GI surgery due to malignant or benign disease received a continuous peripheral all-in-one TPN infusion (30 kcal/kg/day, 0.16 gN/kg/day) or saline infusion for 12 h prior operation. Biopsies from the rectus abdominis muscle were taken at the start of operation for isolation of muscle RNA RNA expression microarray analyses were performed with Agilent Sureprint G3, 8 × 60K arrays using one-color labeling. 447 mRNAs were differently expressed between study and control patients (P < 0.1). mRNAs related to ribosomal biogenesis, mRNA processing, and translation were upregulated during overnight nutrition; particularly anabolic signaling S6K1 (P < 0.01-0.1). Transcripts of genes associated with lysosomal degradation showed consistently lower expression during TPN while mRNAs for ubiquitin-mediated degradation of proteins as well as transcripts related to intracellular signaling pathways, PI3 kinase/MAPkinase, were either increased or decreased. In conclusion, muscle mRNA alterations during overnight standard TPN infusions at constant rate altered mRNAs associated with mTOR signaling; increased initiation of protein translation; and suppressed autophagy/lysosomal degradation of proteins. This indicates that overnight preoperative parenteral nutrition is effective to promote muscle protein metabolism.

Presence of TSH receptors in discrete areas of the hypothalamus and caudal brainstem with relevance for feeding controls-Support for functional significance.

AIMS: Previous studies have shown that brain-derived thyroid-stimulating hormone (TSH) and its receptor (TSHr) are present in hypothalamic extracts. No studies investigating both the anatomical location and functional significance of putative TSHr proteins in specific central nervous system (CNS) nuclei involved in feeding controls have yet been conducted. The aim was thus to determine whether TSHr are present in nuclei associated with feeding behavior, and if such receptors may be functional. METHODS: Brain tissue from adult rats was analyzed for gene expression and receptor protein expression was investigated with immunohistochemistry and western blotting. To investigate whether putative TSHr may be functional, we evaluated food intake of rats given intraparenchymal nanoinjections of TSH into the nucleus of the solitary tract (NTS). RESULTS: RT-qPCR confirmed previous reports that TSHr mRNA is expressed in CNS tissues of the adult rat. Immunohistochemistry showed TSHr-immunoreactivity in the arcuate, the ventromedial, the dorsomedial, and the paraventricular hypothalamic nuclei. We also found TSHr-ir in the dorsal hindbrain to be localized to the area postrema, NTS, dorsal motor nucleus of the vagus, and the hypoglossal motor nucleus. Further protein analysis with western blotting showed 120kDa TSHr-ir proteins present in the hypothalamus and brainstem. Injections of TSH into the NTS reduced food intake similar to the positive control, urocortin. CONCLUSIONS: These data suggest that functional TSHr are present in the caudal brainstem and hypothalamic nuclei of relevance for feeding control as a possibly uncleaved holoreceptor, and highlights a hindbrain component to central TSH inhibition of food intake.

MCG101-induced cancer anorexia-cachexia features altered expression of hypothalamic Nucb2 and Cartpt and increased plasma levels of cocaine- and amphetamine-regulated transcript peptides.

The aim of the present study was to explore central and peripheral host responses to an anorexia-cachexia producing tumor. We focused on neuroendocrine anorexigenic signals in the hypothalamus, brainstem, pituitary and from the tumor per se. Expression of mRNA for corticotropin-releasing hormone (CRH), cocaine- and amphetamine-regulated transcript (CART), nesfatin-1, thyrotropin (TSH) and the TSH receptor were explored. In addition, we examined changes in plasma TSH, CART peptides (CARTp) and serum amyloid P component (SAP). C57BL/6 mice were implanted with MCG101 tumors or sham-treated. A sham-implanted, pair­fed (PF) group was included to delineate between primary tumor and secondary effects from reduced feeding. Food intake and body weight were measured daily. mRNA levels from microdissected mouse brain samples were assayed using qPCR, and plasma levels were determined using ELISA. MCG101 tumors expectedly induced anorexia and loss of body weight. Tumor-bearing (TB) mice exhibited an increase in nesfatin-1 mRNA as well as a decrease in CART mRNA in the paraventricular area (PVN). The CART mRNA response was secondary to reduced caloric intake whereas nesfatin-1 mRNA appeared to be tumor-specifically induced. In the pituitary, CART and TSH mRNA were upregulated in the TB and PF animals compared to the freely fed controls. Plasma levels for CARTp were significantly elevated in TB but not PF mice whereas levels of TSH were unaffected. The plasma CARTp response was correlated to the degree of inflammation represented by SAP. The increase in nesfatin-1 mRNA in the PVN highlights nesfatin-1 as a plausible candidate for causing tumor-induced anorexia. CART mRNA expression in the PVN is likely an adaptation to reduced caloric intake secondary to a cancer anorexia-cachexia syndrome (CACS)­inducing tumor. The MCG101 tumor did not express CART mRNA, thus the elevation of plasma CARTp is host derived and likely driven by inflammation.

Alterations in Tumor DNA Are Related to Short Postoperative Survival in Patients Resected for Pancreatic Carcinoma Aimed at Cure.

OBJECTIVES: Pancreatic ductal adenocarcinomas (PDACs) are found in more than 85% of patients with pancreatic cancer and with 5-year survival of less than 10%. Effective treatment may be radical surgery, which is hampered by rapid relapse. Therefore, our aim was to compare DNA sequence alterations in patients with short and long survival to evaluate if confirmed DNA alterations predict short postoperative survival. METHODS: DNA was extracted from tumor tissue from 59 PDAC patients, analyzed for KRAS mutations, and hybridized to 180 K CGH + SNP microarrays and 450 K methylation arrays. Analyses were based on postoperative survival where less than 12 months was considered to be short survival and more than 18 months was considered long survival. RESULTS: Ninety-three percent of the patients had KRAS mutations in tumor DNA. Great heterogeneity of whole genome DNA sequence alterations were observed among chromosomes within the patient materials. Specific DNA sequence alterations did not directly predict postoperative survival, although short survivors had significantly more and larger DNA amplifications (P < 0.006). Amplifications on chromosome 11 and 21 and deletions on chromosome 2 predicted short postoperative survival (P < 0.03). DNA methylation was not related to survival. CONCLUSIONS: Highly variable genetic differences among DNA regions in PDAC tumors were demonstrated. Postoperative short survival was related to tumor sequence DNA alterations on chromosome 2, 11, and 21.

Food intake, tumor growth, and weight loss in EP2 receptor subtype knockout mice bearing PGE2-producing tumors.

Previous studies in our laboratory have demonstrated that prostaglandin (PG) E2 is involved in anorexia/cachexia development in MCG 101 tumor-bearing mice. In the present study, we investigate the role of PGE receptor subtype EP2 in the development of anorexia after MCG 101 implantation in wild-type (EP2 (+/+)) or EP2-receptor knockout (EP2(-/-)) mice. Our results showed that host absence of EP2 receptors attenuated tumor growth and development of anorexia in tumor-bearing EP2 knockout mice compared to tumor-bearing wild-type animals. Microarray profiling of the hypothalamus revealed a relative twofold change in expression of around 35 genes including mRNA transcripts coding for Phospholipase A2 and Prostaglandin D2 synthase (Ptgds) in EP2 receptor knockout mice compared to wild-type mice. Prostaglandin D2 synthase levels were increased significantly in EP2 receptor knockouts, suggesting that improved food intake may depend on altered balance of prostaglandin production in hypothalamus since PGE2 and PGD2 display opposing effects in feeding control.

Circulating carnosine dipeptidase 1 associates with weight loss and poor prognosis in gastrointestinal cancer.

BACKGROUND: Cancer cachexia (CC) is linked to poor prognosis. Although the mechanisms promoting this condition are not known, several circulating proteins have been proposed to contribute. We analyzed the plasma proteome in cancer subjects in order to identify factors associated with cachexia. DESIGN/SUBJECTS: Plasma was obtained from a screening cohort of 59 patients, newly diagnosed with suspected gastrointestinal cancer, with (n = 32) or without (n = 27) cachexia. Samples were subjected to proteomic profiling using 760 antibodies (targeting 698 individual proteins) from the Human Protein Atlas project. The main findings were validated in a cohort of 93 patients with verified and advanced pancreas cancer. RESULTS: Only six proteins displayed differential plasma levels in the screening cohort. Among these, Carnosine Dipeptidase 1 (CNDP1) was confirmed by sandwich immunoassay to be lower in CC (p = 0.008). In both cohorts, low CNDP1 levels were associated with markers of poor prognosis including weight loss, malnutrition, lipid breakdown, low circulating albumin/IGF1 levels and poor quality of life. Eleven of the subjects in the discovery cohort were finally diagnosed with non-malignant disease but omitting these subjects from the analyses did not have any major influence on the results. CONCLUSIONS: In gastrointestinal cancer, reduced plasma levels of CNDP1 associate with signs of catabolism and poor outcome. These results, together with recently published data demonstrating lower circulating CNDP1 in subjects with glioblastoma and metastatic prostate cancer, suggest that CNDP1 may constitute a marker of aggressive cancer and CC.

Loss of muscle mass in the end of life in patients with advanced cancer.

PURPOSE: Muscle mass depletion is associated with adverse outcomes in cancer patients. There is limited information on the impact of age, sex, tumor type, and inflammation on muscle loss in the end of life of cancer patients. METHODS: Muscle depletion and loss of muscle in the last 2 years of life was estimated in 471 cancer patients from 779 dual-energy X-ray absorptiometry scans. A linear mixed model was used to estimate the impact of age, sex, tumor type, and inflammation. RESULTS: Patients above median age (>71 years) had less muscle mass (-1.1 ± 0.3 kg, P < 0.001). Prevalence of muscle depletion was higher in men than women (59 vs. 28%, P < 0.001). Men lost muscle mass over time (mean, 1.4 ± 0.3 kg/year, P < 0.001) contrary to women (0.3 ± 0.4 kg/year, P = 0.5). Patients with pancreatic cancer had less muscle mass than patients with biliary tract and colorectal cancers (P < 0.02). There were no differences in muscle loss over time in patients grouped by median age or tumor type. The prevalence of elevated C-reactive protein was 61 to 70% during the study. Patients with C-reactive protein >10 mg/L had less muscle mass (0.6 ± 0.2 kg, P < 0.001) and lost muscle mass at an accelerated pace during the disease trajectory (0.7 ± 0.3 kg/year, P = 0.03). CONCLUSIONS: Muscle loss in advanced cancer is related to age, sex, tumor type, and inflammation. The mechanism(s) behind the apparent sexual dimorphism warrants further study.

Expression of major histocompatibility complex class I-related chain A/B (MICA/B) in pancreatic carcinoma.

Major histocompatibility complex class I-related chain A and B (MICA/B) are two stress-inducible ligands that bind to the immunoreceptor NKG2D and play an important role in mediating cytotoxicity of NK and T cells. Release of MIC molecules from the cell surface is thought to constitute an immune escape mechanism of tumor cells and thus could be associated with more aggressive course of tumor growth. In this study, we investigated the expression of MICA/B in ductal pancreatic carcinoma and serum in relation to tumor stage, differentiation and survival. MICA/B expression in tumor tissues and sera from patients with pancreatic cancer were analyzed by immunohistochemical staining (IHC), western blotting and ELISA, respectively. MICA/B expression was present in 17 of 22 (77%) of the tumors but not in normal pancreatic ductal epithelial cells. Poorly differentiated tumors showed more pronounced MICA/B expression compared to differentiated tumors, but did not correlate significantly to other tumor characteristics. MICA/B-negative tumors displayed significantly lower incidence of lymph node metastases (p<0.01), and less mortality within 3 years following resection (p<0.02). In conclusion, tissue levels of MICA/B expression were elevated in pancreatic cancer cells without elevated levels in serum, despite well-recognized acute phase reactants in serum. Poorly differentiated tumors showed high MICA/B expression, which was related to extended tumor lymph node metastases and less frequent long-term survival.