The acute phase protein orosomucoid 1 is upregulated in early lactation but does not trigger appetite-suppressing STAT3 signaling via the leptin receptor.

Dairy cows consume inadequate amounts of feed in early lactation and during conditions and diseases such as excessive fatness, heat stress, and infectious diseases. Affected cows often experience increases in plasma concentrations of acute phase proteins consistent with the negative effect of inflammation on appetite. The acute phase protein orosomucoid 1 (ORM1), also known as alpha-1-acid glycoprotein, was recently reported to reduce appetite in the mouse through its ability to bind the full-length leptin receptor (Ob-Rb) and activate appetite-suppressing signal transducer and activator of transcription 3 (STAT3) signaling. These observations raise the possibility that ORM1 exerts appetite-suppressing effects in dairy cattle during periods of increased inflammatory tone. The applicability of this model was assessed in 2 ways. First, we asked whether ORM1 is regulated during periods of inadequate appetite such as the transition from late pregnancy to early lactation and periods of increased inflammatory tone. Plasma ORM1 was invariant in late pregnancy but increased 2.5-fold between parturition and d 7 of lactation. Gene expression studies showed that liver was the major source of this elevation with little contribution by adipose tissue or mammary gland. Additional studies showed that plasma ORM1 was not increased further by excessive fatness or by reproductive dysfunction in early lactation and was completely unresponsive to inflammatory stimuli such as heat stress or intravascular administration of the endotoxin lipopolysaccharide during established lactation. Second, we tested the ability of ORM1 to trigger STAT3 signaling through Ob-Rb using Chinese hamster ovary K1 (CHO-K1) cells transfected with a STAT3 expression plasmid. In this configuration, CHO-K1 cells did not express Ob-Rb and were incapable of leptin-induced STAT3 phosphorylation. Leptin responsiveness was conferred by co-transfecting with bovine Ob-Rb, with leptin causing increases of 5.7-fold in STAT3 phosphorylation and 2.1-fold in the expression of the STAT3-dependent gene, SOCS3. In contrast, neither bovine or human ORM1 triggered STAT3 phosphorylation irrespective of dose and period of incubation tested. In summary, bovine ORM1 is not increased during periods of increased inflammatory tone except in early lactation and is incapable of Ob-Rb-dependent STAT3 signaling. Overall, these data are inconsistent with ORM1 mediating the appetite-suppressing effects of inflammation in cattle through Ob-Rb.

ALK immunohistochemistry positive, FISH negative NSCLC is infrequent, but associated with impaired survival following treatment with crizotinib.

OBJECTIVE: Metastasized non-small cell lung cancer (NSCLC) with an anaplastic lymphoma kinase (ALK) rearrangement is usually sensitive to a range of ALK-tyrosine kinase inhibitors. ALK-positive NSCLC have been identified in pivotal phase III trials with fluorescence in situ hybridization (ALK FISH+). These tumors are also expressing the fusion product (ALK immunohistochemistry (IHC)+). However, discrepant cases occur, including ALK IHC + FISH-. The aim of this study was to collect ALK IHC + cases and compare within this group response to crizotinib treatment of ALK FISH + cases with ALK FISH- cases. MATERIALS AND METHODS: In this European prospective multicenter research study patients with Stage IV ALK IHC + NSCLC treated with crizotinib were enrolled. Tumor slides were validated centrally for ALK IHC and ALK FISH. RESULTS: Registration of 3523 ALK IHC tests revealed a prevalence of 2.7% (n = 94) ALK IHC + cases. Local ALK FISH analysis resulted in 48 concordant (ALK IHC+/FISH+) and 16 discordant (ALK IHC+/FISH-) cases. Central validation revealed 37 concordant and 7 discordant cases, 5 of which had follow-up. Validation was hampered by limited amount of tissue in biopsy samples. The PFS at 1 year for ALK concordant and discordant was 58% and 20%, respectively (HR = 2.4; 95% CI: 0.78-7.3; p = 0.11). Overall survival was significantly better for concordant cases than discordant cases after central validation (HR=4.5; 95% CI= 1.2-15.9; p=0.010. CONCLUSION: ALK IHC + FISH- NSCLC is infrequent and associated with a worse outcome on personalized treatment. A suitable predictive testing strategy may be to screen first with IHC and then confirm with FISH instead of considering ALK IHC equivalent to ALK FISH according to the current guidelines.

Effects of body condition score on direct and indirect measurements of insulin sensitivity in periparturient dairy cows.

Reductions in insulin sensitivity in periparturient dairy cows develop as a means to support lactation; however, excessive mobilization of fatty acids (FA) increases the risk for peripartal metabolic disorders. Our objectives were to investigate the effect of prepartum body condition score (BCS) on systemic glucose and insulin tolerance, and to compare direct and indirect measurements of insulin sensitivity in peripartal lean and overweight dairy cows. Fourteen multiparous Holstein cows were allocated into two groups according to their BCS at day -28 prepartum: lean (n = 7; BCS ≤ 3.0) or overweight; (n = 7; BCS ≥ 4.0). Liver biopsies were performed on day -27, -14 and 4, relative to expected parturition. Intravenous insulin or glucose tolerances tests were performed following each liver biopsy. Relative to lean cows, overweight cows exhibited lower dry matter intake, lost more BCS and displayed increased plasma FA and ß-hydroxybutyrate concentrations and elevated liver lipid content during peripartum. Glucose clearance rate was lower for all cows postpartum. Prepartum BCS had minimal effects on insulin and glucose tolerance; however, the ability of the cow to restore blood glucose levels following an insulin challenge was suppressed by increased BCS. Glucose-dependent parameters of insulin and glucose tolerance were not correlated with surrogate indices of insulin sensitivity. We conclude that prepartum BCS had minimal effect on systemic insulin sensitivity following parturition. The observed inconsistency between surrogate indices of insulin sensitivity and direct measurements of insulin and glucose tolerance adds support to growing concerns regarding their usefulness as tools to estimate systemic insulin action in periparturient cows.

Methyl donor supplementation suppresses the progression of liver lipid accumulation while modifying the plasma triacylglycerol lipidome in periparturient Holstein dairy cows.

Co-supplementation of methyl donors may lower hepatic lipid content in transition cows. To define the ability of methyl donor supplementation (MDS) to reduce hepatic lipid content and modify the plasma lipidome, 30 multiparous Holstein cows (2.04 ± 0.69 lactations; 689 ± 58 kg of body weight; 3.48 ± 0.10 units of body condition score) were fed a ration with or without rumen-protected methyl donors (22 g/d of Met, 10 g/d of choline chloride, 3 g/d of betaine, 96 mg/d of riboflavin, and 1.4 mg/d of vitamin B12) from d -28 before expected calving through d 14 postpartum. Cows were randomly enrolled based on predefined selection criteria (body condition score and parity). Base diets without MDS were formulated for gestation (15.4% crude protein with a predicted Lys-to-Met ratio of 3.25; 1.44 Mcal of net energy for lactation/kg of dry matter) and lactation (16.6% crude protein with a predicted Lys-to-Met ratio of 3.36; 1.64 Mcal of net energy for lactation/kg of dry matter). Blood sampling occurred from d -28 relative to expected calving through d 14 postpartum. Liver tissue was biopsied at d -28 relative to expected calving and on d 5 and 14 postpartum. In addition to routine analyses, serum AA concentrations on d 10 and 12 were quantified using mass spectrometry. Plasma triacylglycerol (TAG) and cholesteryl esters (CE) were qualitatively measured using time-of-flight mass spectrometry. Data were analyzed using a mixed model with repeated measures. Dry matter intake and milk yield were not modified by MDS. The transition from d -28 relative to expected parturition to d 14 postpartum was characterized by increased plasma fatty acid (0.15 to 0.71 mmol/L) and ß-hydroxybutyrate (0.34 to 0.43 mmol/L) levels and liver lipid content (3.91 to 9.16%). Methyl donor supplementation increased the serum Met level by 26% and decreased the serum Lys-to-Met ratio by 21% on d 10 and 12, respectively. Moreover, the increase in hepatic lipid content from d 5 through 14 postpartum was suppressed with MDS relative to control (3.57 vs. -0.29%). Dietary MDS modified the TAG and CE lipidome. For example, MDS increased plasma TAG 46:3 (carbon number:double bond) by 116% relative to control cows on d 5 postpartum. Moreover, MDS tended to increase plasma CE 34:6. In contrast, MDS lowered plasma TAG 54:8 by 39% relative to control cows on d 5 postpartum. We concluded that in the absence of gains in dry matter intake and milk and milk protein yields, dietary MDS slows the progression of hepatic lipid accumulation and modifies the plasma TAG lipidome in transition cows.

Multi-centre prospective study on diagnosing subtypes of lung cancer by exhaled-breath analysis.

OBJECTIVES: Lung cancer is a leading cause of mortality. Exhaled-breath analysis of volatile organic compounds (VOC's) might detect lung cancer early in the course of the disease, which may improve outcomes. Subtyping lung cancers could be helpful in further clinical decisions. MATERIALS AND METHODS: In a prospective, multi-centre study, using 10 electronic nose devices, 144 subjects diagnosed with NSCLC and 146 healthy subjects, including subjects considered negative for NSCLC after investigation, breathed into the Aeonose™ (The eNose Company, Zutphen, Netherlands). Also, analyses into subtypes of NSCLC, such as adenocarcinoma (AC) and squamous cell carcinoma (SCC), and analyses of patients with small cell lung cancer (SCLC) were performed. RESULTS: Choosing a cut-off point to predominantly rule out cancer resulted for NSCLC in a sensitivity of 94.4%, a specificity of 32.9%, a positive predictive value of 58.1%, a negative predictive value (NPV) of 85.7%, and an area under the curve (AUC) of 0.76. For AC sensitivity, PPV, NPV, and AUC were 81.5%, 56.4%, 79.5%, and 0.74, respectively, while for SCC these numbers were 80.8%, 45.7%, 93.0%, and 0.77, respectively. SCLC could be ruled out with a sensitivity of 88.9% and an NPV of 96.8% with an AUC of 0.86. CONCLUSION: Electronic nose technology with the Aeonose™ can play an important role in rapidly excluding lung cancer due to the high negative predictive value for various, but not all types of lung cancer. Patients showing positive breath tests should still be subjected to further diagnostic testing.

Effects of limonene on ruminal concentrations, fermentation, and lysine degradation in cattle.

Previous in vitro data showed that was inhibited by limonene. We further evaluated effects of limonene on growth of in vitro as well as on ruminal concentrations of in vivo. With in vitro cultivation in anaerobic brain-heart infusion broth, limonene decreased growth of . Thymol also reduced growth of , but it was less effective than limonene. Tylosin effectively reduced growth of in vitro. Although the response over fermentation times and concentrations of antimicrobials differed somewhat between tylosin and limonene, the 2 antimicrobial agents yielded similar inhibitory effects on growth of at concentrations ranging from 6 to 24 mg/L. The effects of limonene on ruminal concentration in vivo were tested in 7 ruminally cannulated heifers (225 kg initial BW) used in a 7 × 4 Youden square design. Treatments included: 1) control, 2) limonene at 10 mg/kg diet DM, 3) limonene at 20 mg/kg diet DM, 4) limonene at 40 mg/kg diet DM, 5) limonene at 80 mg/kg diet DM, 6) CRINA-L (a blend of essential oil components) at 180 mg/kg diet DM, and 7) tylosin at 12 mg/kg diet DM. Each period included 11 d with 10 d washouts between periods. Samples of ruminal contents were collected before treatment initiation and after 4, 7, and 10 d of treatment for measuring by the most probable number method using selective culture medium. Limonene linearly decreased ( = 0.03) ruminal concentration, with the lowest concentration achieved with 40 mg of limonene/kg dietary DM. Limonene tended ( ≤ 0.07) to linearly reduce ruminal molar proportions of propionate and valerate while tending to linearly increase ( ≤ 0.10) those of butyrate and 2-methyl butyrate. Limonene did not affect ruminal NH concentrations or degradation rates of lysine. Neither CRINA-L ( = 0.52) nor tylosin ( = 0.19) affected ruminal concentrations. CRINA-L significantly decreased ruminal concentrations of NH and molar proportions of 3-methyl butyrate, whereas tylosin significantly decreased molar proportions of propionate while increasing those of butyrate and tending to increase those of acetate. Limonene supplementation reduced ruminal concentrations of suggesting that it may have the potential to reduce the prevalence of liver abscesses, although further research is needed to assess the effect of limonene in feedlot cattle.

Different occurrence of CD8+, CD45R0+, and CD68+ immune cells in regional lymph node metastases from colorectal cancer as potential prognostic predictors.

BACKGROUND AND AIMS: To study whether there are differences in the immunohistochemical staining of CD8, CD45R0, and CD68 of immune cells in regional lymph node metastases from colorectal cancer that are of potential interest in prognostic prediction. MATERIALS AND METHODS: Analysis of archival specimens from 93 patients operated on for colorectal cancer (based on monoclonal antibodies, the ABC technique, and semiquantitative classification). RESULTS: There was a significant difference in survival time between patients with respect to the number of positive immune cells. The cancer-specific 5-year survival rate was 77% for patients with high numbers of CD8+ cells, compared to 33% for those with lower numbers. The corresponding figures for patients with CD45R0+ cells were 66% vs. 33%, and for patients with CD68+ cells 60% vs. 38%. Significant differences remained among the 74 patients without adjuvant radio/chemotherapy regarding CD8 and CD45R0 but not CD68. CONCLUSION: The presence of CD8+, CD45R0+, and CD68+ immune cells in regional lymph node metastases may serve as predictors of patients survival in colorectal cancer Dukes' stage C.

The stimulatory effect of L-glutamate and related agents on inositol 1,4,5-trisphosphate production in the cestode Hymenolepis diminuta.

The effects of L-glutamate, acetylcholine, and serotonin (5HT) were examined on generation of inositol 1,4,5-triphosphate [Ins(1,4,5)P3], in membrane preparations of the cestode Hymenolepis diminuta. Only L-glutamate and acetylcholine stimulated a significant elevation in Ins(1,4,5)P3. The response to L-glutamate was stereospecific; D-glutamate or L-aspartate were not as potent. A role for G-protein(s) was supported by the observations that sodium fluoride stimulated Ins(1,4,5)P3 generation, and the L-glutamate response was potentiated by GTP and GTP-S and was suppressed by GDPS. However, studies with pertussis and cholera toxins indicated that the putative G-protein(s) was not pertussis or cholera toxin sensitive. The pharmacological profile of the L-glutamate response was examined partially. Trans-ACPD was a very effective agonist at 10(-5)M. While 10(-3)M L-glutamate, NMDA, and AMPA significantly elevated Ins(1,4,5)P3 levels, quisqualate and kainate did not. The elevation of Ins(1,4,5)P3 levels by L-glutamate and NMDA was antagonized by the specific glutamatergic antagonists AP-5, AP-7, CNQX, and CPP. While the response to ACPD was antagonized by AP5, CPP and CPG, CNQX was without effect. Collectively, the data support the hypothesis that in the cestode H. diminuta, L-glutamate activation of a metabotropic (ACPD) and/or ionotropic-like AMPA/NMDA receptor subtypes proceeds via a G protein(s) to enhance phospholipase C activity, ultimately resulting in the elevation of Ins(1,4,5)P3 levels in the tissues.

Acetylcholine-like immunoreactivity in the cestode Hymenolepis diminuta.

The localization of acetylcholine in tissues of the cestode Hymenolepis diminuta was determined, following derivatization, using an antibody raised against choline-glutaraldehyde-protein. Specific immunoreactivity was observed in the rostellum and beneath the suckers of the scolex, in the longitudinal nerve cords, in cells adjacent to some deep longitudinal muscles, in the genital primordium, in the wall of the cirrus sac, and in the external and internal seminal vesicle. The distribution of acetylcholine-like immunoreactivity in relation to that of serotonin and glutamate, and the distribution of acetylcholinesterase in H. diminuta is discussed.