DNA Mismatch Repair-Deficient Colorectal Carcinoma: Referral Rate for Genetic Cancer Risk Assessment in a Brazilian Cancer Center.

BACKGROUND: Approximately 15% of colorectal cancers (CRCs) are deficient in DNA mismatch repair proteins (dMMR), a characteristic that can occur in both sporadic and hereditary CRC. Due to sparse studies on dMMR CRC in the Brazilian population, we conducted a retrospective analysis of referral rates for Genetic Cancer Risk Assessment of this population and also describing clinical and molecular characterization of these tumors. METHODS: A retrospective, longitudinal, and unicenter study that included patients with dMMR CRC detected by IHC analysis from Pathology Database of our institution, from January 2015 to July 2017. RESULTS: MMR IHC testing was performed in 998 CRC tumors, and 78 tumors (7.8%) had dMMR. The mean age at diagnosis was 56.8 years (17-90), and most patients were female (41 out of 78, 52.6%). Of the 52 patients with right-sided CRC, 40 tumors (77%) had loss of the MLH1 and/or PMS2 expression, and 12 tumors (23%) had loss of MSH2 and/or MSH6 expression (p = 0.005). From 78 patients with dMMR CRC, only 43 patients (55.1%) were referred for genetic counseling (GC), and of them, only 33 patients (76.7%) really went to GC consultation. A total of 21 patients with dMMR CRC performed genetic testing. CONCLUSION: Overall, genetic referral was less than expected in our population. Most of dMMR CRC patients did not receive GC, even in a cancer center, either due to the absence of referral or personal decision and few patients who pursued genetic counseling performed genetic testing.

Contrasting Responses of Wing Morphology of Three Blowfly (Diptera: Calliphoridae) Species to Competition.

Competition influences the expression of morphological, physiological, and behavioral traits and also regulates ecological and evolutionary dynamics. This study aims to identify and characterize changes in wing morphology in response to intra- and interspecific competition in three necrophagous blowfly species. Using geometric morphometry, we analyzed 3,238 wings from Lucilia sericata (Meigen, 1826), Calliphora vicina Robineau-Desvoidy, 1830, and C. vomitoria (Linnaeus, 1758) raised under cloistered and pairwise conditions. The three species reacted similarly to intraspecific competition-reducing wing size with increased competition-but displayed contrasting patterns of response to interspecific competition. Lucilia sericata displayed a directional change in wing shape in response to an interspecific competitor, while C. vicina increased the scattering of individuals across the morphospace, and C. vomitoria displayed no significant change in response to the same stimulus. Our results show that the same stimulus yields distinctive responses; thus, different competition-related strategies are expected to occur in the three species.

Diabetes hinders community-acquired pneumonia outcomes in hospitalized patients.

OBJECTIVES: This study aimed to estimate the prevalence of diabetes mellitus (DM) in hospitalized patients with community-acquired pneumonia (CAP) and its impact on hospital length of stay and in-hospital mortality. RESEARCH DESIGN AND METHODS: We carried out a retrospective, nationwide register analysis of CAP in adult patients admitted to Portuguese hospitals between 2009 and 2012. Anonymous data from 157 291 adult patients with CAP were extracted from the National Hospital Discharge Database and we performed a DM-conditioned analysis stratified by age, sex and year of hospitalization. RESULTS: The 74 175 CAP episodes that matched the inclusion criteria showed a high burden of DM that tended to increase over time, from 23.7% in 2009 to 28.1% in 2012. Interestingly, patients with CAP had high DM prevalence in the context of the national DM prevalence. Episodes of CAP in patients with DM had on average 0.8 days longer hospital stay as compared to patients without DM (p<0.0001), totaling a surplus of 15 370 days of stay attributable to DM in 19 212 admissions. In-hospital mortality was also significantly higher in patients with CAP who have DM (15.2%) versus those who have DM (13.5%) (p=0.002). CONCLUSIONS: Our analysis revealed that DM prevalence was significantly increased within CAP hospital admissions, reinforcing other studies' findings that suggest that DM is a risk factor for CAP. Since patients with CAP who have DM have longer hospitalization time and higher mortality rates, these results hold informative value for patient guidance and healthcare strategies.

Regulation of Anti-Müllerian Hormone and Its Receptor Expression around Follicle Deviation in Cattle.

The anti-Müllerian hormone (AMH) is an important marker of ovarian reserve and for predicting the response to superovulatory treatments in several species. The objective of this study was to investigate whether AMH and its receptor (AMHR2) are regulated in bovine granulosa cells during follicular development. In the first experiment, granulosa cells were retrieved from the two largest follicles on days 2 (before), 3 (at the expected time) or 4 (after deviation) of follicular wave. In the second experiment, four doses of FSH (30, 30, 20 and 20 mg) or saline were administered twice a day starting on Day 2 of the first follicular wave of the cycle. Granulosa cells and follicular fluid were collected from the two largest follicles 12 h after the last injection of FSH or saline. AMH mRNA abundance was similar in granulosa cells of the two largest follicles (F1 and F2) before deviation (Day 2), but greater in dominant (DF) than subordinate follicles (SF) at the expected time (Day 3) and after (Day 4) deviation (p < 0.05). In experiment 1, AMH mRNA levels declined in both DF and SF near the expected time and after deviation when compared to before deviation. There was no difference in AMHR2 mRNA levels before and during follicular deviation (p > 0.05), but they tended to be greater in DFs than SFs (p < 0.1) after deviation. Experiment 2 showed that AMH and AMHR2 mRNA in granulosa cells and AMH protein abundance in follicular fluid were similar (p > 0.05) between both co-dominant follicles collected from the FSH-treated cows. These findings indicate the followings: AMH mRNA levels decrease in both DFs and SFs during follicular deviation; granulosa cells from heathy follicles express more AMH mRNA compared to subordinate follicles undergoing atresia and FSH stimulates AMH and AMHR2 mRNA expression in granulosa cells of co-dominant follicles.

Systemic inhibition of nitric oxide synthesis in non-diabetic individuals produces a significant deterioration in glucose tolerance by increasing insulin clearance and inhibiting insulin secretion.

AIMS/HYPOTHESIS: Endogenous NO inhibits insulin release in isolated beta cells and insulin-degrading enzyme activity in hepatocytes, while NO release from endothelial cells has been suggested to enhance insulin action. We assessed the overall effect of systemic inhibition of endogenous NO synthesis on glucose homeostasis in humans. METHODS: Twenty-four non-diabetic volunteers underwent two hyperglycaemic (+7 mmol/l) clamps with either saline or L-NG-nitroarginine methyl ester (L-NAME, at rates of 2.5, 5, 10 and 20 µg min⁻¹ kg⁻¹) infusion. Another five volunteers underwent an OGTT with either saline or L-NAME (20 µg min⁻¹ kg⁻¹) infusion. Blood pressure and heart rate were measured to monitor NO blockade; during the OGTT, endothelial function was assessed by peripheral arterial tonometry and insulin secretion by C-peptide deconvolution and insulin secretion modelling. RESULTS: Compared with saline, L-NAME at the highest dose raised mean blood pressure (+20 ± 2 mmHg), depressed heart rate (-12 ± 2 bpm) and increased insulin clearance (+50%). First-phase insulin secretion was impaired, but insulin sensitivity (M/I index) was unchanged. During the OGTT, L-NAME raised 2 h plasma glucose by 1.8 mmol/l (p < 0.01), doubled insulin clearance and impaired beta cell glucose sensitivity while depressing endothelial function. CONCLUSIONS/INTERPRETATION: In humans, systemic NO blockade titrated to increase blood pressure and induce endothelial dysfunction does not affect insulin action but significantly impairs glucose tolerance by increasing plasma insulin clearance and depressing insulin secretion, namely first-phase and beta cell glucose sensitivity.

Postprandial insulin resistance in Zucker diabetic fatty rats is associated with parasympathetic-nitric oxide axis deficiencies.

The Zucker diabetic fatty (ZDF) rat is an obesity and type 2 diabetes model. Progression to diabetes is well characterised in ZDF rats, but only in the fasted state. We evaluated the mechanisms underlying postprandial insulin resistance in young ZDF rats. We tested the hypothesis that the overall postprandial action of insulin is affected in ZDF rats as a result of impairment of the hepatic parasympathetic-nitric oxide (PSN-NO) axis and/or glutathione (GSH), resulting in decreased indirect (PSN-NO axis) and direct actions of insulin. Nine-week-old male ZDF rats and lean Zucker rats (LZR, controls) were used. The action of insulin was assessed in the fed state before and after parasympathetic antagonism atropine. Basal hepatic NO and GSH were measured, as well as NO synthase (NOS) and γ-glutamyl-cysteine synthethase (GCS) activity and expression. ZDF rats presented postprandial hyperglycaemia (ZDF, 201.4 ± 12.9 mg/dl; LZR, 107.7 ± 4.3 mg/dl), but not insulinopaenia (ZDF, 5.9 ± 0.8 ng/ml; LZR, 1.5 ± 0.3 ng/ml). Total postprandial insulin resistance was observed (ZDF, 78.6 ± 7.5 mg glucose/kg; LZR, 289.2 ± 24.7 mg glucose/kg), with a decrease in both the direct action of insulin (ZDF, 54.8 ± 7.0 mg glucose/kg; LZR, 173.3 ± 20.5 mg glucose/kg) and the PSN-NO axis (ZDF, 24.5 ± 3.9 mg glucose/kg; LZR, 115.9 ± 19.4 mg glucose/kg). Hepatic NO (ZDF, 117.2 ± 11.4 µmol/g tissue; LZR, 164.6 ± 4.9 µmol/g tissue) and GSH (ZDF, 4.9 ± 0.3 µmol/g; LZR, 5.9 ± 0.2 µmol/g) were also compromised as a result of decreased NOS and GCS activity, respectively. These results suggest a compromise of the mechanism responsible for potentiating insulin action after a meal in ZDF rats. We show that defective PSN-NO axis and GSH synthesis, together with an impaired direct action of insulin, appears to contribute to postprandial insulin resistance in this model.

Understanding postprandial glucose clearance by peripheral organs: the role of the hepatic parasympathetic system.

The hepatic parasympathetic system is one of the major contributors for preserving insulin sensitivity in the postprandial state. Postprandial hepatic vagal control of whole-body glucose clearance and its effect on specific organs remains unknown. Our hypothesis is that, in the postprandial state, the hepatic parasympathetic nerves (HPN) are responsible for a considerable part of extra-hepatic tissue glucose clearance. Two groups of 9-week-old Sprague-Dawley rats were studied, comparing sham-operated versus hepatic parasympathetic denervated animals. Insulin sensitivity was evaluated in the postprandial state by the rapid insulin sensitivity test (RIST). [(3) H]2-deoxy-d-glucose was administered during the RIST. Plasma glucose rate of the disappearance and clearance by skeletal muscle, adipose tissue, liver, pancreas, heart and kidney of this radioisotope was measured. The postprandial denervated group showed a decrease insulin sensitivity of 41.4 ± 5.2%. This group of animals showed a decrease in the rate of plasma [(3) H]2-deoxy-d-glucose disappearance and skeletal muscle, heart and kidney glucose clearance by 45%, 35% and 67%, respectively. These studies show that the major contributor of postprandial whole-body glucose clearance was skeletal muscle; in the range 69-38%, depending on HPN integrity. The results obtained in the present study indicate that HPN are crucial for postprandial action of insulin through a mechanism that is essential for maintenance of skeletal muscle, heart and kidney glucose clearance. These results suggest that hepatic parasympathetic dysfunction could lie at the genesis of type 2 diabetes complications, namely insulin resistance, nephropathy and cardiomyopathy.

Insulin resistance induced by sucrose feeding in rats is due to an impairment of the hepatic parasympathetic nerves.

AIMS/HYPOTHESIS: A considerable proportion of whole-body insulin-stimulated glucose uptake is dependent upon the hepatic insulin-sensitising substance (HISS) in a pathway mediated by the hepatic parasympathetic nerves (HPNs). We tested the hypothesis that a high-sucrose diet leads to the impairment of the HPN-dependent component of insulin action. METHODS: We quantified insulin sensitivity using the rapid insulin sensitivity test, a modified euglycaemic clamp. Quantification of the HPN-dependent component was achieved by administration of a muscarinic receptor antagonist (atropine, 3 mg/kg). RESULTS: Insulin sensitivity was higher in standard-fed than in sucrose-fed Wistar rats (305.6+/-34.1 vs 193.9+/-13.7 mg glucose/kg body weight; p<0.005) and Sprague-Dawley rats (196.4+/-5.9 vs 95.5+/-16.3 mg glucose/kg body weight; p<0.01). The HPN-independent component was similar in the two diet groups. Insulin resistance was entirely due to an impairment of the HPN-dependent component in both Wistar rats (164.3+/-28.1 [standard-fed] vs 26.5+/-7.5 [sucrose-fed] mg glucose/kg body weight; p<0.0001) and Sprague-Dawley rats (111.7+/-9.5 vs 35.3+/-21.4 mg glucose/kg body weight; p<0.01). Furthermore, HPN-dependent insulin resistance in Sprague-Dawley rats was already evident after 2 weeks of a high-sucrose diet (28.5+/-7.6 [2 weeks], 35.3+/-21.4 [6 weeks], 17.9+/-5.4 [9 weeks] mg glucose/kg body weight) and was independent of the nature of sucrose supplementation (12.3+/-4.7 [solid] and 17.9+/-5.4 [liquid] mg glucose/kg body weight). CONCLUSIONS/INTERPRETATION: Our results support the hypothesis that insulin resistance caused by sucrose feeding is due to an impairment of the HPN-dependent component of insulin action, leading to a dysfunction of the HISS pathway.

Hepatic parasympathetic (HISS) control of insulin sensitivity determined by feeding and fasting.

In response to insulin, a hormone [hepatic insulin sensitizing substance (HISS)] is released from the liver to stimulate glucose uptake in skeletal muscle but not liver or gut. The aim was to characterize dynamic control of HISS action in response to insulin and regulation of release by hepatic parasympathetic nerves. Insulin action was assessed by the rapid insulin sensitivity test, where the index is the glucose required (mg/kg) to maintain euglycemia after a bolus of insulin. Blocking HISS release by interruption of the hepatic parasympathetic nerves by surgical denervation, atropine, or blockade of hepatic nitric oxide synthase produced similar degrees of insulin resistance and revealed a similar dynamic pattern of hormone action that began 3--4 min after, and continued for 9--10 min beyond, insulin action (50 mU/kg). HISS action accounted for 56.5 +/- 3.5% of insulin action at insulin doses from 5 to 100 mU/kg (fed). We also tested the hypothesis that HISS release is controlled by the feed/fast status. Feeding resulted in maximal HISS action, which decreased progressively with the duration of fasting.

Tonic activation of A(2A) adenosine receptors unmasks, and of A(1) receptors prevents, a facilitatory action of calcitonin gene-related peptide in the rat hippocampus.

1. We investigated how manipulations of the degree of activation of adenosine A(1) and A(2A) receptors influences the action of the neuropeptide, calcitonin gene-related peptide (CGRP) on synaptic transmission in hippocampal slices. Field excitatory post-synaptic potentials (EPSPs) from the CA1 area were recorded. 2. When applied alone, CGRP (1 - 30 nM) was without effect on field EPSPs. However, CGRP (10 - 30 nM) significantly increased the field EPSP slope when applied to hippocampal slices in the presence of the A(1) receptor antagonist, 1,3-dipropyl-8-cyclopenthyl xanthine (DPCPX, 10 nM), or in the presence of the A(2A) adenosine receptor agonist CGS 21680 (10 nM). 3. The A(2A) receptor antagonist, ZM 241385 (10 nM) as well as adenosine deaminase (ADA, 2 U ml(-1)), prevented the enhancement of field EPSP slope caused by CGRP (30 nM) in the presence of DPCPX (10 nM), suggesting that this effect of CGRP requires the concomitant activation of A(2A) adenosine receptors by endogenous adenosine. 4. The protein kinase-A inhibitors, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 10 microM) and adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS, 50 microM), as well as the inhibitor of ATP-sensitive potassium (K(ATP)) channels, glibenclamide (30 microM), prevented the facilitation of synaptic transmission caused by CGRP (30 nM) in the presence of DPCPX (10 nM), suggesting that this effect of CGRP involves both K(ATP) channels and protein kinase-A. 5. It is concluded that the ability of CGRP to facilitate synaptic transmission in the CA1 area of the hippocampus is under tight control by adenosine, with tonic A(1) receptor activation by endogenous adenosine 'braking' the action of CGRP, and the A(2A) receptors triggering this action.

Shear-induced modulation of vasoconstriction in the hepatic artery and portal vein by nitric oxide.

The effect of shear stress on nitric oxide (NO)-mediated suppression of sympathetic nerve (2-6 Hz)- and norepinephrine (0.5 microgram.kg-1.min-1)-induced vasoconstriction in the hepatic artery (HA) and portal vein (PV) was studied using a perfusion circuit to regulate blood pressure and flow in the cat liver in situ. Holding flow constant resulted in increased shear stress during constriction; holding pressure steady prevented changes in shear stress. When shear stress was allowed to rise, the vasoconstriction (indicated by elevation in perfusion pressure) in response to nerve stimulation and norepinephrine was significantly potentiated after NO synthase blockade using NG-nitro-L-arginine methyl ester (L-NAME, 2.5 mg/kg iv) in both the HA and PV (response to nerves: HA control 28.8 +/- 6.5 mmHg, L-NAME 62.7 +/- 14.6 mmHg; PV control 1.5 +/- 0.5 mmHg, L-NAME 3.3 +/- 0.5 mmHg; response to norepinephrine: HA control 32.4 +/- 9.0 mmHg, L-NAME 60.3 +/- 8.0 mmHg; PV control 1.3 +/- 0.3 mmHg, L-NAME 3.4 +/- 0.7 mmHg). The potentiation was reversed by L-arginine (75 mg/kg). When shear stress was held constant by maintaining constant perfusion pressure, L-NAME did not cause potentiation of vasoconstriction. The data are consistent with the hypothesis that elevated shear stress in the hepatic blood vessels leads to NO-dependent postjunctional modulation of vasoconstriction.

Rapid insulin sensitivity test (RIST).

A rapid insulin sensitivity test (RIST) was recently introduced to assess insulin action in vivo (H. Xie, L. Zhu, Y.L. Zhang, D.J. Legare, and W.W. Lautt. J. Pharmacol. Toxicol. Methods, 35: 77-82. 1996). This technical report describes the current recommended standard operating procedure for the use of the RIST in rats based upon additional experience with approximately 100 tests. We describe the manufacture and use of an arterial-venous shunt that allows rapid multiple arterial samples and intravenous administration of drugs. The RIST procedure involves determination of a stable arterial glucose baseline to define the ideal euglycemic level to be maintained following a 5-min infusion of insulin, with the RIST index being the amount of glucose required to be infused to maintain euglycemia over the test period. Insulin administration by a 5-min infusion is preferable to a 30-s bolus administration. No significant difference was determined between the use of Toronto pork-beef or human insulin. Four consecutive RISTs were carried out in the same animal over 4-5 h with no tendency for change with time. The RIST index is sufficiently sensitive and reproducible to permit establishment of insulin dose-response curves and interference of insulin action by elimination of hepatic parasympathetic nerves, using atropine. This technical report provides the current recommended standard operating procedure for the RIST.

Potentiation to vasodilators by nitric oxide synthase blockade in superior mesenteric but not hepatic artery.

Our objective was to determine the vasodilator effect of adenosine and isoproterenol on the hepatic artery (HA) and superior mesenteric artery (SMA) before and after blockade of nitric oxide (NO) production to evaluate the possibility of organ specificity. Vascular circuits supplied blood flow to the liver or intestine in cats under pentobarbital sodium anesthesia. The NO synthase (NOS) antagonist N(G)-nitro-L-arginine methyl ester (L-NAME; 2.5 mg/kg iv) increased arterial pressure from 106.4 +/- 7.6 to 141.4 +/- 8.1 mmHg and raised basal vascular tone in the SMA but not in the HA. The NOS substrate L-arginine (75 mg/kg) reversed these effects. The decrease in perfusion pressure in response to adenosine was 51.7 +/- 2.9, 135.2 +/- 6.1, and 16.7 +/- 2.4 mmHg, respectively, for control and after L-NAME and L-arginine. Isoproterenol was also potentiated in the SMA. Adenosine and isoproterenol were not potentiated in the HA by L-NAME. Potentiation did not occur when HA or SMA basal tone was elevated by norepinephrine. In conclusion, L-NAME increased basal tone for the SMA and potentiated the dilation induced by adenosine and isoproterenol in the SMA but not in the HA. This study provides evidence that there is a highly organ-specific compensatory mechanism in which the absence of NO promotes potentiation of other vasodilators.

Autoregulatory capacity in the superior mesenteric artery is attenuated by nitric oxide.

The hypothesis that nitric oxide antagonizes pressure-flow autoregulation in the superior mesenteric artery was tested with the use of a nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), and L-arginine. Autoregulation was assessed with the use of two indexes: 1) the autoregulatory index (ARI), expressed as the ratio of change in flow to change in pressure tested over the nonautoregulatory range, 40-70 mmHg, and over the autoregulatory range, 70-140 mmHg; and 2) the slope index, an index of linearity of the pressure-flow curve calculated by dividing the slope of the pressure-flow curve over the autoregulatory range by the slope of the curve over the nonautoregulatory range, expressed as percent. L-NAME significantly increased autoregulation from an ARI of 0.06 +/- 0.05 to 0.28 +/- 0.09 over the autoregulatory range (P < 0.003), and the nitric oxide synthase substrate L-arginine reversed ARI to 0.12 +/- 0.07 (mean +/- SE, n = 7). ARI in the nonautoregulatory range was not altered. The slope index revealed that L-NAME enhanced autoregulation (61 +/- 5.6 compared with control, 93.4 +/- 7.9; P < 0.003), which was reversed by the action of L-arginine (88.5 +/- 5.5, P < 0.007). The data were consistent with the hypothesis that increased flow leads to nitric oxide-induced dilation, which counteracts autoregulation.