Validation of a qualitative immunochromatographic test for the noninvasive assessment of stress in dogs.

Salivary cortisol is regarded as a reliable parameter for the noninvasive assessment of the welfare of animals, because it is strictly related to stress levels. Several methods are available for salivary cortisol measurement in mammals, however rapid diagnostic test for detecting salivary cortisol are confined to humans. The availability of such non invasive diagnostic tools operable in situ would facilitate monitoring of animal welfare. The Cortisol stress™ test provides a simple and rapid tool to discriminate cortisol levels in canine saliva above or below 4ng/ml, which has been suggested as the cut-off value for distinguishing unstressed dogs from those experiencing stress. The test is based on a competitive immunochromatographic assay (ICT) using gold nanoparticles as probes, in which the color intensity of the Test line is inversely correlated to the salivary cortisol level. The qualitative result is obtained by the visual observation of the color formed on the Test line compared to that of the Control line We evaluated the accuracy of the test by determining salivary cortisol in 85 samples of canine saliva belonging to dogs with very variable age, sex, breed, and life history, and comparing the qualitative results to those obtained by a reference ELISA kit. Agreeing results were obtained through the two methods, and the ICT showed high diagnostic sensitivity, specificity and efficiency (100%, 98.4%, and 98.8%, respectively). Furthermore, we evaluated the precision of the test by an experimental design approach, which combines errors due to within-day and between-day variation with the biological variability, and demonstrated that the test could be reliably applied for correctly classifying canine samples, according to their salivary cortisol level. Moreover, we studied the shelf-life of the device in three experimental conditions. We confirmed the stability of the ICT at 4°C and 25°C for at least six months and observed similar results for an accelerated stability study conducted for 7days at 37°C, which suggest that the stability of ICT device could be estimated by the accelerated experiment alternatively to the real-time study.

Circulating cell-free DNA: a promising marker of regional lymphonode metastasis in breast cancer patients.

PURPOSE: We undertook the current study with untreated breast cancer to (1) role the variations in the plasma levels of cfDNA and the size distribution in early stage, (2) determine the frequency in plasma of methylation of three candidate genes, RASSF1A, MAL, and SFRP1, and (3) to determine whether detection of cfDNA variations and methylation changes in plasma might have specific clinical utility. METHODS AND MATERIALS: Thirty-nine patients woman patients (median age 64 years; range, 36-90 years) who underwent surgery for primary BR and 49 healthy females' subjects (control group without any breast lesion) were evaluated. The cfDNA levels were analyzed using quantitative real-time polymerase chain reaction of ß-globin. Based on the ALU repeats, the cfDNA was considered as either total (fragments of 115 bp, ALU115) or tumoral (fragments of 247 bp, ALU247). The association between the levels of the ALU247, ALU115 repeat, and ALU 247/115and the pathologic tumor characteristics was analyzed. Used methylight qPCR method, cfDNA from plasma samples of healthy donors and patients with breast cancer were evaluated for the diagnotic value of the methylation status of three genes (RASSF1A, MAL, SFRP1) frequently methylated in breast cancer. RESULTS: The baseline levels of cfDNA were significantly higher in the patients with cancer, and the level of ALU247 was the most accurate circulating cfDNA marker in discriminating the cancer from non-cancer subjects. A high statistical significance was found by considering the T stage and patients with regional LN metastasis positive cancers showed significantly higher cfDNA level of ALU247. Moreover, patients with methylation of at least one of the gene under investigate showed a higher quantity of cfDNA ALU115 (p< 0.0001) and ALU247 level (p< 0.0001). CONCLUSIONS: We observed that necrosis could be a potential source of circulating tumour-specific cfDNA ALU247; and that cfDNA ALU247 and methylated cfDNA (RASSF1A, MAL and SFRP1) are both a phenotypic feature of tumour biology.

Matt waste from glass separated collection: an eco-sustainable addition for new building materials.

Matt waste (MW), a by-product of purification processes of cullet derived from separated glass waste collection, has been studied as filler for self-compacting concrete and as an addition for newly blended cement. Properties of self-compacting concrete compared to reference samples are reported. They include characteristics at the fresh and hardened states, and the compressive strength and porosity of mortar samples that were formulated with increasing amounts of MW to be used as cement replacement (up to 50wt.%). The effects of matt waste are discussed with respect to the mechanical and microstructural characteristics of the resulting new materials.

Ghrelin stimulates adrenocorticotrophic hormone (ACTH) secretion by human ACTH-secreting pituitary adenomas in vitro.

Ghrelin is a brain-gut peptide with wide-ranging endocrine, metabolic, cardiovascular and neural effects. Ghrelin, like its synthetic counterparts, the growth hormone (GH) secretagogues, has been shown to markedly stimulate adrenocorticotrophic hormone (ACTH) and cortisol secretion in humans and the ACTH-releasing effect of GH secretagogues is even greater in patients with pituitary ACTH-secreting tumours. Furthermore, these tumours synthesize ghrelin itself, suggesting an intrapituitary ghrelin circuit. The aim of the present study was to evaluate the effect of ghrelin on ACTH secretion by human pituitary corticotroph tumours in vitro to test the functionality of this circuit. Nine ACTH-secreting pituitary tumours (four microadenomas, five macroadenomas) were collected during surgery and incubated with 10-100 nM human ghrelin or with 10 nM human corticotrophin-releasing hormone (CRH). Control experiments were performed in rat anterior pituitary primary cultures. ACTH secretion was assessed after 4 h and 24 h incubation by immunometric assay. After 4 h of incubation with ghrelin, medium ACTH concentrations were two- to ten-fold higher compared to ACTH concentrations in unstimulated wells. The ACTH-releasing effect of ghrelin was significantly less than the response elicited by 10 nM CRH (up to 40-fold) Similar results were obtained after 24 h of incubation and a superimposable response pattern was observed in rat anterior pituitary primary cultures. The present study demonstrates that the endogenous GH secretagogue, ghrelin, stimulates ACTH secretion directly from human tumoural corticotrophs, as well as from normal rat pituitary, and indicates that the marked ACTH release elicited by ghrelin in patients with Cushing's disease in vivo is due, at least in part, to its action on the pituitary tumour. However, the reversal of the response pattern reported in vivo, with ghrelin proving a lesser stimulant than CRH in vitro, suggests that additional, suprapituitary mechanisms are involved in the in vivo response. Moreover, these data uphold the concept of a functional intratumoural ghrelin paracrine circuit in human corticotroph adenomas.

Redox regulation of chemokine receptor expression.

Cytokines and reactive oxygen intermediates (ROI) are frequent companions at sites of acute inflammation. We have shown previously that in human monocytes, bacterial lipopolysaccharide, IL-1, and tumor necrosis factor-alpha induce a rapid down-regulation of the monocyte chemotactic protein-1 receptor CCR2 (CC chemokine receptor-2). These stimuli also induce production of ROI. In this paper, we investigate the influence of antioxidants and/or ROI on chemokine-receptor expression. In human monocytes, the antioxidant pyrrolidine dithiocarbamate (PDTC) rapidly inhibited CCR2 (95-100% of inhibition) and CCR5 (77-100% of inhibition) mRNA expression by strongly decreasing transcript stability. CCR2 half-life was decreased from 1.5 h to 45 min; CCR5 half-life was decreased from 2 h to 70 min. This inhibitory activity also included CXCR4 (CXC chemokine receptor-4) but not CXCR2 receptor and, although to a lesser extent, was shared by the antioxidants N-acetyl-l-cysteine and 2-mercaptoethanol. In contrast, the ROI-generating system xanthine/xanthine oxidase increased CCR5 and CXCR4 mRNA expression and counteracted the inhibitory effect of PDTC. Accordingly, H(2)O(2) and the glutathione-depleting drug buthionine sulfoximine increased to different extents CCR2, CCR5, and CXCR4 mRNA expression. The PDTC-mediated inhibition of CCR5 and CXCR4 mRNA expression was associated with decreased chemotactic responsiveness (>90% inhibition) and with a marked inhibition of surface-receptor expression. In contrast, xanthine/xanthine oxidase opposed the bacterial lipopolysaccharide- and tumor necrosis factor-alpha-mediated inhibition of CCR5 and CXCR4 mRNA expression and increased both the CCR5 surface expression and the cell migration (3-fold) in response to macrophage inflammatory protein-1beta. These results suggest that the redox status of cells is a crucial determinant in the regulation of the chemokine system.

Defective expression of the monocyte chemotactic protein-1 receptor CCR2 in macrophages associated with human ovarian carcinoma.

Monocyte chemotactic protein-1 (MCP-1, CCL2) is an important determinant of macrophage infiltration in tumors, ovarian carcinoma in particular. MCP-1 binds the chemokine receptor CCR2. Recent results indicate that proinflammatory and anti-inflammatory signals regulate chemokine receptor expression in monocytes. The present study was designed to investigate the expression of CCR2 in tumor-associated macrophages (TAM) from ovarian cancer patients. TAM isolated from ascitic or solid ovarian carcinoma displayed defective CCR2 mRNA (Northern blot and PCR) and surface expression and did not migrate in response to MCP-1. The defect was selective for CCR2 in that CCR1 and CCR5 were expressed normally in TAM. CCR2 gene expression and chemotactic response to MCP-1 were decreased to a lesser extent in blood monocytes from cancer patients. CCR2 mRNA levels and the chemotactic response to MCP-1 were drastically reduced in fresh monocytes cultured in the presence of tumor ascites from cancer patients. Ab against TNF-alpha restored the CCR2 mRNA level in monocytes cultured in the presence of ascitic fluid. The finding of defective CCR2 expression in TAM, largely dependent on local TNF production, is consistent with previous in vitro data on down-regulation of chemokine receptors by proinflammatory molecules. Receptor inhibition may serve as a mechanism to arrest and retain recruited macrophages and to prevent chemokine scavenging by mononuclear phagocytes at sites of inflammation and tumor growth. In the presence of advanced tumors or chronic inflammation, systemic down-regulation of receptor expression by proinflammatory molecules leaking in the systemic circulation may account for defective chemotaxis and a defective capacity to mount inflammatory responses associated with advanced neoplasia.

Autocrine production of IL-10 mediates defective IL-12 production and NF-kappa B activation in tumor-associated macrophages.

IL-12 is a central cytokine in the activation of inflammation and immunity and in the generation of Th1-type responses. Tumor-associated macrophages (TAM) from mouse and human tumors showed defective production of IL-12. Defective IL-12 production was associated with lack of p50/p65 NF-kappa B activation. TAM produced increased amounts of the immunosuppressive cytokine IL-10. Abs against IL-10 restored the defective capacity of TAM to produce IL-12. Our data suggest that during tumor growth an IL-10-dependent pathway of diversion of macrophage function can be activated into the tumor microenvironment and results in the promotion of the IL-10+ IL-12- phenotype of TAM. Blocking IL-10, as well as other immunosuppressive cytokines present in the tumor microenvironment, such as TGF-beta, may complement therapeutic strategies aimed at activating type I antitumor immune responses.

Divergent effects of dithiocarbamates on AP-1-containing and AP-1-less NFAT sites.

Changes in the redox status of cells affect NF-kappaB and activator protein (AP)-1 nuclear expression and activity. In particular, antioxidants decrease NF-kappaB and increase AP-1 transcriptional activity, thereby regulating gene expression. In T cells, low concentrations of antioxidants enhance IL-2 and inhibit IL-4 expression. Since NFAT binding sites play an essential role in regulating IL-2 and IL-4 gene transcription, we studied the effects of dithiocarbamates, using the pyrrolidine derivative of dithiocarbamate (PDTC), on the activity of the distinct AP-1-containing IL-2 NFAT and AP-1-less IL-4 NFAT enhancers elements. Consistent with the presence of AP-1 proteins within the IL-2 NFAT complex, PDTC strongly enhanced phorbol 12-myristate 13-acetate/phytohemagglutinin-induced NFAT binding to the IL-2 NFAT enhancer and transcriptional activity of a reporter plasmid driven by this NFAT enhancer. In contrast, the activity of the IL-4 NFp enhancer, which does not bind AP-1, was abolished by PDTC treatment. In the Jurkat T cell line treated with PDTC, co-expression of the Ca2+/calmodulin-dependent phosphatase, calcineurin, completely restored the IL-4 NFp enhancer activity. Our data indicate that calcineurin-mediated NFAT activity is a target for antioxidants and provides new insights into the molecular mechanisms controlling differential cytokine gene expression.

Different control mechanisms of growth hormone (GH) secretion between gamma-amino- and gamma-hydroxy-butyric acid: neuroendocrine evidence in Parkinson's disease.

The observation that baclofen stimulates growth hormone (GH) secretion in normal men, but not in parkinsonian patients led us to test the GH releasing effect of other gamma-amino-butyric acid (GABA)ergic agents with different mechanisms of action in Parkinson's disease. For this purpose 10 normal men and 10 de novo parkinsonian patients were tested with sodium valproate (800 mg PO), gamma-hydroxybutyric acid (GHB) (25 mg/kg body weight PO) and baclofen (10 mg PO). All drugs induced a significant increment in serum GH levels in the normal controls. On the other hand, GH secretion in parkinsonian patients did not change after baclofen or sodium valproate administration, whereas it showed normal responsiveness to GHB. These data suggest that the mechanism underlying the GH response to GHB is different from that (or those) mediating sodium valproate and/or baclofen action. In addition, the former, but not the latter mechanism appears to be preserved in the parkinsonian brain.

Bacterial lipopolysaccharide rapidly inhibits expression of C-C chemokine receptors in human monocytes.

The present study was designed to investigate the effect of bacterial lipopolysaccharide (LPS) on C-C chemokine receptors (CCR) expressed in human mononuclear phagocytes. LPS caused a rapid and drastic reduction of CCR2 mRNA levels, which binds MCP-1 and -3. CCR1 and CCR5 mRNAs were also reduced, though to a lesser extent, whereas CXCR2 was unaffected. The rate of nuclear transcription of CCR2 was not affected by LPS, whereas the mRNA half life was reduced from 1.5 h to 45 min. As expected, LPS-induced inhibition of CCR2 mRNA expression was associated with a reduction of both MCP-1 binding and chemotactic responsiveness. The capacity to inhibit CCR2 expression in monocytes was shared by other microbial agents and cytokines (inactivated Streptococci, Propionibacterium acnes, and to a lesser extent, IL-1 and TNF-alpha). In contrast, IL-2 augmented CCR2 expression and MCP-1 itself had no effect. These results suggest that, regulation of receptor expression in addition to agonist production is likely a crucial point in the regulation of the chemokine system.

Is the tantalum Strecker stent suitable for TIPS creation? Short- and mid-term results in 20 consecutive patients.

PURPOSE: To assess the suitability of tantalum Strecker stents for transjugular intrahepatic portosystemic shunt (TIPS) creation. METHODS: TIPS was performed with Strecker balloon-expandable stents in the first 20 patients of our series. A total of 26 prostheses were utilized (average 1.3 per patient). RESULTS: Immediate technical success was achieved in all 20 cases. Nine patients (45%) died during follow-up. The overall average follow-up period was 18.9 months; the 11 survivors were followed for a mean period of 29.8 months. In 4 patients (20%) the stent dislodged towards the hepatic vein during withdrawal of the balloon catheter, and difficulties in recatheterizing the shunt for routine control portal phlebography were frequently encountered. Rebleeding occurred in 5 cases and aggravation of pre-existing encephalopathy occurred in 2 cases. Shunt occlusions or stenosis required further intervention in 4 and 9 patients, respectively. CONCLUSION: In our opinion the tantalum Strecker stent is not particularly suitable for TIPS. Although it has evident advantages, such as high radiopacity and minimal shortening after deployment, the tendency of the device to dislodge and the difficulty in recatheterization during portal phlebography were important drawbacks. Furthermore, the atraumatic ends of the device did not seem to reduce shunt-related complications, which were comparable to those occurring with other types of stent.

[Thyroid metastases: the echographic and computed tomographic aspects]. / Metastasi tiroidee: aspetti con ecografia e Tomografia Computerizzata.

PURPOSE: The modern integrated approach to neoplastic diseases permits to detect metastatic lesions in uncommon sites, such as the thyroid gland. We reviewed the ultrasonographic (US) and Computed Tomographic (CT) patterns of secondary thyroid lesions, comparing imaging findings with primary tumor histopathologic diagnosis. MATERIAL AND METHODS: We reviewed a series of 10 thyroid metastases diagnosed from 1986 to 1995 at staging (4 cases) or follow-up (6 cases) examinations; in the latter, metastases were diagnosed 39 to 89 months (mean: 58 months) after the primary tumor. The patients were 7 women and 3 men, 55 to 94 years old (mean: 65 years). The final diagnosis was made at cytology (4 cases), histology (5 cases) or surgery (1 case). RESULTS: The primary tumors were breast cancer (2 cases), melanoma (2 cases), small cell lung cancer, bronchioalveolar carcinoma, mucoid stomach adenocarcinoma, renal carcinoma, colon carcinoma and leiomyosarcoma (1 case each). The thyroid lesion was unifocal in 6 patients and multifocal in 2; the gland was diffusely involved in 2 patients. Dysphagia was found in all patients. Thyroid function was normal in 6/7 patients and one had hyperthyroidism; normofunctional goiter was found in another patients. US showed focal or diffusely infiltrating hypoechoic lesions; likewise, inhomogeneously hypodense areas with mild contrast enhancement were observed at CT. Thyroid secondary lesions exhibited variable patterns when necrotic, hemorrhagic or calcific areas were included; correlations with primary tumor-pathogenetic and histologic features were often observed. Survival rate was 2-60 months (mean: 19 months). CONCLUSION: Since imaging findings in thyroid metastases are often not specific and sometimes atypical, fine needle biopsy of suspicious lesions should be carried out for both staging and follow-up.

[Assessment of various new predictive parameters of the outcome of mechanical ventilation weaning]. / Valutazione di alcuni nuovi parametri predittivi dell'esito dello svezzamento dalla ventilazione meccanica.

OBJECTIVE: To evaluate new indexes predicting weaning outcome from mechanical ventilation. EXPERIMENTAL DESIGN: Prospective study with two main end-points: a comparison of weaning indexes between successful and unsuccessful groups and an evaluation of their predicting value. ENVIRONMENT: Surgical-Medical Intensive Care Unit. PATIENTS: Patients ventilated for more than 72 hours and subjected to a weaning trial until spontaneous ventilation. MEASUREMENTS: Traditional weaning parameters [respiratory rate (fsb), expiratory minute volume (Vesb), Maximal Inspiratory Pressure (MIP)] along with the new indexes [fsb/Vtsb) (rate to tidal volume ratio), CROP index (Compliance Rate Oxygenation Pressure), P0.1/MIP, IEQ (Inspiratory Effort Quotient), WI (Weaning Index)] were measured before discontinuation of ventilation support. RESULTS: A statistically significant difference was observed between successful and unsuccessful groups for the following parameters: fsb, MIP, fsb/.Vtsb, CROP, Po.1/MIP and WI while no differences were observed for Vesb and IEQ. The sensitivity and specificity for the new indexes were respectively: fsb/Vtsb: 0.65 and 0.58; CROP: 0.70 and 0.63; P0.1/MIP: 0.85 and 0.36; WI: 0.69 and 0.47. CONCLUSION: A statistically significant difference between successful and unsuccessful groups was observed for some but not all new indexes; the diagnostic accuracy of the new indexes were no better than additional parameters.

TIPS (transjugular intrahepatic portosystemic shunt): state of art and personal experience.

After a brief historical view, we describe the basic technique currently used to create percutaneous portosystemic shunt. Between September 1992 and March 1995, TIPS was achieved in 50 out of 52 patients; main indications included bleeding from esophageal or gastric varices and refractory ascites. The mean portal pressure reduction was 14.9 mmHg and the mean residual portosystemic gradient was 10.5 mmHg. The average follow-up time was 11.8 months; in this period the overall mortality rate was 28%, while rebleeding occurred in 8 patients and new onsets of encephalopathy occurred in 4 cases. The major problems were due to shunt related complications observed in 46% of the patients; close follow-up and reintervention are required to keep the shunt previous. TIPS, developed ad an alternative to surgery and endoscopic sclerotherapy, is an effective and relatively safe procedure for the treatment of symptomatic portal hypertensive patient.

[Pneumonia in severe head injury. A prospective study]. / La polmonite nel trauma cranico grave. Uno studio prospettico.

Impairment of the state of consciousness is an important contributing factor in the onset of respiratory tract infections; in this study the data were collected prospectively to investigate the incidence and clinical implications of pneumonia in a population of head injured patients. The study was conducted on all patients treated at our centre throughout 1990. The incidence of pneumonia in the head injured was 10.8% versus 7.3% in the rest of the patients. Mortality in the group with pneumonia was not significantly different from the group without pneumonia. The average time of onset was on the fifth day from admission. The lung injury score (LIS) on the sixth day, the time on artificial ventilation and the length of stay in intensive care were significantly greater in those with pneumonia (1.18, 14.6 days and 21.9 days versus 0.8, 4.2 days and 12.9 days respectively). Staphylococcus was the single most frequently isolated germ. Our study concludes that pneumonia represents a relatively frequent and early complication in patients with head injury, and it is associated with prolonged artificial ventilation and longer staying in ICU.

[Hypercholesterolemia and coronary disease: intervention strategies]. / L'ipercolesterolemia e malattia coronarica: strategie di intervento.

The decision to treat hypercholesterolemia must take into account both the risk of death and myocardial infarction and the risk/benefit ratio of treatment. Nowadays there is convincing evidence that hypercholesterolemia in patients with previous myocardial infarction is an important prognostic factor: furthermore, the excess of risk due to cholesterol is consistently higher than that in the general population. Prospective studies demonstrated that cholesterol lowering treatment obtained either with diet or drugs can improve survival and quality of life in postinfarction patients. Experimental, clinical and angiographic studies suggested that cholesterol lowering therapy can produce both stabilization and regression of the atherosclerotic plaque. In view of this, the evaluation of cholesterol level must be part of the general work-up of patients with previous myocardial infarction. The satisfactory results obtained with diet suggest to try a dietary treatment in post-infarction patients with high cholesterol level before using drugs. Since there are no clear-cut upper values, the end point of treatment should be to lower cholesterol levels to the values recommended for the general population, although we believe that threshold values to be obtained in these subjects should be lower than those requested in primary prevention.

[Blood saving in heart surgery. Personal experience with 232 patients]. / Risparmio di sangue in cardiochirurgia. Esperienze personali su 232 pazienti.

In order to minimize the need of homologous blood transfusion during cardiac surgery four different blood sparing methods have been tested in four groups of patients. In 62 patients (group A) blood from operative field has been collected and processed by the Autotrans (Dideco (R)) apparatus. Intraoperative autotransfusion has been performed in 50 patients (group B). Prostacyclin (Pgh2) has been administered to 36 patients (group C), starting before systemic heparinization, and continuing during extracorporeal circulation. Standard techniques have been employed in a control group of 84 patients (group D). The following parameters have been examined: preoperative blood counts; length of extracorporeal circulation postoperative bleeding (including I.C.U); homologous blood volume transfused; and only in group A the mean number of blood units collected. Significant reduction of bank-blood consumption has been achieved only in B group (intraoperative autotransfusion) while in the remaining groups blood saving was not statistically significant.

Upper visual space neglect and motor deficits after section of the midbrain commissures in the cat.

The neurological deficits following section of the midbrain commissures were studied in the cat. After a lesion of the commissures between the superior and inferior colliculi, with or without involvement of the posterior commissure, the animals showed a long lasting inattention for stimuli in the upper visual space, lack of exploratory head movements towards the neglected space, head ventroflexion and vertical paralysis of gaze. After a lesion of the commissure between the superior colliculi or of its rostral part only, the same symptomatology appeared, but it was short lasting. After a lesion of the posterior commissure, the head was kept dorsiflexed, the exploratory head movements towards the lower visual space were reduced and the stimuli presented in this space were often neglected. There was a paralysis of vertical eye movements. The findings are discussed in the frame of a premotor theory of neglect.