Paraoxonase 1 L55M, Q192R and paraoxonase 2 S311C alleles in atherothrombosis.

Increased oxidative stress is known to play a role in the pathogenesis of atherosclerosis, and polymorphisms in genes encoding for enzymes involved in modulation of oxidant stress, such as paraoxonases (PONs), provide a potentially powerful approach to study the risk of disease susceptibility. Aim of our study is to investigate the possible association among PONs polymorphisms, clinical and metabolic factors, and atherothrombotic events in an Italian population. We evaluated in 105 subjects, with or without atherosclerotic risk factors, the presence of PON1 L55M, PON1 Q192R, and PON2 S311C genetic variants, as well as lipid profile, the concentration of aminothiols (blood reduced glutathione, plasma total glutathione, homocysteine, cysteine, cysteinyl glycine), and malondialdehyde as markers of lipid peroxidation. Clinical, biochemical, and genetic variables were correlated with a history of atherothrombosis. Previous atherothrombotic events were found in 42 patients (40 %): myocardial infarction in 24, stroke or transient ischemic attack in 18. By multiple logistic regression analysis, hypertension (OR = 5.538; 95 % CI 2.202-13.902, P < 0.001), HDL-cholesterol concentration (OR = 0.947; 95 % CI 0.910-0.985, P = 0.007), and the presence of C allele in PON2 gene (OR = 3.595; 95 % CI 1.247-10.361, P = 0.018) were independently associated with atherothrombotic events. Our study sheds light on the role of PON2 as a possible cofactor in determining the risk of events together with the well-known risk markers HDL-cholesterol and hypertension.

Altered vessel signalling molecules in subjects with Downs syndrome.

Downs syndrome (DS) is the most frequent human chromosomal abnormality and is associated with mental retardation. Some evidence indicates that certain inflammatory molecules may be increased in DS. Proinflammatory and vasoactive molecules in the blood of non demented subjects with DS were measured in the present investigation. Plasma levels of interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1) and C reactive protein (CRP) were measured in child (2-14 years), adult (20-50 yrs) and elderly (> 60 yrs) DS subjects. Increased plasma levels of IL-6 and MCP-1 were present in DS. Plasma levels of VEGF were increased only in DS adults. Positive linear correlation between IL-6 and MCP-1 levels was present. However, no subclinical inflammation was apparent in DS, since neopterin and CRP levels were within the normal range. An altered regulation of these molecules might interfere with some processes involved in cognitive performances of DS subjects.

Colony-stimulating factor produced by murine adrenocortical tumor cells.

Conditioned medium obtained from the adrenocortical LAF1 mouse tumor (Y-1) cell cultures was able to stimulate the proliferation and the differentiation of granulocyte-macrophage precursors in the normal murine bone marrow. Colony-stimulating factor (CSF) was spontaneously produced by Y-1 cells also in serum-free cultures. By two cycles of gel chromatography on Sephadex G-150 of concentrated conditioned medium two peaks of colony-stimulating activity were isolated that corresponded to apparent molecular radii of 100,000 and 29,000, respectively. At this step of purification, the two factors gave a similar dose-response curve, showed a remarkable resistance to the heat treatment and pH changes, and were not extracted by ether. Because Y-1 cells resulted in infection by retrovirus, they provide a useful model to investigate the relations between viral coded information and CSF production.

A Novel MGC4607/CCM2 Gene Mutation Associated with Cerebral Spinal and Cutaneous Cavernous Angiomas.

Cerebral cavernous malformations (CCMs) are vascular abnormalities that may cause seizures, headaches, intracerebral hemorrhages, and focal neurological deficits; they can also be clinically silent and occur as a sporadic or an autosomal dominant condition. Three genes have been identified as causing familial CCM: KRIT1/CCM1, MGC4607/CCM2, and PDCD10/CCM3, mapping, respectively, on chromosomes 7q, 7p, and 3q. Here, we report an Italian family affected by CCM due to a MGC4607 gene mutation, on exon 4. All the affected subjects suffered from seizures, and some of them underwent surgery for removal of a cavernous angioma. Brain MRI showed multiple lesions consistent with CCMs in all patients. Spinal and cutaneous cavernous angiomas were present too. This report underlines the need for a careful interdisciplinarity among neurologists, neuroradiologists, neurosurgeons, geneticists, ophthalmologists, and dermatologists for a total evaluation of the different manifestations of familial CCM. This points out that only referral centers are organized to offer a multidisciplinary management of this disease.

Factors produced by macrophages and tumor cells: influence on the granulocytic precursor cells (CFU-C) in normal and tumor-bearing mice.

Some tumors are known to produce colony-stimulating factors (CSF). In mice bearing s.c. Ehrlich tumor (ET) we observed that tumor growth is responsible for hemopoietic perturbations in which the increase of granulocytic-macrophagic precursors (CFU-C) in bone marrow and in the spleen represents a remarkable aspect. CFU-C in bone marrow from ET-bearing mice are more sensitive than CFU-C from normal mice to CSF from L-cells (LC-CM), mouse embryo fibroblasts (MEF-CM) and from the same tumor (ET-CM). Macrophages from normal and from ET-bearing mice exert the same effect on the proliferation of normal bone marrow CFU-C. Bone marrow CFU-C from ET-bearing mice show an increased sensitivity to the inhibitory factors produced by macrophages from ET-bearing mice.

Inefficacy of moving average algorithm as principal quality control procedure on Technicon System H6000.

Bull's algorithm, in its "revisited" formulation, represents one of the main quality control (QC) procedures in several multichannel automatic hematological analyzers. Its efficacy, however, is reduced in the theoretical event that red blood cells (RBC) and hemoglobin (Hgb) undergo a concomitant analytical drift while mean corpuscular volume (MCV) remains unaffected, so that a null effect is registered on the related erythrocytic indices: mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC). This particular phenomenon has been observed on the Technicon System H6000. Routine daily work was kept in control through the use of commercial controls and fresh blood samples: a reproducible positive drift of two directly measured erythrocytic parameters (RBC and Hgb) and of the calculated hematocrit (Hct) was observed proportional to the time of continuous use of the instrument. The usefulness of Bull's QC scheme was greatly reduced: it failed to detect "out of control" situation in 38%, 15%, and 13% of cases in the monitoring of MCV, MCH, and MCHC, respectively, when compared with the traditional 2 SD limits on QC samples.

Myocardial necrosis in ICU patients with acute non-cardiac disease: a prospective study.

OBJECTIVE: To ascertain if, after an episode of hypotension, unnoticed myocardial necrosis could occur in critical care patients with acute non-cardiac illness and to search for signs of cardiac necrosis. DESIGN: A prospective observational study. SETTING: General intensive care unit (ICU) at a tertiary level hospital. PATIENTS: Thirty-one patients in two groups. Group 1 included 19 patients with severe sepsis/septic shock (ACCP/SCCM Consensus Conference). Group 2 included 12 patients with hypovolemic shock. INTERVENTIONS: Biochemical markers of myocardial necrosis (cardiac troponin I (cTnI), creatine kinase (CK), creatine kinase MB mass (CKMB) and myoglobin) were measured at 12 h (T1), 24 h (T2) and 48 h (T3) after enrollment. A standard 12-lead ECG was recorded upon enrollment (T0) and at T2. Anomalous Q-waves or ST segment depression or elevation was considered diagnostic for acute myocardial infarction (AMI). A hypotensive episode (arterial systolic pressure < 90 mmHg at heart rate > 100 bpm) was considered moderate if it lasted 30-60 min or severe if longer than 60 min. MEASUREMENTS AND RESULTS: At T0 none of the patients had AMI on ECG. At T2 a non-Q AMI developed in five patients. Increased levels of troponin I, myoglobin, CK and CKMB were found in 74.2 %, 96.8 %, 74.2 % and 67.7 % of the patients, respectively. Cardiac troponin I increased in 11 out of 19 septic patients and in all hypovolemic patients. There was a significant difference between the groups (p < 0.05). All biochemical markers increased in relationship to the degree of hypotension with cTnI again showing a significant difference. The longer the hypotensive episode was, the greater was the increase (moderate hypotension: median 1.16; quartiles 0.55-3.44 ng/ml, severe hypotension: median 8.53; quartiles 1.1-20.7 ng/ml; p < 0.05). Abnormal levels of cTnI were more frequent in non-survivors than in survivors (p < 0.05). CONCLUSIONS: Hypotension may cause cardiac damage in critically ill patients with acute non-cardiac diseases as shown by abnormal levels of cTnI. It is likely that a high number of these myocardial necroses may go unnoticed on the ECG.

Results of a pilot study of endoscopic screening of first degree relatives of colorectal cancer patients in Italy.

STUDY OBJECTIVE: Screening recommendations for colorectal cancer include sigmoidoscopy in asymptomatic, average risk persons aged 50 and over and colonoscopy every three to five years in high risk groups. Little is known about the eligible population's compliance with endoscopic screening. This is the first Italian report of an endoscopic screening programme for colorectal cancer patients' relatives. DESIGN: In 1986, a pilot project for colorectal cancer screening by endoscopy in high risk subjects was started in the Desio (Milan, Italy) public health service region. The results obtained after seven years are described. SETTING: The names of 536 inhabitants with colorectal cancer diagnosed between January 1975 and December 1984 and their relatives' addresses were obtained from the Regione Lombardia Health System records and from the municipal registry offices respectively. PARTICIPANTS: From October 1986 to October 1993, 778 first degree relatives aged 20-75 were offered colonoscopy. MAIN RESULTS: After seven years, 233 (29.9%) had undergone endoscopic examination, mostly up to the splenic flexure. Being > 60 in age at the start of the programme negatively affected the participation (p < 0.05). Two cancers were detected and adenomatous polyps were found in another 24 of those screened (frequencies 0.9% and 10.3% respectively). Male gender (p < 0.05), increasing age in males (p < 0.01), and two or more affected relatives in females (p < 0.01) positively affected the frequency of polyps detection. CONCLUSION: These results suggest that about 30% of the eligible population would comply at least with sigmoidoscopic screening. The collaboration of family doctors and more widespread public information about the ability to cure colorectal cancer are necessary for better compliance.

A Probabilistic Model to Study Spatial Variations of Primary Productivity in River Impoundments.

A stochastic approach to model the spatial variability of primary productivity in a river impoundment is presented in which data from LANDSAT-TM images of reference data from the field were used as basic inputs to the model. Primary productivity is predicted here by a set of variables (chlorophyll, temperature, and turbidity) combined by means of multivated probabilistic model wherein probability distribution functions were assigned to each variable. Linear regression analysis was used to relate the field reference data to the image data used as input to the model. The model was applied using maps of the variables obtained from the digital imagery. The results was a map of primary productivity probabilities converted to absolute values utilizing the cumulative function for field measurements of primary productivity. The field data were collected over °1.5 yr at 16-d intervals at 12 sample sites. The overall r2 between the model results using field-measured variables and the measured primary productivity carbon values was 0.85 with an RMS (root mean square) error of 16 mg°m-3 °h-1 , where the carbon values ranged from 4 to 300 mg. m-3. h-1 . To test the methodology for the generation of primary productivity maps from LANDSAT imagery, two maps of primary productivity for Kentucky Lake were generated from data collected in August and December 1988. The model output was, in turn, compared to primary production carbon measurements from the reservoir. The r2 s were 0.89 and 0.76 with RMS errors in these carbon values of 10 and 18 mg°m-3 °h-1 . No specific assumptions about Kentucky Lake were required for this approach, so the methodology is applicable to other lakes or reservoirs in similar trophic states.

Increased urinary D-glucaric acid excretion by children living in an area polluted with tetrachlorodibenzoparadioxin (TCDD).

Extremely small doses of TCDD have been shown to induce hepatic microsomal enzymes in animals. Whether levels of environmental exposure to TCDD were sufficient to produce enzyme induction in man, has been investigated in Seveso, where in July 1976 explosion in a factory spread toxic substances, one of which was TCDD, to the surrounding area. The hepatic microsomal enzyme activity was assessed by estimating urinary d-glucaric acid (UGA) excretion in children 6-8 years old. In 31 children, urine samples were collected between August and December 1976; in 67 other children in February 1979. As a control group 60 children living in Busto Arsizio (a small industrial town near Milan) and 26 living Cannero (a non-industrialized village on Lake Maggiore) were chosen. In the first period of collection, children with chloracne (which is considered to be a characteristic manifestation of intoxication with chlorinated products), showed significantly increased levels of UGA compared with children without chloracne. In 1979, children living in the Seveso area showed a statistically significant enhancement of d-glucaric acid excretion compared to the control groups. In conclusion, this study demonstrates that many children living in the Seveso area have an increased activity of hepatic microsomal enzymes, since, although the urinary excretion of d-glucaric acid is only an indirect measure of enzyme activity, studies in man have indicated that it is both sensitive and quantitative. As far as the cause of this increase is concerned, since it is possible to exclude the influence of alcohol, contraceptives, phenobarbitone or other drugs, it is reasonable to conclude that TCDD, a potent inducer agent, could be responsible for this phenomenon.

Steroid receptors in the pathogenesis of chronic subdural hematoma.

Chronic subdural hematoma (CSDH), located between the dura mater and the arachnoid and usually characterized by a well-vascularized external capsule (HEM), has a higher incidence in male patients with elevated urinary estrogens than in female patients. In an attempt to increase our understanding of the physiopathogenesis of CSDH, total estrogen receptor (ER) was measured in HEM specimens from four male patients by a sodium thiocyanate exchange assay and cytosol progesterone receptor (PRc) in three specimens by a dextran-coated charcoal adsorption assay. Although no nuclear ER could be detected, ERc and PRc were found in all three specimens examined. The presence of ER in a mesenchymal tissue like HEM could suggest that, in addition to inducing vascular changes, estrogens might act directly on HEM through a receptor-mediated mechanism more pronounced in men than in women, whose vascular network is adapted to high estrogen values.

Homocysteine, vitamin B12, serum and erythrocyte folate in peritoneal dialysis patients.

Plasma homocysteine (tHcy) is an important risk factor for atherosclerosis in dialysis patients. Few data were reported on the prevalence and severity of hyperhomocysteinemia in peritoneal dialysis (PD) patients. In addition, little attention was paid to the search of factors possibly involved in the pathogenesis of hyperhomocysteinemia in these patients. A cross-sectional study was performed in 107 stable PD patients. None of them was given folate or vitamin B12 supplementation before or during the study. Plasma tHcy, serum vitamin B12, serum and erythrocyte folate were measured by immunoenzymatic methods. Genetic analysis of the methylentetrahydrofolate-reductase thermolabile mutation (tMTHFR) was performed in 61 patients. 97% of patients had tHcy levels higher than normal. tHcy was not different between men and women, patients with or without malnutrition, with or without clinically evident atherosclerotic vasculopathy, with or without anemia. tHcy levels were significantly higher in homozygotes for the tMTHFR mutation than in patients carrying the wild type form. Significant univariate correlation was found between hyperhomocysteinemia and time since the start of dialysis, serum and erythrocyte folate and vitamin B12. The best fitted model equation was log tHcy = 108.53 + 0.1606 (duration of dialysis) -1.1053 (s-F) -0.7980 (age) 0.0215 (vitamin B12). Our results agree with those reported by other authors in hemodialysis patients. Despite the large number of PD patients with normal serum vitamin B12 and folate status, the relation between tHcy and vitamin B12 or folate suggests that the supplementation of these vitamins could be useful irrespective of their serum levels, especially in younger patients or in those treated for a long period of time with peritoneal dialysis.

Effect of tumor-related factors on the in vivo and in vitro colony-forming ability of normal mouse marrow cells.

The effect of normal mouse marrow preincubation with Ehrlich tumor fluid (EAF) and Ehrlich tumor cell-conditioned medium (ET-CM) was investigated both on the in vivo assay for pluripotential stem cells (CFU-S) and on the in vitro assay for granulocytic-macrophagic progenitors (CFU-C). A significant reduction of the number of CFU-S was found when bone marrow cells were incubated with EAF. The morphological analysis showed a relative increase in the granulocytic type colonies besides an absolute increase in undifferentiated ones and an absolute decrease in erythroid colonies. The in vitro data show a decrease in CFU-C production, by stimulation with standard Colony Stimulating Factor (CSF), when the marrow cells are preincubated with EAF and ET-CM. When standard CAF (obtained from L-cells) is used at the higher concentrations, the CFU-C in the ET-CM treated marrow increases to the control values. EAF and ET-CM incubation also affects the CFU-C differentiation by increasing the number of pure granulocytic clones. The experimental data were discussed in order to explain an hypothesis about the activity of tumor-related factors.

Development and evaluation of immunochromatographic rapid tests for screening of cannabinoids, cocaine, and opiates in urine.

The test principle and the optimization of the reactive ingredients are described for the one-step dip and-read immunochromatographic FRONTLINE rapid tests for drugs-of-abuse testing in urine samples. In a multicenter evaluation the rapid tests were compared with FPIA and EMIT immunoassays. Discrepant results were further analyzed by gas chromatography-mass spectrometry methods. In the comparison of the cannabinoids rapid tests versus both immunoassays using clinical and forensic urine samples (399 versus FPIA and 755 versus EMIT), sensitivities and specificities were 97% or better for both comparisons. For cocaine, a sensitivity of 100% versus both routine technologies was obtained, whereas the specificity was reduced somewhat to 91% because of some cross-reactivity with metabolites of methadone and of clozapine. Specificity was very high for the cocaine rapid tests (98-100%) when applied to urine samples of persons not in a methadone maintenance program. Sensitivities and specificities for the opiates rapid tests were 99% or better at all sites when compared with the routine methods. In the screening of about 1200 clinical urine samples for cannabinoids, cocaine or opiates misuse only six samples would have stayed undetected by rapid test analyzes. These results show the FRONTLINE assays allow a reliable and immediate screening for drugs of abuse.

Results of the Italian interlaboratory quality control program for estradiol receptor assay.

This article presents results of the first Italian quality control program for determining the estradiol receptor on lypophilized guinea pig and calf uteri. Despite considerable variability in quantitative terms, the results concur in ability to define samples as positive or negative for receptor content. One of the parameters that most strongly influences accuracy of determination of receptor concentration is protein assay. The evaluation of several lyophilized preparations at scalar concentrations permitted identification, by linear regression, for each laboratory of the systematic and non-systematic variables. More comparable results will be forthcoming when a standardized methodology program has been fully adopted.

Measurement of ionized magnesium with AVL 988/4 electrolyte analyzer: preliminary analytical and clinical results.

Only free magnesium has biological activity: technology for measuring the ionized fraction of magnesium is now available via ion-selective electrodes. We have evaluated an instrument (AVL 988/4) which determines ionized magnesium (cMg2+) with an ion-selective electrode based on the ionophore ETH 7025. The selectivity of the electrode is adequate for the ions normally present in plasma, except for calcium: the interference is automatically corrected by simultaneous measurements of calcium with compensation for the calcium interference to the magnesium signal. We have first verified possible interference caused by sampling procedures: known silicon interference has been avoided by use of glass tubes (BD Vacutainer with no additive, code 7626); heparin interference has been measured and found significant above 20 UI.mL-1 of plasma. Instrument evaluation according to NCCLS protocol gives the following imprecision results on 20 replicated analyses: cMg2+ (mmol.L-1) 1.29, 0.76, 0.23, CVs% (within-run) 0.67, 0.67, 3.00 and CVs% (between-run) 4.06, 3.91, 5.89 respectively. Linearity (in the range 0.23-1.60 mmol.L-1) was: measured cMg2+ = 0.981.(calculated cMg2+) + 0.009 mmol/L; r = 0.999. In healthy adults (n = 103) cMg2+ was in the range 0.46-0.74 mmol.L-1 (with a mean of 0.60 mmol/L and normal distribution). These values represent 57% to 84% of serum total magnesium concentration (TMg) (mean 71%). pH dependence of cMg2+ is present, usually to a lower extent with respect to cCa2+, but it seems different in patients with real or in vitro provoked acidosis and in hemodialyzed patients. Citrate interference on ionized magnesium measurements was found both in vitro and in vivo, whilst that due to lactate was demonstrated only in vitro. On a wide range of cMg2+ (n = 100), a good correlation is obtained both with TMg and ultrafiltrable Mg (UFMg): cMg2+ = 0.723.TMg + 0.008 mmol.L-1, r = 0.978; cMg2+ = 0.912.UFMg + 0.10 mmol.L-1, r = 0.968, respectively. The ionized magnesium in ultrafiltrate was found 25% lower than that in serum. The lifespan of the electrode, evaluated on the basis of both time from installation and on number of measured samples, was estimated longer than 4 months and able to analyze more than 1500 samples, whichever comes first. The four electrodes we used during 18 months behaved all the same way. The correlation between measurements performed in whole blood (WB-cMg2+) and in the corresponding serum (S-cMg2+) was excellent: WB-cMg2+ = 0.954.S-cMg2+ +0.02 mmol.L-1; r = 0.998; n = 60.

Assessment of the role of genetic polymorphism in venous thrombosis through artificial neural networks.

PURPOSE: To assess the role of genetic polymorphisms in venous thrombosis events (VTE) using Artificial Neural Networks (ANNs), a model for solving non-linear problems frequently associated with complex biological systems, due to interactions between biological, genetic and environmental factors. METHODS: A database was generated from a case-control study of venous thrombosis, using 238 patients and 211 controls. The database of 64 variables included age, gender and a panel of 62 genetic variants. Three different ANNs were compared, with logistic regression for the accuracy of predicting cases and controls. RESULTS: ANNs yielded a better performance than the logistic regression algorithm. Indeed, through ANNs models, the 62 variables related to genetic variants were first reduced to a set of 9, and then of 3 (MTHFR 677 C/T, FV arg506gln, ICAM1 gly214arg). CONCLUSIONS: The findings of this study illustrate the power of ANN in evaluating multifactorial data, and show that the different sensitivities of the models of elaboration are related to the characteristics of the data. This may contribute to a better understanding of the role played by genetic polymorphisms in VTE, and help to define, if possible, a test panel of genetic variants to estimate an individual's probability of developing the disease.

Use of radioguided surgery with [111In]-pentetreotide in the management of an ACTH-secreting bronchial carcinoid causing ectopic Cushing's syndrome.

Intraoperative [111In]-pentetreotide scintigraphy with a hand-held gamma detector probe has recently been proposed to increase the intraoperative detection rate of small neuroendocrine tumors and their metastases. We report a case of a 28-yr-old woman with ectopic Cushing's syndrome due to an ACTH-secreting bronchial carcinoid, in whom the use of radioguided surgery improved disease management. At presentation, radiolabeled pentetreotide scintigraphy was the only procedure able to detect the ectopic source of ACTH. After radiologic confirmation, the patient underwent removal of a bronchial carcinoid, with disease persistence. After surgery, pentetreotide scintigraphy showed pathologic uptake in the mediastinum not previously detected at surgery and only subsequently confirmed by radiologic studies. Despite a second thoracic exploration, hormonal, scintigraphic, and radiological evidence of residual disease persisted. Radioguided surgery was then performed using a hand-held gamma probe 48 h after iv administration of a tracer dose of radiolabeled [111In-DTPA-D-Phe1]-pentetreotide, which permitted detection and removal of multiple residual mediastinal lymph node metastases. Clinical and radiologic cure, with no evidence of tracer uptake at pentetreotide scintigraphy, was subsequently observed. The use of an intraoperative gamma counter appears a promising procedure in the management of metastatic ACTH-secreting bronchial carcinoids.