Giuseppe Bellisola

Severe impairment of antioxidant system in human hepatoma

Catalase (CAT), glutathione‐peroxidase (GSH‐Px) activity and reduced glutathione content (GSH) were measured in patients who had hepatocellular carcinoma, and values compared with those of normal liver and liver adjacent to neoplastic tissue. The results showed a remarkable reduction of CAT in tumor and corresponding tumor‐free tissue (P < 0.001 and P < 0.02, respectively). All neoplastic samples had a significant lower activity of CAT than the corresponding adjacent tumor‐free tissue (P < 0.05). The GSH‐Px activity of tumor tissue also was lower than normal (P < 0.001) but similar to that of adjacent tissue. No correlation was noted between the two enzyme activities. Glutathione content was extremely low in tumor (P < 0.001) and even in tumor‐free tissue (P < 0.05) when compared with normal liver. In all cases the content of GSH in neoplastic tissue was lower than that of the corresponding tumor‐free tissue (P < 0.05). Whereas in normal liver the activity of GSH‐Px was positively correlated with the content of GSH, in the neoplastic tissue such a relationship disappeared. All these findings suggest that the antioxidant system of hepatocellular carcinoma cell is severely impaired.

Selenium and glutathione peroxidase variations induced by polyunsaturated fatty acids oral supplementation in humans.

Serum and erythrocyte selenium, erythrocyte and platelet glutathione-peroxidase (GSH-Px) activities, and erythrocyte reduced glutathione (GSH) content were measured in 25 healthy adult individuals before and after daily supplementation with 20 ml of fish oil for 10 weeks. Serum-Se decreased from 0.83 +/- 0.01 mumol/l to 0.75 +/- 0.02 mumol/l (mean +/- S.E.M.) (P less than 0.01); erythrocyte-Se decreased from 4.39 +/- 0.17 nmol/g hemoglobin (Hb) to 2.83 +/- 0.15 nmol/g (P less than 0.001). GSH-Px activities increased both in erythrocytes (6.93 +/- 0.24 iu/g vs 8.18 +/- 0.27 iu/g Hb, P less than 0.01) and in platelets (69.2 +/- 2.8 iu/g vs 90.9 +/- 3.6 iu/g protein, P less than 0.001). The concentration of GSH in erythrocytes fell from 9.56 +/- 0.29 mumol/g Hb to 5.90 +/- 0.30 mumol/g Hb (P less than 0.001). The effects on plasma lipids were evident only for triglycerides (before 1.96 +/- 0.16 mmol/l, after 1.75 +/- 0.14 mmol/l, P less than 0.001). We hypothesise the enrichment of erythrocyte and platelet membranes with polyunsaturated fatty acids (PUFAs), following fish oil intake, can generate increased amounts of lipid peroxides and thus allosterically activate GSH-Px: with time this is harmful for the integrity of the enzyme molecule and Se release may result. We suggest that the Se status of individuals given PUFAs is assessed before and during intake; Se supplements should only be given when serum and/or erythrocyte Se are reduced.

Immunotoxins and other conjugates: preparation and general characteristics.

Targeted toxins represent an invaluable tool offering a wide range of potential applications, both in experimental models and in the clinics. Here we will review several aspects related to the preparation and properties of carrier molecule-toxin heteroconjugates and fusion toxins.

Diagnostic and prognostic value of serum copper and plasma fibrinogen in hepatic carcinoma

To investigate the diagnostic and prognostic value of several biochemical tests in primary liver tumors, the authors studied 36 cases (4 cholangiocarcinomas and 32 hepatocellular carcinomas, 10 of which were associated with cirrhosis) and 47 cases of liver cirrhosis, all with morphologically proven diagnosis. Serum copper (SCu) and plasma fibrinogen (PF) appeared the most useful tests in differential diagnosis between tumors and cirrhosis. In liver tumors, mean SCu level was 200.50, standard deviation (SD) 47.17 μg/dl (121.40, SD 25.90 μg/dl in cirrhosis; P < 0.001). PF level was 461.78, SD 151.25 mg/dl in tumors (275.30 SD, 124.40 mg/dl in cirrhosis; P < 0.001). SCu had a good sensitivity (0.80) and a high specificity (0.92) at a cutoff value of 160 μg/dl; when the cutoff level was raised to 170 μg/dl, the specificity increased to 1, with a sensitivity of 0.77. The combination of SCu and PF improved the diagnostic value slightly. Moreover, with an estimated frequency of tumor in cirrhosis of 10%, SCu had a positive predictive value of 1 (cutoff, 170 μg/dl) and a negative predictive value of 0.97. In nine patients SCu levels decreased after surgical removal of tumor; five other patients, sequentially studied, showed an increase of SCu level that correlated with the progression of the disease. Finally, patients with longer survival had a lower SCu level. These findings suggest that SCu level may be used as a screening test for early detection of neoplastic degeneration, and it is correlated with the extension of tumor mass.

The TSH-dependent variation of the essential elements iodine, selenium and zinc within human thyroid tissues.

Instrumental Neutron Activation Analysis was used in order to measure iodine, selenium and zinc concentration in thyroid samples. A pair of samples of normal and nodular tissue were collected from the thyroid gland from 72 patients selected on the basis of pathological criteria (44 cases of multinodular goiter, 12 of chronic lymphocytic thyroiditis (CLT), 6 of thyroid adenoma (TA) and 12 of thyroid cancer (TC)). The check for tissue homogeneity and sampling error was performed by means of the coefficient of variation (CV%) of the elements in replicate samples of normal and altered tissues. High CV% values (> 15%) for iodine reflected a functional variability in thyroid follicles, while low CV% values (< 10%) for selenium and zinc indicated that the composition of selected tissues was rather homogeneous. The variation of the elements concentration was compared in normal and altered tissues. The mean element concentrations had values close to those already reported in the literature; furthermore, our patients had marginal iodine and selenium deficiency. Both normal and nodular tissues in CLT showed statistically significant lower zinc values as compared with the other thyroid diseases. To evaluate the thyroid function, thyroid stimulating hormone (TSH) and thyroxine (T4) levels were measured in the serum of patients. Two arbitrary serum-TSH threshold levels (TSH < 1.0 and > 4.0 mU/L) were introduced in order to classify, respectively, hyperthyroidism and hypothyroidism, as well as euthyroid conditions (1.0 < TSH < 4.0 mU/L), and each patient was assigned to one of these groups. The influence of TSH in the variation of the concentration of iodine, selenium and zinc in normal and altered human thyroid tissues was significant.

Catalase activity in human hepatocellular carcinoma (HCC).

Liver catalase activity, one of the free-radical scavenger enzymes, has been measured in 22 normal subjects and compared with that of 13 patients suffering from hepatocellular carcinoma. The activity was estimated both in tumor tissue and in tumor-free tissue. A significant reduction of catalase activity was noted in tumor tissue (p less than 0.001) as well as in the adjacent tumor-free tissue (p less than 0.02). In patients with hepatoma, the serum iron level was lower than in normal (p less than 0.01) and was correlated with enzyme activity (r = 0.958). These findings suggest that in hepatocarcinoma the free radical scavenger system is impaired.

Decreased activity of liver glutathione peroxidase in human hepatocellular carcinoma

Glutathione peroxidase (GSH-Px) activity, one of the scavenger enzymes of oxygen active radicals, has been measured in hepatocellular carcinoma (HCC) of 17 patients and the values compared with the activity of adjacent tumor-free tissue and with those of 30 histologically normal livers. The results demonstrate a reduced GSH-Px activity in neoplastic tissue (21.19 vs 33.74 U/g prot.; P less than 0.001). However, the adjacent tumor-free liver also had a reduced activity when compared with normal tissue (23.15 vs 33.74 U/g prot.; P less than 0.01), but this value did not differ from that of HCC tissue. These data suggest that HCC might develop in a GSH-Px-deficient condition.

Rapid recognition of drug-resistance/sensitivity in leukemic cells by Fourier transform infrared microspectroscopy and unsupervised hierarchical cluster analysis

We tested the ability of Fourier Transform (FT) InfraRed (IR) microspectroscopy (microFTIR) in combination with unsupervised Hierarchical Cluster Analysis (HCA) in identifying drug-resistance/sensitivity in leukemic cells exposed to tyrosine kinase inhibitors (TKIs). Experiments were carried out in a well-established mouse model of human Chronic Myelogenous Leukemia (CML). Mouse-derived pro-B Ba/F3 cells transfected with and stably expressing the human p210(BCR-ABL) drug-sensitive wild-type BCR-ABL or the V299L or T315I p210(BCR-ABL) drug-resistant BCR-ABL mutants were exposed to imatinib-mesylate (IMA) or dasatinib (DAS). MicroFTIR was carried out at the Diamond IR beamline MIRIAM where the mid-IR absorbance spectra of individual Ba/F3 cells were acquired using the high brilliance IR synchrotron radiation (SR) via aperture of 15 × 15 μm(2) in sizes. A conventional IR source (globar) was used to compare average spectra over 15 cells or more. IR signatures of drug actions were identified by supervised analyses in the spectra of TKI-sensitive cells. Unsupervised HCA applied to selected intervals of wavenumber allowed us to classify the IR patterns of viable (drug-resistant) and apoptotic (drug-sensitive) cells with an accuracy of >95%. The results from microFTIR + HCA analysis were cross-validated with those obtained via immunochemical methods, i.e. immunoblotting and flow cytometry (FC) that resulted directly and significantly correlated. We conclude that this combined microFTIR + HCA method potentially represents a rapid, convenient and robust screening approach to study the impact of drugs in leukemic cells as well as in peripheral blasts from patients in clinical trials with new anti-leukemic drugs.

Plasma fibronectin in liver cirrhosis and its diagnostic value

Plasma fibronectin (FN) has been measured by immunonephelometric method in 100 cirrhotic patients and compared with that of 77 normal subjects and with that of 57 patients suffering from liver disorders different from cirrhosis. Both, compensated and decompensated cirrhotics had lower plasma FN than controls (31.14 +/- 11.42 and 20.88 +/- 10.43 respectively vs 40.13 +/- 8.58 mg/dl; rho less than 0.02 and rho less than 0.001). FN in ascitic patients was lower than in non-ascitic (rho less than 0.001). These differences were not due to different weight or age of patients. It appears, therefore, that FN parallels in cirrhosis the grade of liver function impairment. No significant difference has been noted between plasma FN of patients with liver diseases different from cirrhosis and control subjects. In cirrhosis, a positive relation has been observed among FN and other parameters of liver function such as serum albumin, cholinesterase activity, fibrinogen and prothrombin time. Plasma FN has a low sensitivity but a high specificity and a good positive predictive value in distinguishing normals and patients with liver disorders different from cirrhosis. This diagnostic value is similar to that of serum albumin.

Detection of CFTR protein in human leukocytes by flow cytometry

Leukocytes have previously been shown to express detectable levels of the protein cystic fibrosis transmembrane conductance regulator (CFTR). This study aims to evaluate the application of flow cytometric (FC) analysis to detect CFTR expression, and changes thereof, in these cells. Aliquots (200 μL) of peripheral whole blood from 12 healthy control volunteers (CTRLs), 12 carriers of a CFTR mutation (CFC), and 40 patients with cystic fibrosis (CF) carrying various combinations of CFTR mutations were incubated with specific fluorescent probes recognizing CFTR protein expressed on the plasma membrane of leukocytes. FC was applied to analyze CFTR expression in monocytes, lymphocytes, and polymorphonuclear (PMN) cells. CFTR protein was detected in monocytes and lymphocytes, whereas inconclusive results were obtained from the analysis of PMN cells. Mean fluorescence intensity (MFI) ratio value and %CFTR‐positive cells above a selected threshold were the two parameters selected to quantify CFTR expression in cells. Lowest variability and the highest reproducibility were obtained when analyzing monocytes. ANOVA results indicated that both parameters were able to discriminate monocytes of healthy controls and CF individuals according to CFTR mutation classes with high accuracy. Significantly increased MFI ratio values were recorded in CFTR‐defective cells that were also able to improve CFTR function after ex vivo treatment with PTC124 (Ataluren), an investigative drug designed to permit the ribosome to read through nonsense CFTR mutations. The method described is minimally invasive and may be used in the monitoring of responses to drugs whose efficacy can depend on increased CFTR protein expression levels.