Mario Governa

Decrease in peripheral blood polymorphonuclear leukocyte chemotactic index in endometriosis: role of prostaglandin E2 release

Objective To investigate the effect of disease on peripheral blood polymorphonuclear leukocyte chemotactic index and natural killer cell cytotoxicity and to provide additional information concerning the cell-mediated immune function in endometriosis. Methods Chemotactic index of peripheral blood polymorphonuclear leukocytes, natural killer cell activity, and plasma estradiol (E2) and plasma prostaglandin (PG) E2 levels were evaluated in 46 women who underwent laparoscopy or laparotomy for pelvic pain, infertility, and/or benign adnexal masses. Results The 20 women (43%) with endometriosis showed a decrease in peripheral blood polymorphonuclear leukocyte chemotactic index, related to advanced disease stage (P < .001). A significant inverse correlation was observed between plasma PGE2 levels and chemotactic index in stage III and IV endometriosis (r = −.73, P = .004). Similarly, natural cytotoxicity was decreased significantly with respect to the stage of endometriosis (P = .004) and related inversely to plasma PGE2 levels (r = −.74, P = .003). A direct relationship was observed between PGE2 and plasma E2 levels (r = .59, P = .006). Conclusion Advanced endometriosis is associated with decreased peripheral blood polymorphonuclear leukocyte chemotactic index and natural killer cytotoxicity, which may be related to plasma PGE2 and E2 levels.

Profiling Tumor-Associated Markers for Early Detection of Malignant Mesothelioma: An Epidemiologic Study

Improved detection methods for diagnosis of asymptomatic malignant mesothelioma (MM) are essential for an early and reliable detection and treatment of this type of neoplastic disease. Thus, focus has been on finding tumor markers in the blood that can be used for noninvasive detection of MM. Ninety-four asbestos-exposed subjects defined at high risk, 22 patients with MM, and 54 healthy subjects were recruited for evaluation of the clinical significance of 8-hydroxy-2′-deoxyguanosine (8OHdG) in WBCs and plasma concentrations of soluble mesothelin-related peptides (SMRPs), angiogenic factors [platelet-derived growth factor β, hepatocyte growth factor, basic fibroblast growth factor, and vascular endothelial growth factor β (VEGFβ)], and matrix proteases [matrix metalloproteinase (MMP) 2, MMP9, tissue inhibitor of metalloproteinase (TIMP) 1, and TIMP2] for potential early detection of MM. The area under receiver operating characteristic (ROC) curves indicate that 8OHdG levels can discriminate asbestos-exposed subjects from healthy controls but not from MM patients. Significant area under ROC curve values were found for SMRPs, discriminating asbestos-exposed subjects from MM patients but not from healthy controls. Except for platelet-derived growth factor β, the hepatocyte growth factor, basic fibroblast growth factor, and VEGFβ can significantly differentiate high-risk individuals from healthy control and cancer groups. No diagnostic value was observed for MMP2, MMP9, TIMP1, and TIMP2. In addition to the diagnostic performance defined by the ROC analysis, the sensitivity and specificity results of markers with clinical significance were calculated at defined cutoffs. The combination of 8OHdG, VEGFβ, and SMRPs best distinguished the individual groups, suggesting a potential indicator of early and advanced MM cancers. The combination of blood biomarkers and radiographic findings could be used to stratify the risk of mesothelioma in asbestos-exposed populations. (Cancer Epidemiol Biomarkers Prev 2008;17(1):163–70)

Effects of lead on polymorphonuclear leukocyte (PMN) functions in occupationally exposed workers

Previous in vitro experiments have shown that lead can inhibit PMN chemotaxis, phagocytosis and super-oxide formation. Moreover, we have observed an inhibition of PMN chemotaxis in workers occupationally exposed to lead with a mean blood lead concentration of 3.06 (μmol/l. The present study was carried out to evaluate locomotion and luminol assisted chemiluminescence (CL) of polymorphonuclear leukocytes (PMN) harvested from ten lead occupationally exposed workers with blood lead concentrations of 1.59 μmol/l (SD 0.27 μmol/l). Since lipids affect PMN activity and lipid composition is modified in erythrocytes of lead workers, PMN lipids were also studied. Ten healthy male subjects of the same age were taken as controls. Chemotaxis, i.e. locomotion stimulated through a specific membrane receptor, was impaired in the PMN of lead workers, but random migration, i. e. unstimulated cell locomotion, and respiratory burst were both unmodified. Cholesterol and phospholipids were not changed, but the percentage of arachidonic acid was significantly increased. The release of LTB4, generated by the oxidative metabolism of arachidonic acid, was increased. CL, which detects reactive oxygen species (ROS), was unmodified, but this lack of change could be the result of an increase in ROS, due to the augmentated percentage of arachidonic acid, and of a decrease in ROS, due to a direct inhibitory effect of lead on ROS generation. On the basis of the results from these ex vivo experiments, the conclusion that chemotaxis is the PMN function primarily affected by lead was confirmed. PMN are considered to be one of the first cellular targets for the action of lead; low exposure to lead modifies their activity and mainly modifies chemotaxis and LTB4 production.

Urinary excretion of 2,5-hexanedione and peripheral polyneuropathies in workers exposed to hexane

Forty shoe factory workers who were exposed to hexane were investigated to see if there was a correlation between electroneuromyographic changes indicative of neuropathy and urinary excretion of 2,5-hexanedione. Urinary samples were analyzed for the presence of the metabolic products of n-hexane and its isomers. Electrodiagnostic examination was carried out following the urinary sampling. A rating scale was used to obtain a cumulative numeric index of electrodiagnostic findings. 2,5-Hexanedione and gamma-valerolactone were discovered in all cases, while 2-hexanol was found in 11 cases. 2,5-Hexanedione was the main metabolite in most cases (39 of 40). Only in 1 case was a low level of 2-methyl-2-pentanol detected; 3-methyl-2-pentanol was never detected. Metabolic products of cyclohexane were present in about one-fifth of the cases, while trichloroethanol, a metabolic product of trichoroethylene, was nearly always present, all at very low concentrations. Electromyographic abnormalities significant for early detection of toxic polyneuropathy were found in 14 cases. A statistically significant correlation of the electroneuromyographic scoring on the urinary concentrations of measured metabolites was observed only with 2,5-hexanedione and gamma-valerolactone, both derived from n-hexane. Since gamma-valerolactone is probably not a true metabolite of n-hexane, our results support the hypothesis that polyneuropathies in shoemakers are due to 2,5-hexanedione. For practical purposes the urinary concentration of 2,5-hexanedione can serve as a predictive measurement for early detection of neurotoxic lesions at preclinical states.

Role of iron in asbestos-body-induced oxidant radical generation.

Asbestos bodies (AB) were harvested from human lung tissue digests and isolated from uncoated asbestos fibers. Samples containing 1000 AB were added to a reactive solution to investigate the ability of AB to oxidize deoxy-D-ribose and generate reactive oxygen species (ROS) in the presence of ascorbate and hydrogen peroxide as determined by formation of thiobarbituric acid (TBA)-reactive products. Three types of asbestos fibers were tested for comparison, since they are known to be able to produce ROS. The absorbance values measured with 1000 AB were significantly higher than those observed with 1000 fibers of the three types of asbestos. Since in our reaction system the only source of transition metals was the iron-rich AB, data suggest iron derived from the ferritin coating of AB was involved in oxidant generation. Addition of iron to AB enhanced TBA-reactive product formation, while chelation of Fe with deferoxamine reduced this reaction. Hydroxyl radical scavengers 1,3-dimethyl-2-thiourea (DMTU) and mannitol (MN) also effectively blocked TBA-reactive product generation. Data indicate the importance of Fe in AB-induced oxidant damage. With the addition of polymorphonuclear leukocytes (PMN) to AB, incubation in the reactive solution gave very high amounts of TBA-reactive products, but using a reactive solution devoid of ascorbate, very low amounts of TBA-reactive products were generated. In the latter condition, the superoxide of cell membranes probably reduced and removed iron from AB-coating ferritin, but less effectively than ascorbate. Further after the possible reoxidation of Fe2+, Fe3+ could be coordinated by lactoferrin. Since such availability of reductant is never approached in living systems, the iron in the AB coating is unlikely to function as a catalyst of Fenton-type reactions in vivo.

In vitro impairment of human granulocyte functions by lead.

Chemotaxis and receptor independent phagocytosis of human polymorphonuclear leukocytes (PMNs) exposed to lead in vitro (concentrations between 1.2 μM and 115 μM) were studied. Chemotaxis was measured in Boyden chambers and phagocytosis was investigated using latex beads.Additional methods were also applied. Superoxide anion formation from PMNs activated with preopsonized zymosan was quantified as superoxide dismutase-inhibitable reduction of ferricytochrome c. Steady state fluorescence polarization was performed using trimethylammonium diphenylexatriene (TMA-DPH).Lead concentrations were highly correlated both with decreased chemotactic activity r=0.70 p<0.01) and with decreased phagocytosis (r=0.68 p<0.01). Ferricytochrome c reduction was not significantly affected. An increase in fluorescence polarization was recorded at the highest concentration of lead used, i.e. 57.6 μM and 115 μM, both in unstimulated PMNs and in PMNs activated with N-formyl-methionyl-leucyl-phenylalanine chemotactic peptide (n-FMLP). Moreover, an increase in the fluorescence polarization was observed in PMNs pretreated with a microtubule disrupting drug, exposed to lead concentrations of 14.4 μM and 57.6 μM and then activated with n-FMLP; no increase was recorded at the lowest lead concentrations used, i.e. 1.2 μM and 3.6 μM.The possible interaction of lead with the membrane — cytoskeleton apparatus is discussed.

Biological effects and comparative cytotoxicity of thermal transformed asbestos-containing materials in a human alveolar epithelial cell line

Asbestos fibres can be transformed into potentially non-hazardous silicates by high-temperature treatment via complete solid-state transformation. A549 cells were exposed to standard concentrations of raw cement asbestos (RCA), chrysotile and cement asbestos subjected to an industrial process at 1200 degrees C (Cry_1200 and KRY.AS, respectively), raw commercial grey cement (GC). Cell growth rate and viability (MTT test) were detected in vitro. RCA and KRY.AS subjected to comprehensive microstructural study by electron microscopy were further in vitro assayed to compare their cytotoxic potential by morphostructural studies, proliferation index (Ki-67 antigen), apoptosis induction (AO/EB staining) assays and detection of intracellular reactive oxygen species (ROS) with the fluorescent DCFA dye. More severe cytotoxic damage was induced by RCA than by KRY.AS after each incubation period. Exposure to KRY.AS and GC resulted in comparable cell growth rates and cytotoxic effects. Cells incubated with RCA showed greater apoptotic induction and ROS production and a lower cell proliferation index than those exposed to KRY.AS. Chrysotile asbestos and RCA subjected to heat treatment underwent complete microstructure transformation. The final product of heat treatment of cement asbestos, KRY.AS, was considerably more inert and had lower cytotoxic potential than the original asbestos material in all in vitro tests.

Changes of membrane fluidity in chemotactic peptide-stimulated polymorphonuclear leukocytes

Although the phenomenon of stimulus-response coupling in polymorphonuclear leukocytes involves a series of membrane events the influence of stimulation on membrane fluidity is to clarify. In our experiments we have used 1-(4-trimethylaminophenyl) 6-phenyl-1,3,5-hexatriene and 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization technique to evaluate membrane fluidity in living polymorphonuclear leukocytes after stimulation with N-formyl-methyonil-leucyl-phenylalanine peptide which has a well defined membrane receptor on the plasma membrane. We report that polymorphonuclear leukocytes stimulation increases 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene polarization, only when colcemid, a microtubule disrupting drug, is added to polymorphonuclear leukocytes. This can be viewed as an indirect evidence that microtubules are involved in the control of polymorphonuclear leukocytes membrane fluidity. On the contrary no changes have been observed with 1,6-diphenyl-1,3,5-hexatriene. This study indicates the potential use of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene to evaluate the involvement of plasma membrane physical state during intact cell activity.

In vitro biological effects of clay minerals advised as substitutes for asbestos

We studied one sample of commercial sepiolite and two samples of commercial vermiculite—clay minerals proposed as replacements for asbestos—and testedin vitro their abilities to activate complement, to lyse erythrocytes, and to elicit the production of reactive oxygen species (ROS) with human polymorphonuclear leukocytes (PMN) or bovine alveolar macrophages (AM); their behavior was compared with that of asbestos fibers obtained from the Union International Contra Cancer (UICC) as reference standards, as well as with kaolinite and illite, main members of the clay mineral family.Since in short-termin vitro tests the biological activity of mineral particles seems especially related to the active sites on their surface, we first measured the specific surface area of each mineral. Sepiolite was unreactive in two of the three tests we used (complement activation and ROS production) and able to lyse a minimal percentage of red blood cells. Vermiculite was shown to be incapable of activating complement, to have a moderate hemolytic activity and a high ability to elicite ROS production, although lower than that of chrysotile. Sepiolite, therefore, might be of more interest than vermiculite, given the low level of biological effects detected during the tests used to compare both clay minerals with asbestos fibres. The ROS production does not seem to require phagocytosis. A high ROS production was observed with kaolinite: this result casts doubt on the ability of pathogenic mineral dustsin vitro to induce a greater release of ROS than nonpathogenic mineral dusts.

Increased membrane heterogeneity in stimulated human granulocytes

TMA‐DPH fluorescence decay in human PMN before and after stimulation with FMLP was studied using frequency domain fluorometry. Membrane heterogeneity was assessed by the width of the continuous distributions of lifetime values of Lorentzian shape used to describe the fluorescence decay. In non‐stimulated granulocytes TMA‐DPH fluorescence decay is characterized by two distributions of lifetime values centered at 6.5 and 1.0 ns and full width at half maximum of 0.3 and 1.2 ns, respectively. Within 15 min after stimulation, the center values of the two distribution components were 5.1 and 0.8 ns and the distribution width was 0.8 and 0.6 ns, respectively. These results indicate changes of membrane domain organization which can be ascribed to compositional changes redistribution of membrane components.