Attilia Giuliani

Contribution to characterization of oxidative stress in HIV/AIDS patients

Infection by human immunodeficiency virus (HIV) causes persistent chronic inflammation. Viral Tat protein plays a role in the intracellular increase of reactive oxygen species (ROS) thus increasing apoptotic index, mostly the one mediated by FAS/CD95, and depleting CD4+ T lymphocytes. The aim of this study was to investigate whether there is a relationship between an extensive array of redox status indices (glutathione (GSH), malondialdehyde (MDA), peroxidation potential, total antioxidant status, glutathione peroxidase (GPx), superoxide dismutase (SOD), total hydroperoxide (TH), DNA fragmentation) and relative CD4, CD95, CD38/CD8 T lymphocyte counts in HIV/AIDS patients compared to healthy subjects. Blood samples from 85 HIV/AIDS patients and 40 healthy subjects were tested by spectrophotometric techniques in order to measure oxidative stress indices, and by flow cytometry to quantify T cell subsets. Patients were divided in two groups according to CDC 1993 guidelines. CD95 and CD38 increase paralleled the severity of HIV infection. Both a reduction of GSH levels and an increase in MDA and TH levels were detected in the plasma of HIV+ patients. These patients also showed an increase of DNA fragmentation in lymphocytes as well as a significant (P<0.05) reduction of GPx and an increase in SOD activity in erythrocytes. Relatively to the control group, HIV-infected patients had significantly differences in global indices of total antioxidant status. These results corroborate that substantial oxidative stress occurs during HIV infection. To our knowledge this study is the first relating oxidative stress indices with both CD38/CD8 and CD95 lymphocytes subsets.

Effects of ozone oxidative preconditioning on nitric oxide generation and cellular redox balance in a rat model of hepatic ischaemia-reperfusion.

Background: Many studies indicate that oxygen free‐radical formation after reoxygenation of liver may initiate the cascade of hepatocellular injury. It has been demonstrated that controlled ozone administration may promote an oxidative preconditioning or adaptation to oxidative stress, preventing the damage induced by reactive oxygen species and protecting against liver ischaemia–reperfusion (I/R) injury.

Changes in the Ceramide Composition of Rat Forebrain Gangliosides with Age

Abstract: Five major gangliosides (GM1, GD1a, GDlb, GTlb, and GQlb) were extracted and isolated by normal‐phase HPLC from the forebrain of Sprague‐Dawley rats of ages ranging from 3 days to 24 months. Each ganglioside was fractionated by reverse‐phase HPLC into the molecular species carrying a single long‐chain base moiety. At all ages, the C18:1 and C20:l long‐chain base species predominated, whereas the C18:0 and C20:0 ones represented 1–3% of the total. The C18:l long‐chain base species, predominant at 3 days (91–96%), diminished with age and reached, at 2 years, 73%, 65%, 61%, 59%, and 45% of the total for GDla, GM1, GTlb, GDlb, and GQlb, respectively. The content of the C20:1 long‐chain base species, low at birth (4–9%), increased with age in all gangliosides and reached, at 2 years, 27–55% of the total. The developmental behavior of the ganglioside species containing the C18:1 long‐chain base was characterized by the following: (a) a biphasic profile with a maximum around 15 days for GD1a, the most abundant ganglioside at all ages; (b) an increase until 6 months for GM1; (c) a sharp decrease until 30 days, followed by leveling for GTlb; and (d) a low, constant level for GDlb and GQlb. All the ganglioside species containing the C20:1 long‐chain base showed a constant increase during development, the increase being more marked in the first 30 days. The molecular species of all gangliosides carrying the C18:1 long‐chain base were virtually devoid of 20:0 fatty acids and carried a higher content of 18:0 fatty acids than the corresponding C20:l long‐chain base species (average 80 versus 57%). Moreover, in the C18:1 long‐chain base species, the 18:0 fatty acid content diminished with age from 89 to 72%, with a concurrent increase of 16:0 and 18:1 fatty acids, whereas the C20:l long‐chain base species had an age‐constant fatty acid composition.

Neuroprotective efficacy of nimesulide against hippocampal neuronal damage following transient forebrain ischemia

Cyclooxygenase-2 is involved in the inflammatory component of the ischemic cascade, playing an important role in the delayed progression of the brain damage. The present study evaluated the pharmacological effects of the selective cyclooxygenase-2 inhibitor nimesulide on delayed neuronal death of hippocampal CA1 neurons following transient global cerebral ischemia in gerbils. Administration of therapeutically relevant doses of nimesulide (3, 6 and 12 mg/kg; i.p.) 30 min before ischemia and at 6, 12, 24, 48 and 72 h after ischemia significantly (P<0.01) reduced hippocampal neuronal damage. Treatment with a single dose of nimesulide given 30 min before ischemia also resulted in a significant increase in the number of healthy neurons in the hippocampal CA1 sector 7 days after ischemia. Of interest is the finding that nimesulide rescued CA1 pyramidal neurons from ischemic death even when treatment was delayed until 24 h after ischemia (34+/-9% protection). Neuroprotective effect of nimesulide is still evident 30 days after the ischemic episode, providing the first experimental evidence that cyclooxygenase-2 inhibitors confer a long-lasting neuroprotection. Oral administration of nimesulide was also able to significantly reduce brain damage, suggesting that protective effects are independent of the route of administration. The present study confirms the ability of cyclooxygenase-2 inhibitors to reduce brain damage induced by cerebral ischemia and indicates that nimesulide can provide protection when administered for up to 24 h post-ischemia.

Anti-inflammatory activity and lipid peroxidation inhibition of iridoid lamiide isolated from Bouchea fluminensis (Vell.) Mold. (Verbenaceae).

Anti-inflammatory activity of an ethanolic extract from Bouchea fluminensis leaves was demonstrated. From de ethanolic extract, the active compound was isolated and characterized as the iridoid lamiide. The activity of lamiide on rat-brain phospholipid peroxidation showed a powerful effect (IC(50)=0.92+/-0.01 mM) and an anti-inflammatory activity in carrageenin-induced paw edema test (ED(50)=62.3+/-7 mg/kg weight).

New chemical trends in ganglioside research

A report is given of recent progress in the methodology for isolation of gangliosides from natural sources, for the preparation of molecular species of gangliosides homogeneous in both the oligosaccharide and ceramide portions of the molecule, for chemical manipulation and derivatization of gangliosides, and for the preparation of gangliosides radiolabelled in different parts of the molecule. Particular emphasis has been given to: high performance liquid chromatographic procedures capable to separate gangliosides on the basis of their oligosaccharide or ceramide moieties and yielding completely homogeneous compounds, that is gangliosides with a single oligosaccharide, a single long chain base and a single fatty acid; two-dimensional thin-layer chromatographic procedures, provided with a fully computerized quantification system, particularly suitable to identifying gangliosides containing alkali-labile linkages, including ganglioside lactones; chemical procedures of high yield for reducing gangliosides at the double bond of long chain base, for selective removal of the fatty acyl moiety and replacement with a novel fatty acid, and for the synthesis of ganglioside lactones; chemical procedures for inserting fluorescent, paramagnetic or photoreactive probes at the fatty acyl part of the ganglioside molecule; procedures for chemical isotopic radiolabelling of gangliosides at the level of sialic acid acetyl group and at the fatty acid moiety. Examples are provided evidencing the significance and potential use of a variety of ganglioside derivatives in the study of ganglioside metabolism and functional implications.

Patterns of some lysosomal enzymes in the plasma and of proteins in urine of workers exposed to inorganic mercury

SummaryIn a population of workers (81 subjects) exposed to inorganic mercury vapors in a chlorine alkali plant (airborne mercury concentration between 0.06 and 0.3 mg/m3) and in a group (104 subjects) of people never exposed to mercury the plasma level of β-galactosidase, β-glucuronidase, β-N-acetylglucosaminidase, and β-glucosidase was determined.The results are as follows:1.The plasma level of the four acid lysosomal hydrolases are higher in the group of workers exposed to mercury than in the control group. The difference is significant at the P < 0.001 level for all studied enzymatic activities.2.There is a significant correlation between the increase of the plasma levels of these enzymatic activities and the degree of exposure. These results suggest the use of this procedure for detecting an undue mercury absorption in workers exposed to the metal before any clinical signs.RBC and plasma cholinesterase activities were not affected. The results of the study of urinary proteins were not useful for detecting an early impairment of the kidney function. However, in some cases we observed a marked glomerular proteinuria without any clinical explanation: this fact makes one very cautious about denying the existence of a kidney impairment in workers exposed to the metal before any clinical sign is present.

Behaviour of several enzymes of lysosomal origin in human plasma during pregnancy

The following enzymes of lysosomal origin were fluorimetrically determined in maternal plasma from the second to the ninth month of pregnancy at 1-mth intervals: beta-D-N-acetylglucosaminidase (EC 3.2.1.30), beta-D-glucuronidase (EC 3.2.1.31), beta-D-glucosidase (EC 3.2.1.21), beta-D-galactosidase (EC 3.2.1.22), alpha-D-galactosidase (EC 3.2.1.23), alpha-L-fucosidase (EC 3.2.1.51) and alpha-D-mannosidase (EC 3.2.1.24) (pH 4.0). As reference microsomal alpha-D-mannosidase (pH 5.7) was also studied. Thirty-eight healthy women, aged 18-37 yr, who had a normal pregnancy followed by normal parturition, were studied. All enzymes, with the only exception of beta-D-galactosidase, showed a progressive and statistically significant increase of activity throughout pregnancy. At the end of pregnancy, the increase ranged from a maximum of 5.6-fold for beta-D-N-acetylglucosaminidase to a minimum of 0.55-fold for alpha-D-mannosidase, pH 5.7. In the case of beta-D-N-acetylglucosaminidase, the level at the fifth month of pregnancy was significantly higher than that at the third month, and from the sixth to the ninth month each level significantly differed from that of the month immediately preceding.

Effect of Increase of Dietary Micronutrient Intake on Oxidative Stress Indicators in HIV/AIDS Patients

Several recent studies in human immunodeficiency virus (HIV) patients have identified micronutrient deficiencies as affecting progression to acquired immunodeficiency syndrome (AIDS) and death. Although the mechanisms are not known, micronutrient deficiencies may exacerbate the oxidative stress induced by HIV. In addition, infection and its evolution likely lead to an increased requirement for nutritional micronutrients, especially antioxidants. To evaluate this, 40 relatively healthy, institutionalized HIV-infected individuals were recruited for assessment before or three months after fresh fruit and vegetable supply were increased due to seasonal supply. Seven-day dietary records were recorded at the beginning (December) and end of the three-month study period (March). Oxidative stress indices and CD4+, CD38+/CD8+, and CD95+ T-lymphocyte subsets were also measured at these times. No significant differences were found in calorie or protein intake across the study period, but vitamin A, C, and E intakes all increased. A number of redox indicators were modified (increase: total antioxidant status, glutathione peroxidase, and glutathione; and decrease: superoxide dismutase) during the study period. However, no change in malondialdehyde, hydroperoxides, or DNA damage was noted but a significant reduction in CD38+/CD8+ relative count was seen. Within the context and limitations of this study, the increase of dietary fruits and vegetables intake for three months had some beneficial effects on nutrition, systemic redox balance, and immune parameters in HIV-infected persons.

Laser-light scattering study of size and stability of ganglioside-phospholipid small unilamellar vesicles

Abstract The effect of the presence of ganglioside GM1, up to 20% by mol, on the size, stability upon aging, and changes with dilution, of small unilamellar vesicles of egg phosphatidylcholine (EPC) was studied by static and dynamic laser-light scattering technique. Standardised conditions were preliminarily set up for the preparation of small unilamellar vesicles by the sonication-ultra-centrifugation method. Under these conditions the presence of ganglioside caused a progressive decrease of lipid concentration in the upper 2.2 ml of high speed supernatant, from the original 9 μmol/ml to 1.3 μmol/ml reached with vesicles containing 20% of GM1. The hydrodynamic radius ( R H ), apparent molecular weight ( M ) and polydispersity index (ν) decreased in the presence of ganglioside from the values of 16 nm, 3.3 × 10 6 and 0.15, respectively, to 11 nm, 1.9 × 10 6 and 0.10, in vesicle preparations with 20% of GM1. R H and ν of EPC vesicles increased by only 3% and 12%, respectively, upon aging at 37°C for 6 h. The variations of the same parameters in vesicles containing up to 15% of GM1 were still lower, indicating that the presence of ganglioside tends to increase the stability upon aging of small unilamellar vesicles. Dilution of vesicle dispersions down to 0.1 μmol/ml caused a modest, but significant increase of R H , M and ν. The extent of increase was similar for EPC and EPC/GM1 vesicles with a GM1 proportion lower than 20%. Passage of GM1 containing vesicles on molecular sieve chromatographic columns, caused changes of R H , M and ν, which were consistent with the dilution operated during chromatography.