Silvia Ghezzi

Polymerase chain reaction for Toxoplasma gondii DNA in the cerebrospinal fluid of AIDS patients with focal brain lesions

ObjectiveTo study the accuracy of polymerase chain reaction (PCR) for Toxoplasma gondii DNA in the cerebrospinal fluid (CSF) of AIDS patients for the diagnosis of T. gondii encephalitis. PatientsEighty-two AIDS patients with brain lesions. At autopsy, 19 patients (group A) had toxoplasmic encephalitis and 33 (group B) primary brain lymphoma or other infections. Brain histology was not available for 30 patients; cerebral lesions improved after anti-Toxoplasma therapy in 16 (group C), but there was no improvement in 14 patients (group D). MethodsT. gondii RH strain was serially diluted in microplate wells. After heat denaturation, nested PCR was performed on diluted tachyzoites and on 10 μl CSF with primers flanking the B1 repetitive region of T. gondii genome. ResultsDNA from one to five tachyzoites was detected in each experiment. PCR was positive in eight (42.1 %) out of 19 group A samples, none of the group B samples, 10 (62.5%) out of 16 group C samples and none of the group D samples. Among group A and C patients, PCR was positive in all 11, and in seven out of 24 (29.1%; P < 0.04) patients who had received anti-Toxoplasma therapy for less or more than 1 week at the time of rachicentesis, respectively. ConclusionsNested PCR for T. gondii in CSF may improve early differential diagnosis of AIDS-associated focal brain lesions. Higher diagnostic accuracy was achieved when lumbar puncture was performed in the first week of anti-Toxoplasma therapy.

Different patterns characterize Omega 6 and Omega 3 long chain polyunsaturated fatty acid levels in blood from Italian infants, children, adults and elderly

Long chain polyunsaturated fatty acids (LC-PUFA), especially the Omega 3, modulate key functions in the body. Their circulating levels are representative of their status, and may vary at different ages. We have compared the FA status in Italian subjects from neonates to adulthood, assessed through FA analysis of blood drops from fingertips. Data from four cohorts of Italian subjects (total number 1835), have been pooled in four age-groups: neonates (4 days, n=81), children (2-9 years, n=728), adults (40-59 years, n=434) and elderly (60-79 years, n=592). LC-PUFA of both series (Omega 3 and 6) are higher in the blood of neonates than at subsequent ages, reflecting the efficient transfer of these FA from mothers to the fetus. In contrast, the lowest levels of Omega 3 PUFA, especially of DHA, are found in children, probably reflecting inadequate dietary intakes, with possible consequences on the health status at subsequent ages.

Relative potencies of statins in reducing cholesterol synthesis and enhancing linoleic acid metabolism.

Simvastatin enhances the conversion of linoleic acid to their long chain polyunsaturated fatty acid derivatives, e.g. arachidonic acid, in addition to typically inhibiting the de novo cholesterol synthesis, in cultured cells. The dose-response relationships for the above effects show that simvastatin, atorvastatin and fluvastatin affect linoleic acid conversion and the delta5 desaturase step more potently than the synthesis of cholesterol, simvastatin being the most effective in inhibiting sterol synthesis, whereas atorvastatin in stimulating the conversion of linoleic acid.

Whole blood fatty acid analysis with micromethod in cystic fibrosis and pulmonary disease.

OBJECTIVESnTo assess fatty acid (FA) profiles in whole blood of 90 cystic fibrosis patients (CF) and 30 control subjects (C) and to correlate FA changes to the severity of respiratory disease.nnnMETHODSnWhole blood FA were assessed by GC with a micromethod-based analysis.nnnRESULTSnSaturated and monounsaturated FA are higher, whereas polyunsaturated FA are lower in CF versus C with reduction of total n-6 FA, 22:5n-3 and 22:6n-3 (DHA). The product of linoleic acid (LA) x DHA, proposed as a marker for the disease, is 30% lower in CF than in C. Correlations with the severity of the respiratory disease are present for different FA and for the LA x DHA product. There is a reduction of Delta5 desaturase activity in CF, greater in severe disease, suggesting a basic metabolic alteration.nnnCONCLUSIONSnThe micromethod-based analysis of blood FA facilitates the assessment of the FA status while confirming alterations of FA profiles already reported in specific blood compartments of CF.

Effects of cigarette smoke on cell viability, linoleic acid metabolism and cholesterol synthesis, in THP-1 cells.

Cigarette smoke (CS) contains thousands of substances, mainly free radicals that have as a target the polyunsaturated fatty acids (PUFA). Long chain PUFA are produced through elongation and desaturation reactions from their precursors; the desaturation reactions are catalyzed by different enzymes: the conversion of 18:2n−6 (linoleic acid, LA) to 18:3n−6 by Δ6 desaturase, while that of 20:3n−6 to 20:4n−6 by Δ5 desaturase. The aim of this work is to evaluate the effect of serum exposed to cigarette smoke (SE-FBS) on (1) cell viability and proliferation, (2) [1-14C] LA conversion and desaturase activities in THP−1 cells, a monocytic cell line. In THP−1, CS inhibits cell proliferation dose-dependently, by producing a modification in the cell cycle with a reduced number of cells in synthesis and mitosis phases at higher concentrations. CS also decreases [1-14C] LA conversion to its derivatives in a concentration-dependent manner, inhibiting the activities of Δ6 and mainly Δ5 desaturase. In addition, CS does not modify the incorporation of LA into various lipid classes but it reduces cholesterol synthesis from radiolabelled acetate, and increases free fatty acid, TG and CE levels. In conclusion, CS affects lipid metabolism, inhibiting LA conversion and desaturase activities. CS also shifts the “de novo” lipid synthesis from free cholesterol to TG and CE, where LA is preferentially esterified.

Pharmacological modulation of fatty acid desaturation and of cholesterol biosynthesis in THP-1 cells

In THP-1 cells, simvastatin decreases, in a concentration-dependent manner, cholesterol synthesis and increases linoleic acid (LA) conversion to its long-chain derivatives, in particular to arachidonic acid, activating Δ6 and Δ5 fatty acid (FA) desaturases. The intermediates in cholesterol synthesis, mevalonate and geranylgeraniol, partially reverse the effects of simvastatin on the LA conversion. The aims of this work were to evaluate: (i) the correlation between cholesterol synthesis and desaturase activity and (ii) the possible involvement of protein isoprenylation in desaturase activity, assessed through pharmacological treatments. THP-1 cells were incubated with [1-14C]LA or with [1-14C]di-homo-γ-linolenic acid (DHGLA) and treated with simvastatin or with curcumin and nicardipine, inhibitors of desaturases. Curcumin was more active than nicardipine in inhibiting LA and DHGLA conversion: 20 μM curcumin, alone or with simvastatin, totally inhibited Δ6 and Δ5 desaturation steps; 10 μM nicardipine only partially inhibited the enzymes, being more active on Δ5 desaturase. Simvastatin treatment decreased the incorporation of acetate in cholesterol (−93.8%) and cholesterol esters (−70.2%), as expected. Curcumin and nicardipine also decreased cholesterol synthesis and potentiated simvastatin. Finally, the isoprenylation inhibitors (perillic acid and GGTI-286) neither affected the conversion of LA nor inhibited the Δ5 desaturase activity. In conclusion, our results indicate that there is no direct relationship between cholesterol synthesis and desaturase activity. In fact, simvastatin decreased cholesterol synthesis and enhanced LA conversion (mainly Δ5 desaturation), whereas curcumin and nicardipin decreased Δ5 desaturation, with a limited effect on cholesterol synthesis.

The in vitro effects of cigarette smoke on fatty acid metabolism are partially counteracted by simvastatin

BACKGROUNDnStatins enhance the synthesis of long-chain polyunsaturated fatty acids (LC-PUFAs) from their precursors both in vitro and in vivo. In particular, an increased conversion of linoleic acid (LA) and of alpha-linolenic acid to their derivatives is observed in cultured cells. On the contrary, cigarette smoke (CS) negatively and dose-dependently affects the LC-PUFA production.nnnAIMnTo evaluate the effects of CS alone or with simvastatin, on [1-(14)C] LA metabolism in THP-1 cells.nnnRESULTSnCS inhibits LA conversion; after co-incubation, simvastatin nullifies the effects of CS, maintaining LA conversion comparable to controls. However, at the highest CS concentration, simvastatin is unable to counteract the effects of CS. Changes of LA conversion reflect the modulation of desaturase activities by simvastatin and CS.nnnCONCLUSIONnCS decreases PUFA conversion and its effects are modulated by the opposite effect of statins. It can be speculated that statin treatments in smoking patients may provide some beneficial effects on PUFA metabolism in addition to lowering cholesterol levels.

Immobilized Pig Brain NAD Glycohydrolase for the Preparation of NAD Analogues

Among the enzymes whose activities are affected by pyridine nucleotides, the dehydrogenases are widely studied under the biochemical and physiological point of view. Their technological importance is based on the development of methods for clinical analyses as well as on the preparation of particular biosensors. In addition to dehydrogenases, other enzymes related to NAD and NADP play an important role in reactions where the pyridine nucleotides work as substrates instead of coenzymes. Some of these reactions are connected with pathological infections, such as bacteria toxins, whereas other enzymes, named NADases or NAD glycohydrolases, catalyze reactions normally occurring in fungi, mammalian seminal plasma, mammalian organs, and other tissues, leading to the breaking of the nicotinamide N-ribosidic bond in NAD(P). The enzymes from bacteria, fungi, and mammalian seminal plasma (EC 3.2.2.5) show a pure hydrolytic activity-5 and yield nicotinamide and adenosine diphosphate ribose (ADPR) according to the following reaction: