Survey of Lyme disease in Abruzzo (Italy).

This study reports the diagnostic activity for the serological diagnosis of Lyme Disease (LD) in the patients of Abruzzo, a central region of Italy. During the period from August 1994 to July 1999, serological examinations for anti-Borrelia burgdorferi antibodies were performed on 1089 samples from 769 patients with symptomatology consistent with LD. Using an immunoenzymatic technique which was confirmed with Western Blot, 29 patients were diagnosed positive. Twenty-five of these patients contracted the disease in Abruzzo, two during a trip to the USA, one was from Molise and one from Marche. Overall the patients were young, 64% were women and residents of costal areas who frequently engaged in naturalistic activities. The most common symptoms were articular and one patient presented Bannwarth Syndrome. The various antibiotic therapies used gave good results in most cases. These are the first cases reported in literature for this region and for Molise. We believe that LD is underestimated, especially due to the favorable climatic and environmental conditions present in this region. Therefore, we suggest an intensification of clinical and epidemiological controls.

Formation of 22 and 24 carbon 6-desaturated fatty acids from exogenous deuterated arachidonic acid is activated in THP-1 cells at high substrate concentrations.

Deuterated arachidonic acid (AA, [2H8]20:4 n-6) 1-25 microM, is converted to other fatty acids, as evaluated by gas chromatography-mass spectrometry, in THP-1 cells. The major products, in the 1 to 10 microM range, are 22:4 (elongated) and 20:3 (reduced in 5). At 25 microM, 24:4, 24:5 and 22:5 accumulate, with [2H8]/[2H0] ratios higher than in AA. At high AA concentration preferential conversion to elongated fatty acids with 5 unsaturations, through a 6 desaturase takes place and the 4-desaturated 22:5 appears to be formed through beta-oxidation of 24:5.

Eicosanoid and inositol phosphate response to platelet-activating factor (PAF) and to a PAF antagonist in rat astroglial cells.

The effects of different concentrations of exogenous platelet-activating factor (PAF) on the formation of arachidonic acid-cyclooxygenase metabolites and on the production of inositol phosphates have been investigated in a primary culture of rat astroglial cells. The cells were used at confluence and the purity was checked by immunostaining of the culture with specific antibodies against glial fibrillary acidic protein. Incubation of the cells with PAF (range 10(-9) to 10(-6) M) resulted in maximal accumulation of total inositol phosphate (620 +/- 60% increment over basal values, P < 0.001) at the concentration of 10(-8) M, after 1 min of stimulation. Smaller inositol phosphate accumulation occurred at higher concentrations of the agonist and at longer stimulation time. After 1 min of stimulation with PAF, the accumulation of the cyclooxygenase metabolites, thromboxane B2 (630 +/- 58 vs 20 +/- 2 pg/mg protein in non-stimulated samples) and 6-keto-prostaglandin F1 alpha (132 +/- 15 vs 55 +/- 7 pg/mg protein in non-stimulated samples) was also maximal at 10(-8) M concentration of the agonist. When the cultures were stimulated with PAF or Ca(2+)-ionophore after preincubation with equimolar concentration of the PAF inhibitor BN 52021, a significant inhibition in the synthesis of both inositol phosphates and cyclooxygenase metabolites occurred only in the PAF-stimulated cells.

n-6 and n-3 fatty acid accumulation in thp-1 cell phospholipids.

The human monocytic leukemia cell line THP-1 is depleted of the long-chain n-6, AA, when compared to human monocytes. This reflects the low availability of this FA in the growth medium generally used for cultured cells. The effects of AA, as well as EPA, supplementation of THP-1 cells on the incorporation of these FA in cell PL, especially in PC and PE, was investigated. In addition the incorporation of labeled AA in PL from THP-1 cells was compared to that in human monocytes. Measurements were done through HPLC separation of PL, detected by UV absorption and radioactivity, FA analysis by GC and characterization of PC subclasses by FAB-MS. Marked differences were observed in the incorporation of the two FA in cell PL, particularly two PC subclasses, and in the accumulation in individual PL after supplementation of THP-1 cells. Accumulation of AA and EPA in THP-1 cells appeared to be mutually independent. The incorporation of AA was also quite different in THP-1 from that in monocytes. Thus, characterization of the FA content in lipids of cultured cells is an essential requirement for optimal utilization of these cells.

Modulation of lipid derived mediators by polyunsaturated fatty acids.

Cell stimulation by a number of agonists triggers the formation of products of lipid hydrolysis, which act either as intracellular mediators of signal transduction or as modulators of cell-cell interactions. This process is mediated by the activation of hydrolytic enzymes, the phospholipases (PLase), especially the A2 and C, acting on cell phospholipids (PL). Among the major products being formed, the following: a) the inositol phosphates (IP), especially IP3, and diacylglycerols (DAG) generated intracellularly from phosphoinositides through PLase C, b) the eicosanoids, the arachidonic acid (AA) metabolites produced through combined PLase A2 and (cyclo- and lip-) oxygenase activities, and released from cells, c) the ether lipid PAF, derived from alkylacyl phosphatidylcholine through PLase A2, have attracted the attention of investigators for their important biological roles. Interest has also been recently developed towards products of sphingolipid hydrolysis, sphingosine and ceramide, which are generated by various cell types after stimulation, and exert biological activities. Cell glycerophospholipids are rich in long-chain polyunsaturated fatty acids (PUFA) of the n-6, namely AA 20:4 n-6, and n-3, mainly docosahexaenoic acid (DHA) 22:6, series. These compounds are differentially distributed among the various PL classes and their levels in cells are modulated through the intake with the diet of either the 18-C fatty acids (FA), precursors, linoleic 18:2 n-6, and, alpha-linolenic 18:3 n-3, respectively--followed by conversion to their long-chain PUFA derivatives, or through the intake of the performed compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

In human monocytes interleukin-1 stimulates a phospholipase C active on phosphatidylcholine and inactive on phosphatidylinositol.

Interleukin-1 (IL-1) can initiate the synthesis of prostaglandins which in turn act as endogenous modulators of IL-1 production. The human monocyte/macrophage synthesizes various eicosanoids through the activation of the cellular phospholipase system. Cell stimulation results in the activation of phospholipase A2 (PLA2) whose major substrate is phosphatidylcholine (PC) and the release of the eicosanoid precursor arachidonic acid (AA) from PC. Another pathway is the stimulation of a phospholipase C (PLC) mainly active on phosphoinositides and the resulting formation of inositol phosphates (IPs) and diacylglycerol (DAG). Phospholipids other than phosphoinositides can also be hydrolysed by PLC to give rise to DAG. Studies have shown that IL-1 does not activate the IP pathway, but it primarily stimulates a PLC linked to phosphatidylethanolamine in cultured rat mesangial cells, and a PLC linked to PC in Jurkart cells. We have stimulated human monocytes with IL-1 and calcium ionophore A23187 and we have observed their effect on the phospholipase system. The results indicate that IL-1 does not activate the formation of IPs in cells labeled with [3H]myo-inositol. In contrast, in cells labeled with [3H]AA, IL-1 causes the formation of DAG associated with the hydrolysis of PC. Moreover, after stimulation with IL-1 there is no accumulation of free AA which would indicate that there has been no activation of PLA2, which occurs instead with A23187 stimulation. These data suggest that, in monocytes, IL-1 does not directly stimulate a PLA2 or a PLC active on phosphatidylinositol; instead it primarily stimulates a PLC active on PC.

Increments of dietary linoleate raise liver arachidonate, but markedly reduce heart n-6 and n-3 fatty acids in the rat.

Four diets containing 20% of energy (en%) as fat and with linoleic acid contents of 1.9, 3.1, 7.7 and 10.1 en%, respectively, were fed to one-month-old male rats for three months. The fatty acid profiles and the levels of the major n-6 and n-3 fatty acids in the lipids of plasma, liver, heart and kidney were measured. We found that with increasing concentrations of 18:2n-6 in the diet, linoleic acid rose in plasma and in all organs, but long-chain n-6 and n-3 fatty acids responded differently. In liver, arachidonic acid increased and n-3 fatty acids were not significantly affected; in heart, both arachidonic and docosahexaenoic acids were progressively reduced; and in kidney, there was no change of n-6 and n-3. The results indicate that incremental changes in dietary linoleate affect the levels of polyunsaturated fatty acids in liver and extrahepatic organs differently.

Metabolic changes in the hippocampus after prolonged epileptic discharge.

Sustained epileptic seizures were induced in cats by means of penicillin (PCN). After a three hour period tissue from the archicortex was removed, frozen, and extracted for metabolic studies. The concentration of ATP, ADP, AMP, phosphocreatine, glucose, glucose-6-phosphate, pyruvate, lactate, glutamate and aspartate were determined. There was a 50% decrease in phosphocreatine concentration, a slight decrease in the level of ATP and a slight increase in the levels of ADP and AMP. There was a decrease in the total adenine nucleotide and the ATP/ADP and ATP/AMP ratios. The absence of a significant change in adenylate energy charge potential reflects the remarkable ability of the brain to stabilize its energy state even after intense seizure activity. A reflection of increased glycolysis is the presence of decreased glucose (nearly 50%), and increased lactate, concentrations. The metabolic changes observed in the archicortex are comparable to those observed by others in the neocortex, indicating perhaps the relative metabolic uniformity of these two types of cortex.

The cross-linking of tubulin with imidoesters.

Tubulin was reacted with a monofunctional imidoester, ethyl acetimidate, and three bifunctional imidoesters ranging in extension from 5 to 10 A. Extensive cross-linking was found to occur with the three bifunctional reagents resulting in the formation of dimers, trimers, tetramers, pentamers, and hexamers. In addition, a similar cross-linking patterns was observed with the monofunctional reagent. This type of cross-linking is rarely seen when proteins are reacted with imidoesters. Given that the cross-linking span of ethyl acetimidate is only 3 A it is reasonable to infer that there are nucleophilic groups in close proximity within the tubulin dimer. Amidinated tubulin is still capable of assembling into microtubules.

Stability of microtubule protein over the pH range: 6.9--9.5.

The pH stability range of a microtubule protein preparation has been investigated between 6.9 and 9.5. Microtubule protein was exposed to various pH values in this range and then returned to pH 6.9. The appearance of microtubules as verified by electron microscopy and sedimentation analysis under polymerizing conditions was taken as an indication of a conformationally stable protein. Between pH 6.9 and pH 8.0 the loss in the ability to form microtubules was found to be reversible, at pH 8.2 it was partially reversible, above pH 8.2 it was irreversible. Tubulin and the microtubule-associated protein fraction were separately exposed to high pH. It was observed that tubulin exposed to high pH can still form microtubules in the presence of untreated microtubule-associated protein. On the other hand, microtubule-associated protein exposed to high pH could not initiate microtubule assembly with untreated tubulin. It was concluded from these observations that the loss in the ability of a microtubule protein preparation to assemble at high pH is due to a change in the microtubule-associated protein fraction and that tubulin is conformationally stable even after exposure to pH 9.5.