Increased cerebrospinal fluid levels of cytokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1ß (MIP-1ß) in patients with amyotrophic lateral sclerosis. / Incremento de las citoquinas proteína quimiotáctica de monocitos-1 (MCP-1) y proteína inflamatoria macrofágica-1ß (MIP-1ß) en líquido cefalorraquídeo de pacientes con esclerosis lateral amiotrófica.

INTRODUCTION: Neuroinflammation has recently been described in amyotrophic lateral sclerosis (ALS). However, the precise role of such proinflammatory cytokines as monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1ß (MIP-1ß) in ALS has not yet been determined. In this study, we determined cerebrospinal fluid (CSF) MCP-1 and MIP-1ß levels and assessed their association with the duration and severity of ALS. METHODS: Concentrations of MCP-1 and MIP-1ß were determined in the CSF of 77 patients diagnosed with ALS and 13 controls. Cytokine levels were analysed in relation to ALS duration (<12months vs. >12months) and severity (<30points vs. >30points on the ALS Functional Rating Scale administered at hospital admission). RESULTS: Higher CSF MIP-1ß (10.68pg/mL vs. 4.69pg/mL, P<.0001) and MCP-1 (234.89pg/mL vs. 160.95pg/mL, P=.011) levels were found in the 77 patients with ALS compared to controls. There were no differences in levels of either cytokine in relation to disease duration or severity. However, we did observe a significant positive correlation between MIP-1ß and MCP-1 in patients with ALS. CONCLUSIONS: The increase in MIP-1ß and MCP-1 levels suggests that these cytokines may have a synergistic effect on ALS pathogenesis. However, in our cohort, no association was found with either the duration or the clinical severity of the disease.

Synthesis and in vitro antiprotozoal activity of 5-nitrothiophene-2-carboxaldehyde thiosemicarbazone derivatives.

Several thiosemicarbazone derivatives of 5-nitrothiophene-2-carboxaldehyde were prepared by the simple process in which N(4)-thiosemicarbazone moiety was replaced by aliphatic, arylic and cyclic amine. Among these thiosemicarbazones compound 11 showed significant antiamoebic activity whereas compound 3 was more active antitrichomonal than the reference drug.

Synthesis, characterization and antiamoebic activity of benzimidazole derivatives and their vanadium and molybdenum complexes.

Reaction of [MoO(2)(acac)(2)] (where, acac=acetyl acetone) and KVO(3) with 2-(salicylidieneimine) benzimidazole lead to form new complexes [MoO(2)(sal-BMZ)(2)] and K [VO(2)(sal-BMZ)(2)] [where, sal-BMZ=2-(salicylidieneimine) benzimidazole], which showed the monobasic bidentate nature of the ligand in which the phenolic oxygen and the imine nitrogen of the ligand are coordinated to the metal ion. These complexes were characterized along with nine other complexes of oxoperoxovanadium (V), molybdenum (Vl) and tungsten (Vl) with benzimidazole derivatives and screened in vitro by micro dilution technique for their amoebicidal activity with a view to search for a more effective agent against Entamoeba histolytica suggests that compound 2 and 3 might be endowed with important antiamoebic properties since they showed IC(50 )values in a microM range.

Synthesis, characterisation and antiamoebic activity of new thiophene-2-carboxaldehyde thiosemicarbazone derivatives and their cyclooctadiene Ru(II) complexes.

Reaction of new thiosemicarbazones (1-4) derived from thiophene-2-carboxaldehyde and cycloalkylaminothiocarbonylhydrazine with [Ru(eta(4)-C8H12)(CH3CN)2Cl2] leads to form complexes (1a-4a) of the type [Ru(eta(4)-C8H12)(TSC)Cl2] (where TSC=thiosemicarbazone). All the compounds have been characterised by elemental analysis, IR, 1H NMR, electronic spectra and thermogravimetric analysis. It is concluded that the thionic sulphur and the azomethine nitrogen atom of the ligands are bonded to the metal ion. In vitro antiamoebic screening against (HK-9) strain of Entamoeba histolytica indicated that the Ru(II) complexes of thiophene-2-carboxaldehyde thiosemicarbazones were found more active than the thiosemicarbazones.

Auxotrophy to lipoproteins of Entamoeba histolytica cultivated under axenic conditions.

Entamoeba histolytica grows in media without serum but with a mixture of aminoacids, vitamins, lipoproteins, free cholesterol, phospholipids and fatty acids called PACSR. The ability of lipoproteins and free lipids to support growth of three E. histolytica strains (HK9, HMI:IMSS and HM3:IMSS) was analysed. Tubes containing 5 ml culture medium, amino acids, vitamins and either 120-1,200 microg lipoproteins/ml or 0.017-0.10 mg free lipids/ml (predissolved in absolute ethanol) were inoculated with 1x10(4) trophozoites/ml and incubated at 37 degrees C for 72 h. Amoebae died within 12 h in the presence of any free lipid combination, while those having 240-480 mg lipoproteins/ml reached densities similar to or higher than those of controls (depending on strain). The addition of ethanol (0.1%) to the media produced stable lipid solutions and did not show significant adverse effects. Accordingly, E. histolytica is auxotrophic to lipoproteins and unable to use free cholesterol, phospholipids or fatty acids.

Detection of a factor released by L5178Y lymphoblasts that inhibits mouse macrophage-activation induced by lipopolysaccharides.

BACKGROUND: Several factors inhibit cellular immune response by deactivating macrophages, but very few, such as those described here, prevent macrophage activation. METHODS: Ascites liquid from 12-day-old BALB/c mice bearing 5178Y lymphoma tumors was collected, and cell-free ascites liquid (CFAL) was separated from lymphoblasts. The supernatant (S1) was obtained from the homogenized and centrifuged lymphoblasts. Then, macrophage cultures containing 0.2 x 10(6) cells from lymphoma-bearing or healthy mice were added to 10 microL of CFAL or S1, plus 5 micrograms of lipopolysaccharides (LPS)/mL, 40 U interferon-gamma or a blend of both. Macrophages were incubated with CFAL or S1 prior to or after adding the activators to investigate whether any of the previously mentioned lymphoma fractions inhibited macrophage activation or whether they deactivated them. The effect of CFAL or S1 was estimated as the diminution of the amount of nitric oxide released by the experimental macrophage cultures with respect to controls (activated macrophages treated with none of the lymphoma fractions). RESULTS: LPS, IFN-gamma, and the LPS/gamma blend activated macrophages from both lymphoma-bearing and healthy mice. None of the lymphoma fractions deactivated macrophages. CFAL, but not S1, inhibited the macrophage activation, i.e., the percentage of inhibition of nitric oxide releasing 76.7% and 78.1% in macrophages from healthy and lymphoma-bearing mice, respectively. In addition, CFAL was unable to inhibit the macrophage-activation effect of IFN-gamma or the LPS/IFN-gamma blend. CONCLUSIONS: Mouse L5178Y lymphoma releases a factor that in vitro inhibits the macrophage activation induced by LPS, but not by IFN-gamma controls.

Comparable growth of a Trichomonas vaginalis strain in PEHPS and TYI-S-33 media.

PEHPS medium, developed for axenic cultivation of Entamoeba histolytica and E. invadens, was also capable of supporting the growth of a Trichomonas vaginalis strain, with an inoculum of 1 to 100 trichomonads/ml. The logarithmic growth phase in PEHPS or in TYI-S-33 medium lasted 72 h; yield (3.33 +/- 0.56 x 10(6) trichomonads/ml), duplication time (4.27 h), number of duplications (16.85), or increase ratio (33,328) in PEHPS medium showed no significant differences with those obtained in TYI-S-33 under similar culture conditions. Accordingly, PEHPS medium might be used for the axenic cultivation of T. vaginalis.

Orally administered gossypol has no effect on eight hamster erythrocytic enzymes.

The effectiveness of gossypol as an antifertilizing agent is due to the severe injuries or death that this drug produces on spermatozoa and spermatides. Several in vitro and in vivo studies have shown that spermatozoal lactic and malic dehydrogenases are inhibited by gossypol; and that these are more susceptible than the somatic enzymes. Notwithstanding, the in vivo effects on other somatic enzymes have been poorly analyzed. The present study shows that gossypol did not produce toxic effects on eight erythrocytic enzymes of male hamsters that were fed daily with 20 mg of gossypol/kg, for 1, 3, 5 or 10 days. The enzymatic activities analyzed were: adenylate kinase, hexokinase, glucose-6-phosphatase, glucose phosphoisomerase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglyceratokinase and pyruvate kinase.

Identification of Entamoeba histolytica intracellular phospholipase A and lysophospholipase L1 activities.

Entamoeba histolytica phospholipase A and lysophospholipase activities from a vesicular subcellular fraction (P30) were analyzed. The products, obtained using specific substrates labeled with 14C or 3H, indicated the presence of phospholipase A1 and A2 as well as lysophospholipase L1 activities. The enzymes detected could participate in phospholipid metabolism and the alkaline phospholipase A2 may contribute to E. histolytica cytopathogenicity.

Differential effects of the (+)- and (-)-gossypol enantiomers upon Entamoeba histolytica axenic cultures.

The in-vitro anti-amoebic effects of (+/-)-, (+)-, (-)-gossypol and emetine were tested against axenic trophozoites from five Entamoeba histolytica strains. The (-)-isomer was more active than the racemate and the (+)-isomer. These results indicate that the gossypol anti-amoebic activity is mainly due to its content of (-)-gossypol in all strains tested.

Entamoeba histolytica: inhibition of malic enzyme and alcohol dehydrogenase by (+/- )-, (+)-, and (-)- gossypol.

Gossypol, a natural racemic mixture with action on NADP- and NAD-oxidoreductases from diverse species, has been proposed as a possible antiamebic medication considering several of its pharmacological properties. In this study it was found that malic enzyme and alcohol dehydrogenase from Entamoeba histolytica are strongly inhibited by (+/-)-gossypol, and both (+)- and (-)- enantiomers. The inhibition was of the noncompetitive type among their respective substrates in all cases. The (+/-), (+)-, (-)-gossypol half-maximal inhibitory concentrations (IC50) for the malic enzyme were 3.71, 13.37 and 1.03 microM, and against the alcohol dehydrogenase 79.64, 124.43 and 42.56 microM, respectively. Therefore, the (-) enantiomer resulted 3.6 and 13.0 times more potent than the racemic mixture and (+)-gossypol, respectively, to inhibit the malic enzyme, and 1.9 times and 2.9 times more potent than the racemic mixture and (+)-gossypol, respectively, against the alcohol dehydrogenase. Accordingly, one possible mechanism of the antiamebic effect of gossypol could be the inhibition of vital NADP-dependent enzymes as those analyzed in this study.

Entamoeba histolytica cysts with a defective wall formed under axenic conditions.

Axenic HK9 Entamoeba histolytica strain amoebae, maintained in PEHS medium, displayed several cystic characteristics that involve an active process of cystic wall formation, cellular volume and density diminution, and one or two nuclear divisions. The differentiation process was asynchronic, beginning after the logarithmic growth phase. The axenic cysts, which were maintained in a 50 mOsm/kg medium at 4 degrees C for 72 h, produced growing trophozoites within 1-7 days of incubation at 36 degrees C in fresh medium. Negative results were obtained with trophozoites submitted to the above treatment, and with axenic cysts maintained in double-distilled water at 4 degrees C for 24 h, or in 0.1% sarkosyl, for 10 min at room temperature instead of 55 mosmol/kg medium. Thus, the HK9 E. histolytica strain, cultured in PEHPS, produced under axenic conditions a small proportion of mature, metabolically active cysts, but with an immature or abnormal wall.

Further studies on the in vitro activity of gossypol as antiamebic agent.

Gossypol has an in vitro antiamebic effect, which is 11,39, and 980 times more potent than emetime, metronidazole and diiodohydroxyquinolein, respectively, on five Entamoeba histolytica axenic strains. The trophozoite NADP-dependent malic enzyme and alcohol dehydrogenase are inhibited by (+/-)-gossypol, in a noncompetitive manner with respect to the substrate. From the (-)- and (+)- enantiomers, which integrate the natural gossypol mixture, the first one is three and four times more active in inhibiting both the amebic culture growth and the mentioned oxidoreductases, respectively. The above results justify the analysis of gossypol as an antiamebic drug in experimentally infected animals.