BIOMED-1 concerted action report: flow cytometric characterization of CD7+ cell subsets in normal bone marrow as a basis for the diagnosis and follow-up of T cell acute lymphoblastic leukemia (T-ALL).

The European BIOMED-1 Concerted Action was initiated in 1994 to improve and standardize the flow cytometric detection of minimal residual disease (MRD) in acute leukemia (AL). Three different protocols were defined to identify the normal subsets of B, T and myeloid cells in bone marrow (BM), and were applied to the different types of AL in order to study aberrant immunophenotypes. Using sensitive acquisition methods ('live gate') T cell subsets in normal BM could be identified with five triple-stains: CD7/CD5/CD3, CD7/CD4/CD8, CD7/CD2/CD3, CD7/CD38/CD34 and TdT/CD7/surface or cytoplasmic (cy)CD3 (antibodies conjugated with FITC/PE/PECy5 or PerCP, respectively). The identification of T cell subsets in BM allowed definition of 'empty spaces' (ie areas of flow cytometric plots where normally no cells are found). All studied T-ALL cases (n = 65) were located in 'empty spaces' and could be discriminated from normal T cells. The most informative triple staining was TdT/CD7/cyCD3, which was aberrant in 91% of T-ALL cases. In most cases, two or more aberrant patterns were found. Apparently the immunophenotypes of T-ALL differ significantly from normal BM T cells. This is mostly caused by their thymocytic origin, but also the neoplastic transformation might have affected antigen expression patterns. Application of the five proposed marker combinations in T-ALL contributes to standardized detection of MRD, since cells persistent or reappearing in the 'empty spaces' can be easily identified in follow-up BM samples during and after treatment.     

Design, synthesis and activity of ascorbic acid prodrugs of nipecotic, kynurenic and diclophenamic acids, liable to increase neurotropic activity.

To improve the entry of certain drugs into brain, ascorbic acid (AA) conjugates of these drugs were synthesized and their capacity to interact with SVCT2 ascorbate transporters was explored. Kinetic studies clearly indicate that all of the conjugates were able to competitively inhibit ascorbate transport in human retinal pigment epithelial cells (HRPE). In vivo studies, in a mouse model system, demonstrate that conjugate 3 is better absorbed compared to the nonconjugated parent drug.     

Serum insulin/glucose ratio and urinary loss of insulin after trauma in young animals: effect of adrenergic blockade

Hyperglycemia and relative hypoinsulinemia are characteristic in the post-traumatic period. The altered insulin kinetics in this phase are dependent on many factors, which include stimulation of adrenoreceptors of pancreatic beta-cells and increased urinary clearance of insulin. Using the young rabbit as an experimental model, two series of experiments were designed. In study 1 (effect of trauma on serum insulin/glucose [I/G] ratio and urinary loss of insulin) two groups of animals (control and trauma) on parenteral nutrition were studied during 4 days. In study 2, in an acute 3-h period the effects of alpha- and beta-adrenoceptor blockers on insulin secretion were investigated. The animals were divided into four groups: control, trauma, trauma + phentolamine, and trauma + propranolol. In study 1 it was demonstrated that after trauma there was a decrease in I/G ratio and an increase in urinary loss of insulin, but no alteration in absolute levels of serum insulin. In study 2 we could demonstrate that treatment with adrenoceptor blockers did not prevent the lowering effect of trauma on I/G ratio. It is suggested that the diabetes-like state of the post-injury period is a complex phenomenon that is dependent on other important factors, as well as catecholamine inhibition of insulin secretion.     

Abnormal platelet aggregation in patients with Raynaud''s phenomenon.

Platelet aggregation in vitro to several aggregating agents (serotonin (5-HT), adenosine diphosphate, adrenaline and collagen) was studied in 16 patients with primary and secondary Raynaud's phenomenon and compared with that in 13 normal volunteers. Platelets from patients with Raynaud's phenomenon had significantly greater responses to all the 5-HT concentrations tested (p less than 0.001 for 10 microM; p less than 0.01 for 1 microM; p less than 0.05 for 0.1 microM; p less than 0.02 for 0.025 microM) and to low doses of adenosine diphosphate (p less than 0.01 for 1 microM; p less than 0.02 for 0.5 microM) but normal responses to collagen, adrenaline, and high doses of adenosine diphosphate. Patients with secondary Raynaud's phenomenon were significantly more hypersensitive to 0.5 microM adenosine diphosphate than patients with primary Raynaud's phenomenon. In patients with secondary Raynaud's phenomenon there was a significant correlation between the extent of 5-HT aggregation and the duration of the disease. The finding that platelets from patients with Raynaud's phenomenon have enhanced responses to 5-HT and adenosine diphosphate, but normal responses to adrenaline and collagen, is consistent with a role for 5-HT in this disease.     

Central dopamine deficiency in pure autonomic failure

Objective Pure autonomic failure (PAF) and Parkinson’s disease (PD) share several clinical laboratory abnormalities; however, PAF is not associated with parkinsonism. In this study, we tested the hypothesis that preservation of nigrostriatal dopaminergic innervation explains the absence of motor dysfunction in PAF. Methods Patients with PAF (N = 5) or PD (N = 21) and control subjects (N = 14) had brain 6-[¹⁸F]fluorodopa positron emission tomographic scanning and cerebrospinal fluid catechol measurements. A patient with PAF and another with PD had rapid postmortem striatal, nigral, and sympathetic ganglion sampling, with assays of catechols and tyrosine hydroxylase activity. Results The PAF and PD groups had similarly low mean substantia nigra (SN):occipital (OCC) ratios of 6-[¹⁸F]fluorodopa-derived radioactivity and similarly low cerebrospinal fluid dihydroxyphenylacetic acid and DOPA levels. Only the PD group, however, had low PUT:OCC, caudate:OCC, or PUT:SN ratios. The PAF and PD cases had similarly low SN tissue concentrations of dopamine and tyrosine hydroxylase activity, but the PD patient had tenfold lower PUT dopamine and the PAF patient 15-fold lower myocardial norepinephrine concentrations. Conclusions Surprisingly, PAF and PD entail similarly severe nigral and overall central dopaminergic denervation. There is more severe loss of striatal dopaminergic terminals in PD than in PAF and more severe loss of sympathetic noradrenergic terminals in PAF than in PD. These differences explain the distinctive clinical manifestations of the two Lewy body diseases. Parkinsonism appears to reflect striatal dopamine deficiency rather than loss of nigral dopaminergic neurons per se.     

Potential toxicity of toluene and xylene evoked by mitochondrial uncoupling.

Toluene and xylene are chemicals present in various laboratory and other industrial products. Their toxicity to the nervous system and to the liver has been well documented. In the present work, we have studied in vitro effects of toluene and xylene on the respiration of succinate-energized isolated rat liver mitochondria, membrane potential, Ca2+ release, reactive oxygen species (ROS), and ATP levels. Also Ca2+-dependent, cyclosporine A-sensitive mitochondrial swelling, an indicator of mitochondrial permeability transition (MPT), was studied. At 0.5-2.5 and 0.25-1mM concentrations respectively, toluene and xylene stimulated state 4 respiration in apparent association with mitochondrial membrane potential dissipation and Ca2+ release; these actions of both solvents are consistent with mitochondrial uncoupling. At higher concentrations (2.5 and 5mM, respectively) toluene and xylene also inhibited state 3 respiration. At 0.1-1mM concentrations, xylene elicited significant increase of ROS generation and partly Ca2+-dependent cyclosporine A-sensitive mitochondrial swelling. At 1 mM concentration, toluene or xylene caused depletions of mitochondrial ATP, amounting to 66.3% and 40.3%, respectively; depletions were only slightly dependent on Ca2+. It was concluded that mitochondrial uncoupling via ATP depletion might be responsible for the cell toxicity of toluene described earlier and in particular, of xylene. In the case of xylene, mitochondrial ROS generation and MPT also appear to be involved.     

Mangifera indica L. extract (Vimang) and its main polyphenol mangiferin prevent mitochondrial oxidative stress in atherosclerosis-prone hypercholesterolemic mouse

Atherosclerosis is linked to a number of oxidative events ranging from low-density lipoprotein (LDL) oxidation to the increased production of intracellular reactive oxygen species (ROS). We have recently demonstrated that liver mitochondria isolated from the atherosclerosis-prone hypercholesterolemic LDL receptor knockout (LDLr(-/-)) mice have lower content of NADP(H)-linked substrates than the controls and, as consequence, higher sensitivity to oxidative stress and mitochondrial membrane permeability transition (MPT). In the present work, we show that oral supplementation with the antioxidants Mangifera indica L. extract (Vimang) or its main polyphenol mangiferin shifted the sensitivity of LDLr(-/-) liver mitochondria to MPT to control levels. These in vivo treatments with Vimang and mangiferin also significantly reduced ROS generation by both isolated LDLr(-/-) liver mitochondria and spleen lymphocytes. In addition, these antioxidant treatments prevented mitochondrial NAD(P)H-linked substrates depletion and NADPH spontaneous oxidation. In summary, Vimang and mangiferin spared the endogenous reducing equivalents (NADPH) in LDLr(-/-) mice mitochondria correcting their lower antioxidant capacity and restoring the organelle redox homeostasis. The effective bioavailability of these compounds makes them suitable antioxidants with potential use in atherosclerosis susceptible conditions.