[Telemonitoring in continuous positive airway pressure-treated patients with obstructive sleep apnoea syndrome: An algorithm proposal]. / Télésuivi des patients traités par pression positive continue pour un syndrome d'apnées/hypopnées obstructives du sommeil : proposition d'un arbre décisionnel.

Most of the continuous positive airway pressure (CPAP) devices currently in use allow telemonitoring of observance, leaks and the apnoea-hypopnoea index (AHI). La Société française de recherche et de médecine du sommeil (SFRMS) and La Société de pneumologie de langue française (SPLF) workgroup offer to CPAP prescribers and to home care providers a scientific document which has the following purposes: to underline the relevance of the telemonitoring of leaks and the AHI, to define alert thresholds, to describe the principal mechanisms generating excessive leaks and high AHI, and to propose a diagnostic algorithm.

Identification of distinct pathological signatures induced by patient-derived α-synuclein structures in nonhuman primates.

Dopaminergic neuronal cell death, associated with intracellular α-synuclein (α-syn)-rich protein aggregates [termed "Lewy bodies" (LBs)], is a well-established characteristic of Parkinson's disease (PD). Much evidence, accumulated from multiple experimental models, has suggested that α-syn plays a role in PD pathogenesis, not only as a trigger of pathology but also as a mediator of disease progression through pathological spreading. Here, we have used a machine learning-based approach to identify unique signatures of neurodegeneration in monkeys induced by distinct α-syn pathogenic structures derived from patients with PD. Unexpectedly, our results show that, in nonhuman primates, a small amount of singular α-syn aggregates is as toxic as larger amyloid fibrils present in the LBs, thus reinforcing the need for preclinical research in this species. Furthermore, our results provide evidence supporting the true multifactorial nature of PD, as multiple causes can induce a similar outcome regarding dopaminergic neurodegeneration.

[Search for the T790M mutation: The need to persevere]. / Recherche de la mutation T790M : savoir persévérer.

In cases of advanced EGFR mutation-positive non-small cell lung cancer, first or second generation EGFR-tyrosine kinase inhibitors (TKI-EGFR 1G or TKI-EGFR 2G) are recommended as first line treatment. Inexorably, progressive disease occurs and, in 50-60% of the cases, is secondary to a T790M resistant mutation. The prescription of osimertinib (TKI-EGFR3G) in second line is dependent on identification of the T790M mutation. We report 7 cases in which the identification of the T790M mutation required repeated analyses of cell free DNA and/or biopsies over a period of time. In some cases, a positive result was obtained a long time after progressive disease had been diagnosed during treatment with first or second generation EGFR-TKI. We discuss here the different modalities of screening for the T790M mutation and we encourage persevering in this search when no alternative mechanism of resistance has been identified.

Anthracyclines trigger apoptosis of both G0-G1 and cycling peripheral blood lymphocytes and induce massive deletion of mature T and B cells.

The anthracyclines daunorubicin and doxorubicin were shown to induce apoptosis of hematopoietic cell lines. Here we report that they induce apoptosis of both nonactivated and phytohemagglutinin-activated human peripheral blood lymphocytes. Apoptosis demonstrated by surface expression of phosphatidylserine and typical nuclear alterations reached a maximum after 48 h of incubation with these agents. In contrast to topoisomerase inhibitors (etoposide and camptothecin) and antimetabolites (methotrexate and 5-fluorouracil) that induced apoptosis of activated cells only, daunorubicin and doxorubicin triggered apoptosis of cells in the G0-G1 phases of the cell cycle. In agreement with in vitro data, a single i.p. injection of daunorubicin or doxorubicin in BALB/c mice induced T- and B-cell depletion in spleen, lymph nodes, and to a lesser extent in the thymus. Soluble Fas-Fc, CD95 antagonistic antibodies, as well as the p55 tumor necrosis factor receptor-immunoglobulin fusion protein, did not inhibit drug-induced apoptosis. The level of reactive oxygen species was significantly increased in the presence of daunorubicin or doxorubicin only in nonactivated lymphocytes. However, antioxidants such as N-acetyl-L-cysteine or glutathione did not prevent apoptosis. Activation of caspase-3 after daunorubicin or doxorubicin treatment of either nonactivated or activated lymphocytes was demonstrated by the cleavage of poly(ADP-ribose) polymerase, which was, as apoptosis, inhibited by the peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Finally, daunorubicin and doxorubicin induced a rapid production of ceramides. These data indicate that anthracyclines may induce major peripheral T-cell deletion, a property not shared by many cytotoxic agents.

[Noninvasive ventilation. The 2015 guidelines from the Groupe Assistance Ventilatoire (GAV) of the Société de Pneumologie de Langue Française (SPLF)]. / Ventilation non invasive. Les conseils 2015 du Groupe assistance ventilatoire (GAV) de la Société de pneumologie de langue française (SPLF).

A task force issued from the Groupe Assistance Ventilatoire (GAV) of the Société de Pneumologie de Langue Française (SPLF) was committed to develop a series of expert advice concerning various practical topics related to long-term non invasive ventilation by applying the Choosing Wisely® methodology. Three topics were selected: monitoring of noninvasive ventilation, the interpretation of data obtained from built-in devices coupled to home ventilators and the role of hybrid modes (target volume with variable pressure support. For each topic, the experts have developed practical tips based on a comprehensive analysis of recent insights and evidence from the literature and from clinical experience.

Caspase-8 is an effector in apoptotic death of dopaminergic neurons in Parkinson's disease, but pathway inhibition results in neuronal necrosis.

Caspase-8 is a proximal effector protein of the tumor necrosis factor receptor family death pathway. In the present human postmortem study, we observed a significantly higher percentage of dopaminergic (DA) substantia nigra pars compacta neurons that displayed caspase-8 activation in Parkinson's disease (PD) patients compared with controls. In an in vivo experimental PD model, namely subchronically 1,2,3,6-tetrahydropyridine-treated mice, we also show that caspase-8 is indeed activated after exposure to this toxin early in the course of cell demise, suggesting that caspase-8 activation precedes and is not the consequence of cell death. However, cotreatment of 1-methyl-4-phenylpyridinium-intoxicated primary DA cultures with broad-spectrum and specific caspase-8 inhibitors did not result in neuroprotection but seemed to trigger a switch from apoptosis to necrosis. We propose that this effect is related to ATP depletion and suggest that the use of caspase inhibitors in pathologies linked to intracellular energy depletion, such as PD, should be cautiously evaluated.

Phosphatidic acid stimulates the rolipram-sensitive cyclic nucleotide phosphodiesterase from rat thymocytes.

The role of phospholipid metabolites in the modulation of cyclic AMP degradation during the early response of rat thymic lymphocytes to mitogenic stimulation was investigated by measuring their in vitro effect on the activity of five different cyclic nucleotide phosphodiesterase forms separated from thymocyte cytosol by means of an HPLC technique. Arachidonic acid was found to markedly inhibit four of the enzyme forms, with IC50 ranging from 14 to 60 microM, while its hydroperoxy and hydroxy derivatives proved inefficient. Dioctanoylglycerol, a biologically active diacylglycerol, was weakly inhibitory while phosphatidic acid, the diacylglycerol phosphorylated derivative, markedly stimulated the two cyclic-AMP-specific type-IV forms identified in thymocyte cytosol, by 50 and 70%. In intact cells labelled with tritiated arachidonate, the mitogenic lectin concanavalin A induced a rapid 4-5-fold increase in radiolabelled phosphatidic acid which peaked at 1 min, and remained elevated for at least 30 min. These observations suggest that phosphatidic acid formed during the mitogenic stimulation of T-cells might be responsible for an early activation of cyclic AMP degradation with, as a consequence, a lowering of cyclic AMP level, which is reported to be necessary for the occurrence of the first steps of mitogenesis.

The topoisomerase inhibitors camptothecin and etoposide induce a CD95-independent apoptosis of activated peripheral lymphocytes.

The effect of etoposide and camptothecin, two topoisomerase inhibitors directed against topoisomerases II and I, respectively, was evaluated on human peripheral blood lymphocytes. Etoposide and camptothecin induced apoptosis of mitogen-activated but not resting CD4+ and CD8+ T lymphocytes. Cell sensitivity to these agents required G1 to S-phase transition of the cell cycle. Conversely, daunorubicin, an intercalating agent and topoisomerase II inhibitor, induced apoptosis of both resting and activated lymphocytes. Although etoposide and camptothecin induced CD95-ligand mRNA expression, drug-induced apoptosis of activated human lymphocytes was not inhibited by CD95 antagonists. Drug-induced cell death was also not inhibited by p55 TNFR-Ig fusion protein. Activation of the caspases cascade was suggested by the partial inhibitory effect of the tripeptide zVAD-fmk and documented by activation of caspase 3. Finally etoposide and camptothecin induced a rapid production of ceramide in activated but not resting peripheral blood lymphocytes, suggesting that ceramide might initiate the signaling apoptotic cascade in sensitive cells.

Activation of a cyclic nucleotide phosphodiesterase 4 (PDE4) from rat thymocytes by phosphatidic acid.

The cytosolic cyclic nucleotide phosphodiesterase (PDE) activity from rat thymocytes was resolved into five peaks by HPLC. Only two forms of the cAMP-specific PDE4 were found to be sensitive to physiologically relevant phosphatidic acid (PA) concentrations. PA activated the PDE4-peak 3 form, the fatty acid composition and unsaturation degree determining the efficiency of PA. The PDE4-peak 2 form was inhibited only by PA with saturated fatty acyl groups. PDE4 activation was specific of anionic phospholipids, a free phosphate group in the phospholipid molecule being required for maximum activation. These results suggest that PA may contribute to the lowering of cAMP level required in the early steps of a lympho-proliferative response, thus regulating immune functions through PDE4 activation.

Human lymphocytes stimulate prostacyclin synthesis in human umbilical vein endothelial cells. Involvement of endothelial cPLA2.

Prostacyclin (PGI2) contributes to the maintenance of a nonadhesive luminal surface in blood vessels due to its anti-platelet and vasodilatory properties. Here, we sought to determine whether peripheral blood lymphocytes (PBL) may regulate the PGI2 production of human umbilical vein endothelial cells (HUVEC). Cell-cell contact between HUVEC and lymphocytes markedly enhanced PGI2 synthesis as a function of the number of lymphocytes added. This stimulated synthesis was totally suppressed when lymphocytes and HUVEC were separated by a microporous insert. It was not due to prostaglandin H synthase up-regulation. The pretreatment of lymphocytes with the PGI2 synthase inhibitor tranylcypromine partially inhibited PGI2 synthesis (47%), suggesting a transcellular metabolism of the endothelial prostaglandin endoperoxide PGH2 by the lymphocyte PGI2 synthase. Experiments using [14C]arachidonate-labeled lymphocytes coincubated with unlabeled HUVEC, and [14C]arachidonate-labeled HUVEC coincubated with unlabeled lymphocytes showed that the arachidonic acid used for PGI2 synthesis was totally of endothelial origin. Furthermore, the PGI2 synthesis was strongly inhibited by the cytosolic phospholipase A2 inhibitor, MAFP and totally suppressed by the combination of the calcium chelators, BAPTA and EGTA. Collectively, these results suggest that lymphocytes trigger an outside-in signaling in endothelial cells involving cPLA2 activation. Overall, the switch-on for PGI2 synthesis induced by lymphocytes might serve as a protection against atherothrombogenesis.

Relationships between phosphatidic acid and cyclic nucleotide phosphodiesterases in activated human blood mononuclear cells.

We have previously shown that mitogenic activation of human PBMC rapidly increases both the intracellular phosphatidic acid (PA) level and cyclic nucleotide phosphodiesterase (PDE) activity, with time-course responses, suggesting a causative relationship between the two events. PA also directly stimulated cAMP-PDE activity in acellular systems. Thus the mitogenic properties of PA night be due to its ability to lower the level of cAMP, a negative effector of lymphocyte activation, through PDE activation. In this study, human PBMC were stimulated either with the mitogenic lectin ConA, the anti-CD3 mAb OKT3, or the phorbol ester TPA. All three agonists increased the radiolabeled PA level and the PA mass in treated cells and simultaneously increased cytosolic and particulate cAMP- and cGMP-PDE activities, with significant positive correlations between PA accumulation and PDE activities. Furthermore, the ConA-induced PDE activation was dose-dependently reduced by treatment of PBMC with the diacylglycerol-kinase inhibitor R59022. This compound also dose-dependently lowered the PA level and inhibited the proliferative response to ConA. In addition, TPA-induced PDE activation was totally abolished by ethanol, which strongly reduced PA accumulation in response to the phorbol ester. These data suggest that PA increase may be linked to mitogen-induced PDE activation. Experiments performed in the presence of rolipram indicated that ConA and TPA stimulated both the rolipram-sensitive PDE4 and the rolipram-insensitive PDE activities, OKT3 being more active on PDE4. All three agonists stimulated the cGMP-specific PDE5. These results suggest that PA is an important component of the mechanisms that maintain a low level of cyclic nucleotides, which is a prerequisite for an optimal lymphoproliferative response.

Arachidonic acid induces differentiation of uterine stromal to decidual cells.

Fatty acids have been involved in the proliferation and differentiation of numerous cells, as mediated via peroxisome proliferator-activated receptors (PPARs) or lipid metabolites (prostaglandins, diacylglycerol). In the present study, we have investigated the effect of arachidonic acid (AA), docosahexaenoic acid (DHA) and its precursor eicosapentaenoic acid (EPA) on the differentiation of a rat uterine stromal cell line, UIII. As markers of decidualization, we have investigated morphological changes, monitored by inverted light and scanning electron microscopy. The induction of 3 proteins, desmin, hsp-25 and prolactin, which are all considered to be markers of decidualization, were analyzed by immunocytochemistry or Western blotting. Addition of AA (30 microM) to the medium of cultured cells for 48h induced cell spreading and flattening. Cells became enlarged (x 2.5) and some of them were binucleated. Using scanning electron microscopy, we confirmed these morphological changes and showed that the enlargement of the cells was followed by numerous extracellular processes, leading to an increase in cell surface area and intercellular communications. Immunocytochemistry showed that this treatment also induced the expression of desmin, which seems to direct morphological changes, beginning as a perinuclear ring and extending to the cell membrane. The time course of desmin expression was studied by Western blotting. No desmin expression was present before 4h of AA treatment. Desmin induction was maximum at 24h of treatment and plateaued thereafter. DHA and EPA (30 microM), added to the medium, failed to induce any change. However, in cells previously differentiated with AA and expressing desmin, treatment with DHA or EPA (30microM) reversed partially the action of AA, EPA being the most effective. AA also induced hsp-25, though all cells did not express this protein. A prolactin (PRL)-like factor was induced by AA, as recognized by an antibody against pituitary rPRL, and migrated as the standard. Moreover, a fragment of 16 kDa was also revealed by this antibody, suggesting that the PRL-like factor cleaved, was similar to PRL and that the PRL-like factor could be identical to PRL. In conclusion, these results show that AA is able to specifically induce the decidualization of uterine stromal cells in vitro.

Cyclic nucleotide phosphodiesterase activity in the quail oviduct: hormonal regulation and involvement in estrogen-induced growth.

A previous study has shown that cAMP was involved in estrogen-activated growth in the quail oviduct. The present study was undertaken to investigate the hormonal regulation of 3',5'-cyclic nucleotide phosphodiesterase activity in the oviduct. Tamoxifen, an antiestrogen compound, and 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase, were also used to determine the relationship between estradiol-induced cell proliferation and cAMP phosphodiesterase activity. Cyclic nucleotide phosphodiesterase was almost completely restricted to the cytosolic fraction (108,000 X g supernatant) of the quail oviduct homogenate. By affinity chromatography on immobilized calmodulin, we separated and partially characterized three different forms of the enzyme. They differed in their cyclic nucleotide specificities, kinetics, and sensitivity to calmodulin. In vivo, estradiol benzoate (EB) modulated crude cytosolic phosphodiesterase activity. cAMP and cyclic-GMP hydrolyzing activities increased between 12 and 48 h after a single injection of EB and then declined to return to control value by 96 h. Estrone, 17 alpha-estradiol, progesterone, and testosterone were ineffective, while estriol slightly increased cyclic-GMP hydrolyzing activity. When administered with EB, tamoxifen drastically increased oviduct cAMP concentration while it completely inhibited oviduct growth and the activation of cAMP phosphodiesterase induced by EB alone. Moreover, 3-isobutyl-1-methylxanthine produced a dose-dependent inhibition of oviduct cell proliferation when given with EB. These results demonstrate that the activation of cAMP phosphodiesterase after an injection of EB and the subsequent decrease in oviduct cAMP concentration are necessary for the epithelial cells to achieve their proliferative cycle.

Effect of estrogen on adenosine 3'5',cyclic monophosphate in quail oviduct: possible involvement in estradiol-activated growth.

Previous studies have shown that estradiol indirectly stimulated the proliferation of oviduct epithelial cells in the quail. The present study was undertaken to investigate the effects of estradiol and other steroid hormones on the cAMP concentration. The ability of forskolin, a specific activator of the catalytic subunit of adenylate cyclase, to induce oviduct cell proliferation and specific protein synthesis (progesterone receptor) in the absence of estrogen was also tested. Administration of estradiol benzoate (EB) to immature female quails produced a transient surge in oviduct cAMP concentration. After EB injection, cAMP concentration increased by 36.7% after 6 h and returned to control values after 12 h. This rise in oviduct cAMP concentration preceded the beginning of DNA synthesis. The same effect was observed even in the absence of increased plasma estradiol, when the hormone was perfused through the hepatic portal vein. Estriol, estrone, and testosterone failed to elevate cAMP concentrations. After repeated EB injections, the oviduct cAMP concentration declined below the control value (-66% after 72 h). A similar drop in the cAMP concentration was observed in developing quails during the proliferative phase of the luminal epithelial and glandular cells. Administration of forskolin to immature female quail pretreated with EB rapidly increased the oviduct cAMP concentration, induced a burst of DNA synthesis, and shortened the prereplicative period. In addition, forskolin administration did not increase the progesterone receptor concentration. These results demonstrate that cAMP is involved in the mechanism by which estradiol indirectly stimulates oviduct epithelial cell proliferation in the quail. The events that may take place during the prereplicative period and the antiproliferative effect of progesterone through a sustained increase in the cAMP concentration in the oviduct are discussed.

Calpastatin immunoreactivity in the monkey and human brain of control subjects and patients with Parkinson's disease.

Parkinson's disease is characterized by a selective loss of dopaminergic neurons in the nigrostriatal pathway. However, not all dopaminergic neurons degenerate in this disease, and calcium has been suspected of playing a role in this differential vulnerability. An overexpression of the calcium-dependent protease calpain II has recently been reported in the parkinsonian substantia nigra, suggesting that a rise in intracellular calcium concentrations may be involved in the mechanism leading to cell death. The proteasic activity of calpain is regulated by an endogenous inhibitory protein called calpastatin. Because little is known about the distribution of calpastatin in the primate brain, we first analyzed immunohistochemically the calpastatin expression in normal human and monkey brain. A ubiquitous distribution of calpastatin immunostaining was observed in both cases, but its expression was variable from one region to another. In the basal ganglia, staining was intense in the striatum, in the pallidal complex, and in some nuclei of the thalamus. The cerebellum was stained intensely, particularly in the granular and Purkinje cell layers. A dense, heterogeneous staining was observed in the hippocampal formation, mostly in the pyramidal and granular layers. The distribution of staining was similar in the different cerebral cortices studied, and it was most intense in layer V. In the brainstem, staining was particularly prominent in the substantia nigra pars reticulata and compacta, the central gray substance, the superior colliculus, and the cuneiform nucleus, and staining was moderate in the tegmenti pedonculopontinus nucleus and the griseum pontis. In the second part of the study, the authors compared calpastatin expression in the mesencephalon between patients with Parkinson's disease and control subjects. Sequential double staining revealed that some dopaminergic neurons coexpress calpastatin, the proportion of double-stained neurons ranging between 52% and 76% among the different dopaminergic cell groups. Quantitative analysis of the number of calpastatin-stained neurons evidenced a loss of both calpastatin-positive and calpastatin-negative neurons in the substantia nigra of patients with Parkinson's disease. These data suggest that calpain II overexpression in Parkinson's disease is not compensated for by a concomitant increase in calpastatin expression.

Parkin immunoreactivity in the brain of human and non-human primates: an immunohistochemical analysis in normal conditions and in Parkinsonian syndromes.

The etiology of Parkinson's disease is unknown, but the gene involved in an autosomic recessive form of the disease with early onset has recently been identified. It codes for a protein with an unknown function called parkin. In the present study we produced a specific polyclonal antiserum against human parkin. Immunohistochemical analysis showed that parkin is expressed in neuronal perikarya and processes but also in glial and blood vessels in the primate brain (human and monkey). Electron microscopy indicated that parkin immunoreactivity is mostly located in large cytoplasmic vesicles and at the level of the endoplasmic reticulum. Parkin was expressed heterogeneously in various structures of the brain. It was detectable in the dopaminergic systems at the level of the perikarya in the mesencephalon but also in the striatum. However, parkin was also expressed by numerous nondopaminergic neurons. The staining intensity of parkin was particularly high in the hippocampal formation, the pallidal complex, the red nucleus, and the cerebellum. Comparison of control subjects with patients with Parkinson's disease and control animals with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated animals revealed a loss of parkin-immunoreactive neurons only in the substantia nigra pars compacta. Furthermore, the surviving dopaminergic neurons in the parkinsonian state continued to express parkin at a level similar to that observed in the control situation. These data indicate that parkin is a widely expressed protein. Thus, the degeneration of dopaminergic neurons in familial cases of Parkinson's disease with autosomal recessive transmission cannot be explained solely in terms of an alteration of this protein.

FADD: A link between TNF family receptors and caspases in Parkinson's disease.

Fas-associating protein with a death domain (FADD) is a proximal adaptor protein of the tumor necrosis factor (TNF) receptor family death pathway. This human postmortem study showed a significant decrease in the percentage of FADD-immunoreactive dopaminergic (DA) neurons in the substantia nigra pars compacta of patients with PD compared with controls (-24.8%). This decrease correlated with the known selective vulnerability of nigral DA neurons in PD, suggesting that this pathway contributes to the susceptibility of DA neurons in PD to TNF-mediated apoptosis.

[Cyclic nucleotide phosphodiesterase in vertebrates]. / La nucléotide cyclique phosphodiestérase chez les vertébrés.

The cellular concentration of cyclic nucleotides is largely dependent upon the activity of the enzymatic system responsible for their degradation: cyclic nucleotide phosphodiesterase. This enzymatic system thus plays a crucial role in the regulation of the multiple functions which are modulated by cyclic nucleotides in the organism. Many methodological problems, as well as the complexity of the phosphodiesterase system have long maintained a confusion in this field. Recent progresses (purification to homogeneity of some enzymatic forms, discovery of regulatory mechanisms, particularly) have brought a considerable evolution in the knowledge of the system. It is now well established that cyclic nucleotide phosphodiesterase exists under several isoenzymatic forms, the properties and distribution of which largely differ from a tissue to another. Some of these forms are relatively well characterized, while the representativity of others is still discussed. The significance of this multiplicity of isoenzymes, and their interrelationships are presently under study. A very interesting aspect in the study of this enzymatic system is that it is submitted to several physiological regulatory processes. Recent studies on this point suggest that phosphodiesterase might play a major role in the response of the organism to several hormones. These fundamental studies of phosphodiesterase system find a most interesting application in the pharmacological field. Indeed, numerous synthetic compounds which inhibit the enzyme present a strong pharmacological interest.

Pentasubstituted quercetin analogues as selective inhibitors of particulate 3':5'-cyclic-AMP phosphodiesterase from rat brain.

Some penta-O-substituted analogues of quercetin were synthesized and tested for the inhibition of cytosolic and particulate rat brain cyclic AMP and cyclic GMP phosphodiesterase activities. Ten of these compounds are potent and highly selective inhibitors of cAMP hydrolysis with respect to cGMP hydrolysis. They inhibit more potently the particulate enzyme than the cytosolic preparation. The highest selectivity was observed with penta-O-ethylquercetin and analogue 6d, which proved to be more selective and more potent inhibitors than the reference compound Ro 20-1724. Some structure-activity relationships are discussed.

Increased M-calpain expression in the mesencephalon of patients with Parkinson's disease but not in other neurodegenerative disorders involving the mesencephalon: a role in nerve cell death?

Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra and, to a lesser extent, the ventral tegmental area and catecholaminergic cell group A8. However, among these dopaminergic neurons, those expressing the calcium buffering protein calbindin are selectively preserved, suggesting that a rise in intracellular calcium concentrations may be involved in the cascade of events leading to nerve cell death in Parkinson's disease. We therefore analysed immunohistochemically the expression of the calcium-dependent protease calpain II (m-calpain) in the mesencephalon of patients with Parkinson's disease, progressive supranuclear palsy or striatonigral degeneration, where nigral dopaminergic neurons degenerate, and matched controls without nigral involvement. Calpain immunoreactivity was found in fibers and neuronal perikarya in the substantia nigra, the ventral tegmental area, catecholaminergic cell group A8 and the locus coeruleus. In patients with Parkinson's disease but not with the other neurodegenerative disorders, m-calpain immunoreactivity was detected in fibers with an abnormal morphology and in Lewy bodies. Sequential double staining revealed that most of these m-calpain-positive fibers and neuronal perikarya co-expressed tyrosine hydroxylase, indicating that most m-calpain neurons are catecholaminergic. Quantitative analysis of m-calpain staining in the substantia nigra and locus coeruleus revealed an increased density of fibers and neuronal perikarya in parkinsonian patients in both structures. These data suggest that increased calcium concentrations may be associated with nerve cell death in Parkinson's disease.