[The years in pictures; medical milestones since 1970]. / De tijd in beeld; medische mij lpalen sinds 1970.

The end of the year is always a perfect moment to look back. However, looking back over the past year alone is not enough if you want a complete picture of developments in medical science, which is why the Dutch Journal of Medicine (NTvG) has posted a timeline on its website charting medical milestones over the past 45 years. Some of these milestones are illustrated in the infographic in this number, and some interesting patterns can be observed. In this article we discuss various trends, from the increasing tendency to quantify to the rise of evidence-based medicine.

[The Six Million Dollar Man: from fiction to reality]. / De Man van Zes Miljoen: van fictie naar werkelijkheid.

The term 'bionic' has been in existence since 1958, but only gained general recognition from the television series 'The Six Million Dollar Man'. Following a crash, the central figure in this series - test pilot Steve Austin - has an eye, an arm and both legs replaced by prostheses which make him stronger and faster than a normal person. This story is based on the science fiction book 'Cyborg' by Martin Caidin. In the world of comic books and films there are a number of examples of people who are given superhuman powers by having technological gadgets built in. Although the latter is not yet possible, the bionic human has now become reality.

Astrocyte-enhanced neuronal survival is mediated by scavenging of extracellular reactive oxygen species.

The survival of cultured neurons is promoted by the presence of antioxidants or astrocytes. This indicates that extracellular reactive oxygen species (ROS) impair neuronal survival and suggests that astrocytes exert their survival-enhancing effect through inactivation of these toxicants. However, to our knowledge, data supporting this hypothesis are lacking. Previously, we showed that loss of the antioxidant glutathione abolishes the neuronal survival-stimulating action of astrocytes in cocultures, consisting of rat striatal astrocytes and mesencephalic, dopaminergic neurons. Using uptake of [3H]dopamine as marker of neuronal survival, we presently investigated whether this effect of glutathione depletion is mediated by extracellular ROS. For this purpose, we incubated glutathione-depleted cocultures with superoxide dismutase, catalase or both. Whereas superoxide dismutase had no effect and catalase only partially protected, addition of the enzymes together completely prevented the impairment of neuronal survival caused by glutathione loss. No change in neuronal survival occurred upon exposure of control cocultures to superoxide dismutase and/or catalase. These data strongly implicate scavenging of extracellular ROS in astrocyte-stimulated neuronal survival and moreover suggest a crucial role for glutathione in this process.

Establishment of human adult astrocyte cultures derived from postmortem multiple sclerosis and control brain and spinal cord regions: immunophenotypical and functional characterization.

We have successfully established highly enriched astrocyte cultures upon passaging of primary cultures derived from various regions of postmortem human adult brain and spinal cord. Tissues were collected at autopsies with relatively short postmortem times (3-9 hr) from multiple sclerosis (MS) and (normal) control cases. Immunocytochemical analysis showed that primary cultures were composed of colonies of oligoclonal cells that expressed the intermediate filament proteins glial fibrillary acidic protein (GFAP), vimentin, as well as glutamine synthetase (GS). Passaging the astrocytes did not affect their proliferating capacity as monitored by bromodeoxyuridine (BrdU) incorporation. Astrocyte-specific markers were stably expressed for at least 12 passages per individual tissue sample. Large numbers of GFAP-positive astrocytes were obtained from each sample and could be stored frozen and recultured. Very few macrophages/microglial cells (1-3%) were present in the human adult astrocyte cultures, using a panel of macrophage-specific markers. However, the monoclonal antibodies (mAbs KP1, EBM1, 25F9) and lysozyme antiserum directed against lysosomal antigens strongly immunostained cultured astrocytes derived from MS and control cases, implicating that expression of these lysosomal antigens is not restricted to macrophages/ microglial cells in human glial cell cultures. Interestingly, astrocytes derived from active demyelinated MS lesions showed an increased proliferating capacity compared to astrocytes derived from non-lesioned and normal brain and spinal cord regions, as shown with a microculture tetrazolium assay (MTT assay).

Anethole dithiolethione prevents oxidative damage in glutathione-depleted astrocytes.

Astrocytes protect neurons against reactive oxygen species such as hydrogen peroxide, a capacity which reportedly is abolished following loss of the antioxidant glutathione. Anethole dithiolethione, a sulfur-containing compound which is used in humans, is known to increase cellular glutathione levels and thought thereby to protect against oxidative damage. In the present study we found that anethole dithiolethione increased the glutathione content of cultured rat striatal astrocytes. This effect was abolished by coincubation with the glutathione synthesis inhibitor buthionine sulfoximine. Nevertheless, in the presence of buthionine sulfoximine, despite the lack of an increase in the lowered glutathione level, anethole dithiolethione fully protected the astrocytes against the enhanced toxicity of hydrogen peroxide. Thus, apparently other mechanisms than stimulation of glutathione synthesis are involved in the compound's protective action in astrocytes. Considering the occurrence of lowered glutathione levels in neurodegenerative syndromes, we conclude that further evaluation of the therapeutic potential of anethole dithiolethione is warranted.

The antiproliferative effect of 8-chloro-adenosine, an active metabolite of 8-chloro-cyclic adenosine monophosphate, and disturbances in nucleic acid synthesis and cell cycle kinetics.

8-Chloro-adenosine, the dephosphorylated metabolite of the antineoplastic agent 8-chloro-cyclic AMP, has been proposed to act on the regulatory subunits of cyclic AMP-dependent protein kinase. 8-Chloro-adenosine has a growth-inhibitory effect, the mechanism of which is unclear. We investigated the effects of 8-chloro-cyclic AMP and 8-chloro-adenosine on nucleic acid synthesis and cell cycle kinetics in two human glioma cell lines. These effects were compared to those of the cyclic AMP analogue 8-(4-chlorophenyl)-thio-cyclic AMP (8-CPTcAMP), which is less susceptible to dephosphorylation. Whereas 8-CPTcAMP almost completely inhibited RNA and DNA synthesis, both 8-chloro-adenosine and 8-chloro-cyclic AMP only partly inhibited synthesis of RNA and DNA at growth-inhibitory concentrations, as demonstrated by using [5-1H] uridine and [14C]thymidine incorporation. Therefore, the growth-inhibitory effect of 8-chloro-cyclic AMP is not (or not completely) due to activation of cyclic AMP-dependent protein kinase nor to the inhibition of nucleic acid synthesis. Flow cytometric analysis revealed that 8-chloro-cyclic AMP and 8-chloro-adenosine probably block cell cycle progression at the G2M phase. The effects of 8-chloro-cyclic AMP on nucleic acid synthesis and cell cycle progression were largely prevented by adenosine deaminase, which inactivates 8-chloro-adenosine. This indicates that the effects of 8-chloro-cyclic AMP were at least in part due to its metabolite 8-chloro-adenosine. Incorporation of 8-chloro-adenosine into RNA and DNA might contribute to the disturbance of the cell cycle kinetics and growth-inhibitory effect of 8-chloro-adenosine.

Astrocyte-mediated enhancement of neuronal survival is abolished by glutathione deficiency.

Astrocytes promote the survival of neurons. Conditions characterized by loss of neurons, such as aging and aging-related neurodegenerative disorders, are accompanied by both disturbances in astrocyte-neuron interactions and signs of oxidative damage. Neuronal glutathione, a major antioxidant in the brain, is maintained by astrocytes and brain levels of glutathione are reduced in named conditions. Therefore, we focused on a possible link between glutathione deficiency and loss of astrocyte-derived neuronal support. For this purpose, we used a coculture system consisting of rat striatal astrocytes and mesencephalic, dopaminergic (DAergic) neurons. Using tyrosine hydroxylase immunocytochemistry and radiolabeled dopamine uptake as parameters, an increase in the number and outgrowth of DAergic neurons was noted in cocultures as compared to cultures of mesencephalic neurons alone. This enhanced survival of DAergic neurons in cocultures was abolished following depletion of glutathione with buthionine sulfoximine. As demonstrated by glial fibrillary acidic protein immunocytochemistry and a microtiter tetrazolium assay, under these conditions no change in astrocyte survival occurred. However, glutathione depletion in cocultures was accompanied by loss of astrocyte-mediated neuroprotection against hydrogen peroxide toxicity. Thus, our results indicate that glutathione is important for the maintenance of the neuronal support function of astrocytes and that glutathione deficiency in the brain may lead to enhanced vulnerability of neurons to (oxidative) damage.

Glutathione is involved in the granular storage of dopamine in rat PC 12 pheochromocytoma cells: implications for the pathogenesis of Parkinson's disease.

Parkinson's disease (PD) is characterized by degeneration of dopamine (DA)-containing nigro-striatal neurons. Loss of the antioxidant glutathione (GSH) has been implicated in the pathogenesis of PD. Previously, we showed that the oxidant hydrogen peroxide inhibits vesicular uptake of DA in nigro-striatal neurons. Hydrogen peroxide is scavenged by GSH and, therefore, we investigated a possible link between the process of vesicular storage of DA and GSH metabolism. For this purpose, we used rat pheochromocytoma-derived PC12 cells, a model system applied extensively for studying monoamine storage mechanisms. We show that depletion of endogenous DA stores with reserpine was accompanied in PC12 cells by a long-lasting, significant increase in GSH content the extent of which appeared to be inversely related to the rate of GSH synthesis. A similar increase in GSH content was observed after depletion of DA stores with the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine. In the presence of alpha-methyl-p-tyrosine, refilling of the DA stores by exogenous DA reduced GSH content back to control level. Lowering of PC12 GSH content, via blockade of its synthesis with buthionine sulfoximine, however, led to a significantly decreased accumulation of exogenous [3H]DA without affecting uptake of the acetylcholine precursor [14C]choline. These data suggest that GSH is involved in the granular storage of DA in PC12 cells and that, considering the molecular characteristics of the granular transport system, it is likely that GSH is used to protect susceptible parts of this system against (possibly DA-induced) oxidative damage.

Presence of glutathione immunoreactivity in cultured neurones and astrocytes.

Although glutathione (GSH) is considered an important antioxidant in the brain, its cellular localization is unclear. In general, neurones are supposed to contain considerably less GSH than astrocytes. We determined biochemically and immunocytochemically the presence of GSH in cultured neurones and astrocytes from the cortex, mesencephalon and striatum. Cortical neurones contained less GSH than astrocytes whereas GSH levels in neurones from the striatum and mesencephalon were comparable to those in astrocytes. Immunocytochemistry showed significant GSH staining in neurones. Fluorescent double staining of GSH and tyrosine hydroxylase revealed that dopaminergic neurones also contained GSH, although apparently at a lower level than other mesencephalic neurones.

Differential sensitivity to hydrogen peroxide of dopaminergic and noradrenergic neurotransmission in rat brain slices.

Oxidative stress, induced by hydrogen peroxide, has been implicated in the pathogenesis of Parkinson's disease. Only scarce information is available if and how hydrogen peroxide, a side product of catecholamine (CA) breakdown, interferes with CAergic neurotransmission. Therefore, we investigated the effect of hydrogen peroxide on the release of [3H]dopamine (DA) and [3H]noradrenaline (NA) from rat striatal and cortical tissue slices, respectively. Hydrogen peroxide (0.01-1 mM) stimulated the spontaneous release of [3H]DA from striatal slices. Its effect on [3H]NA release from cortical slices, however, was much smaller than on DA release and occurred only in concentrations above 0.1 mM. Furthermore, only in concentrations of 1 mM or higher did a stimulation of spontaneous release of radioactivity from striatal slices incubated with [3H]choline occur. Omission of calcium significantly enhanced the effect on DA release, and an increase of calcium significantly reduced it. Blockade of vesicular storage with reserpine (0.3 microM) almost completely abolished [3H]DA release induced by hydrogen peroxide. Following incubation of striatal slices with [3H]NA in the presence of the NA (re)uptake blocker desmethylimipramine (0.3 microM), NA release was observed at a concentration (0.1 mM) at which no effect occurred in cortical slices. Moreover, under these conditions [3]NA and [3H]DA release from striatal slices reached comparable levels. Our results show that hydrogen peroxide induces a nonexocytotic release of DA and NA by interfering with the vesicular uptake and/or storage of these CAs. However, the striatal DA storage system, irrespective of the presence of either DA or NA, appeared to be substantially more sensitive to this effect than its cortical equivalent for storage of NA.

Cultured rat striatal and cortical astrocytes protect mesencephalic dopaminergic neurons against hydrogen peroxide toxicity independent of their effect on neuronal development.

Reactive oxygen species (ROS), including hydrogen peroxide, are supposed to be involved in the degeneration of dopaminergic neurons in Parkinson's disease. The potential role of astrocytes against neurotoxic effects of ROS was studied in cocultures of rat mesencephalic neurons and rat striatal or cortical astrocytes. Neuronal [3H]dopamine uptake, a marker of dopaminergic neuron integrity, was enhanced by striatal astrocytes, but not by cortical astrocytes, compared to uptake in mesencephalic neurons cultured alone. Whereas hydrogen peroxide at concentrations up to 100 microM reduced the [3H]dopamine uptake in neuronal cultures, no reduction of the uptake was observed in cocultures, regardless of the origin of the supporting astrocytes. These results suggest that astrocyte mediated protection of neurons against hydrogen peroxide induced toxicity is not directly related to a region-specific neurotrophic effect.

Dopamine D1 receptor agonists display a different intrinsic activity in rat, monkey and human astrocytes.

Measuring dopamine D1 receptor stimulated cyclic AMP production in cultured astrocytes from rat, monkey and human brain, we demonstrate that the 'classical' drug SKF 38393 (7,8-dihydroxy-1- phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine) is a partial agonist with particularly low intrinsic activity in primates. Furthermore, its analogue SKF 81297 (6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5- tetrahydro-1 H-3-benzazepine) is shown to be a full agonist in rats but a partial, albeit more efficacious, agonist in primates, whereas the benzopyran A 68930 ((1R,3S)-1-aminomethyl-5,6- dihydroxy-3-phenyl-isochroman HCl) displays full efficacy in both species. The data suggest that cultured astrocytes provide a good model to study species differences in the pharmacological characteristics of dopamine D1 receptor agonists and indicate that SKF 38393 is not suited to study dopamine D1 receptor function in primates.

Effect of suramin on adenylate cyclase and protein kinase C.

The trypanocidal drug, suramin, has been shown to possess antitumour activity, both in vitro and in vivo. Its mechanism of action, however, remains unclear although an effect on signal transduction has been proposed. We therefore studied the in vitro effect of suramin on protein kinase C (PKC), on adenylate cyclase and on the intracellular calcium concentrations [Ca2+]i in human cancer cell lines. Ca(2+)- and phospholipid-dependent PKC was isolated from a normal rat spleen, and compared with that of the human cancer cell lines MCF-7 (breast cancer) and PC3 (prostate cancer). PKC was inhibited by 50% at 55, 40 and 27 microM suramin in the three PKC sources, respectively, while 300 microM of suramin gave 97, 95 and 99% inhibition. With 50 nM staurosporine, a known PKC inhibitor, we observed 80, 99 and 96% inhibition in these three different sources of PKC. Six day exposure of these cell lines to suramin, causing 50% growth inhibition, decreased the Ca(2+)- and phospholipid-dependent PKC activity in MCF-7 cells to 52% of the control and in PC3 cells to 48% at equitoxic concentrations (45 and 150 microM suramin, respectively). These concentrations of suramin slightly increased (approximately 2-fold) the adenylate cyclase activity in MCF-7 cells, but not in PC3 cells. In MCF-7 and PC3 cells, we measured the [Ca2+]i using Fura-2 fluorescence and observed a decrease in MCF-7 cells from 126 to 99 nM when the cells were exposed for 6 days to 45 microM suramin. In PC3 cells, [C2+]i decreased from 131 to 117 nM after exposure to 150 microM suramin. In conclusion, suramin inhibited the Ca(2+)- and phospholipid-dependent PKC activity in both cell lines in a dose-dependent manner. Only in the more sensitive MCF-7 cell line was a significant effect of suramin on intracellular Ca2+ and adenylate cyclase observed, indicating that one of the mechanisms of action of suramin could be mediated by perturbations of intracellular signalling pathways.

8-Chloro-cyclic adenosine monophosphate, a novel cyclic AMP analog that inhibits human glioma cell growth in concentrations that do not induce differentiation.

Cyclic AMP is supposed to play a role in cell growth and differentiation via activation of protein kinase A. The cAMP signal transduction pathway may therefore be used as a target for the development of anticancer drugs. We compared the effects of 8ClcAMP, a newly developed cAMP analog, to the effects of more commonly used cAMP analogs on the morphology and the proliferation of three human glioma cell lines. 8ClcAMP was the most potent growth inhibitor, exhibiting an IC50 of approximately 10 microM and inducing growth arrest in all three glioma cell lines at a concentration of 100 microM. The cAMP analogs 8CPTcAMP, dibutyryl cAMP, and 8BrcAMP were much less potent. If used in concentrations that induce growth arrest, both 8CPTcAMP and IBMX, but not 8ClcAMP, induced morphological differentiation of the glioma cells. Apparently, the growth-inhibiting effect of 8ClcAMP is not paralleled by its ability to induce morphological differentiation. The explanation for this phenomenon may be that 8ClcAMP does not exert its growth-inhibiting effect via activation of cAMP-dependent protein kinase. Two alternative mechanisms of action are discussed. Since 100 microM 8ClcAMP retarded the growth of normal rat astrocytes only to a marginal extent, without cytotoxic effects, it is concluded that 8ClcAMP may offer interesting perspectives in the treatment of malignant glioma.

Growth inhibition of human glioma cells induced by 8-chloroadenosine, an active metabolite of 8-chloro cyclic adenosine 3':5'-monophosphate.

8-Chloroadenosine 3':5'-monophosphate (8ClcAMP) inhibits the growth of human glioma cell lines at much lower concentrations than more commonly used cyclic AMP analogues, without inducing morphological differentiation. The mechanism by which 8ClcAMP exerts this effect is not fully understood. We examined whether the growth-inhibitory effect of this compound is due to an active metabolite, using a sulforhodamine protein stain assay to determine the proliferation rate of the WF human glioma cell line. 8-Chloroadenosine, one of the metabolites, inhibited the proliferation of WF human glioma cells more potently than 8ClcAMP. In the presence of adenosine deaminase, which converts 8-chloroadenosine into 8-chloroinosine, 8-chloroadenosine no longer inhibited human glioma cell growth. Addition of adenosine deaminase also largely reduced the growth-inhibitory effect of 8ClcAMP, but not of 8-(4-chlorophenylthio)cAMP. High performance liquid chromatography analysis revealed that at least part of the 8ClcAMP in the culture medium is converted into 8-chloroadenosine. We concluded that 8ClcAMP exerts its growth-inhibitory effect through its active metabolite 8-chloroadenosine. Adenylate cyclase assays showed that 8-chloroadenosine does not affect the intracellular cAMP production through adenosine A1 or A2 receptor activation, which makes it unlikely that 8-chloroadenosine inhibits glioma cell growth by increasing the intracellular cyclic AMP concentration.

Implication of glucocorticoid receptors in the stimulation of human glioma cell proliferation by dexamethasone.

Dexamethasone is frequently used in the therapy of brain tumor patients. We investigated the effect of dexamethasone on the proliferation of three short-term and four established human glioma cell lines in vitro, using a microculture tetrazolium assay to determine growth rates. In one short-term culture and in one established cell line dexamethasone consistently stimulated the proliferation in a concentration-dependent way. The proliferation was maximally enhanced at a concentration of approximately 0.1 microM. In these two cell lines a relatively high level of glucocorticoid receptors was present, whereas low levels of glucocorticoid receptors were found in the other cell lines. In addition, we demonstrated that the stimulatory effects of dexamethasone on the proliferation of the glioma cell lines can be antagonized by the antiglucocorticoid RU38486. The results demonstrate unequivocally that the glucocorticoid receptor plays a role in the growth stimulating effect of dexamethasone.

Influence of beta-aminoproprionitrile (BAPN) on cell growth and elastic fiber formation in cultures of auricular chondrocytes.

Auricular chondrocytes isolated from 4-day-old rabbits and grown in vitro for 14 days, proliferated rapidly and produced a conspicuous network of elastic fibers. Beta-aminoproprionitrile (BAPN), which in vivo inhibits cross-linking of elastin, decreased the formation of elastic fibers at a concentration of 10-20 micrograms/ml and prevented formation at 40 micrograms/ml. At a concentration of 5 micrograms/ml only the so-called patches of elastin appeared to be absent. The inhibitory effect of BAPN on cell growth did not exceed 10%, which indicates that BAPN is only slightly harmful to auricular chondrocytes and can safely be used in studies on elastin deposition by these cells in vitro.