Effects of coffees before and after special treatment procedure on cell membrane potentials in stomach cells.

Coffee, one of the most excessively used beverages worldwide, commences the risk of gastroesophageal reflux (GER), which may lead to gastric ulcers and increase the risk of gastric cancer. Many attempts have been made by the coffee industry to diminish the irritating effect on mucosa by means of altering the extraction methods concerning gerbic acids and the roasting processes. This paper describes the effect of differently produced coffees involving two brands of Darboven and two brands of other coffee roasters. The aim of this study was to prove the results of gastric potential measurements we found in literature by using human AGS gastric epithelial cells (human adenocarcinoma). All four coffee extracts tested differentially affected the membrane resting potential of AGS cells. Coffees no. 1 and no. 2 depolarized the cells, presumably by increasing the cation entry into the cytosol. In marked contrast, coffee no. 4 hyperpolarizes the cells, possibly by H(+) extrusion and/or Cl(-) influx, suggesting that this coffee might increase acidity in the stomach, which might negatively affect the stomach, especially in people with gastroesophageal reflux symptoms. Overall, our data suggest that different roasting methods of coffees affect the membrane potentials of AGS stomach cells, resulting in increased influx of H+ possibly resulting in decreased stomach acidity and thus reducing GER. These results are in good accordance with clinical pharmacological results from potential difference measurements in healthy volunteers we found in the literature.

Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia.

Minocycline, a semisynthetic tetracycline derivative, protects brain against global and focal ischemia in rodents. We examined whether minocycline reduces excitotoxicity in primary neuronal cultures. Minocycline (0.02 microm) significantly increased neuronal survival in mixed spinal cord (SC) cultures treated with 500 microm glutamate or 100 microm kainate for 24 hr. Treatment with these excitotoxins induced a dose-dependent proliferation of microglia that was associated with increased release of interleukin-1beta (IL-1beta) and was followed by increased lactate dehydrogenase (LDH) release. The excitotoxicity was enhanced when microglial cells were cultured on top of SC cultures. Minocycline prevented excitotoxin-induced microglial proliferation and the increased release of nitric oxide (NO) metabolites and IL-1beta. Excitotoxins induced microglial proliferation and increased the release of NO metabolites and IL-1beta also in pure microglia cultures, and these responses were inhibited by minocycline. In both SC and pure microglia cultures, excitotoxins activated p38 mitogen-activated protein kinase (p38 MAPK) exclusively in microglia. Minocycline inhibited p38 MAPK activation in SC cultures, and treatment with SB203580, a p38 MAPK inhibitor, but not with PD98059, a p44/42 MAPK inhibitor, increased neuronal survival. In pure microglia cultures, glutamate induced transient activation of p38 MAPK, and this was inhibited by minocycline. These findings indicate that the proliferation and activation of microglia contributes to excitotoxicity, which is inhibited by minocycline, an antibiotic used in severe human infections.

Interleukin-6-associated inflammatory processes in Alzheimer's disease: new therapeutic options.

The cytokine interleukin-6 is consistently detected in the brains of Alzheimer's disease patients but not in the brains of nondemented elderly persons. Until recently it was unclear whether an interleukin-6-associated inflammatory mechanism is an early or late event in the pathological cascade of Alzheimer's disease. We investigated whether interleukin-6 could be detected in plaques of Alzheimer's disease patients prior to the onset of neuritic degeneration. We found interleukin-6 mostly in plaques where neuritic pathology has not yet developed. This indicates that the appearance of interleukin-6 may precede neuritic changes and is not just a consequence of neuritic degeneration. Therefore, one may hypothesize that activation of inflammatory mechanisms may cause neuritic degeneration in plaques. A suppression of interleukin-6 synthesis could, therefore, be of therapeutic value. Upon screening a number of substances, we found that a small number of nonsteroidal antiinflammatory drugs, including tenidap, were able to inhibit interleukin-6 synthesis in cultured human astrocytoma cells. These substances may be therapeutically useful in Alzheimer's disease and should be evaluated in clinical studies.

Inflammation and Alzheimer's disease.

Inflammation clearly occurs in pathologically vulnerable regions of the Alzheimer's disease (AD) brain, and it does so with the full complexity of local peripheral inflammatory responses. In the periphery, degenerating tissue and the deposition of highly insoluble abnormal materials are classical stimulants of inflammation. Likewise, in the AD brain damaged neurons and neurites and highly insoluble amyloid beta peptide deposits and neurofibrillary tangles provide obvious stimuli for inflammation. Because these stimuli are discrete, microlocalized, and present from early preclinical to terminal stages of AD, local upregulation of complement, cytokines, acute phase reactants, and other inflammatory mediators is also discrete, microlocalized, and chronic. Cumulated over many years, direct and bystander damage from AD inflammatory mechanisms is likely to significantly exacerbate the very pathogenic processes that gave rise to it. Thus, animal models and clinical studies, although still in their infancy, strongly suggest that AD inflammation significantly contributes to AD pathogenesis. By better understanding AD inflammatory and immunoregulatory processes, it should be possible to develop anti-inflammatory approaches that may not cure AD but will likely help slow the progression or delay the onset of this devastating disorder.

Pathways of inflammatory activation in Alzheimer's disease: potential targets for disease modifying drugs.

In the human brain several cell types are capable of initiating and amplifying a brain specific inflammatory response involving the synthesis of cytokines, prostaglandins and oxygen free radicals. In Alzheimer's disease (AD), signs of an inflammatory activation of microglia and astroglia are present inside and outside amyloid deposits. Cell culture and animal models suggest an interactive relationship between inflammatory activation, reduced neuronal functioning and deposition of amyloid. The activation of inflammation-associated enzymes such as p38 mitogen-activated protein kinase (p38 MAPK) and cycloxygenase-2 (COX-2) is not restricted to glial cells but also found in neurons and may contribute to intraneuronal damage. Epidemiological studies have shown a reduced risk of AD among users of anti-inflammatory drugs. Therefore, anti-inflammatory drugs have become the focus of several new treatment strategies. Small clinical trials with non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and diclofenac showed a trend for a disease modifying effect, while clinical trials with steroids did not show a beneficial effect. NSAIDs may not only act on COX-2 but also inhibit COX-1 activity or activate peroxisome proliferator-activated receptor gamma (PPAR gamma). Among promising new strategies to reduce the inflammatory activation in the CNS interfering with intracellular pro-inflammatory pathways has been shown to be effective in various cell culture and animal models. Inhibitors of p38MAPK and PPAR gamma agonists may be suitable agents to suppress inflammatory activation in AD.

Carbamazepine-induced upregulation of adenosine A1-receptors in astrocyte cultures affects coupling to the phosphoinositol signaling pathway.

The anticonvulsant and antibipolar drug carbamazepine (CBZ) is known to act as a specific antagonist at adenosine A1-receptors. After a 3-week application of CBZ, A1-receptors are upregulated in the rat brain. We have investigated the consequences of this upregulation for the A1-receptor-mediated signal transduction in primary astrocyte cultures from different regions of the rat brain. CBZ treatment for 10 days had no effect on adenosine A1-receptor mRNA expression in cultures with high basal A1-receptor mRNA levels, but increased A1-receptor mRNA in cultures exhibiting low basal A1-receptor mRNA levels. This upregulation of A1-receptor mRNA was accompanied by an upregulation or induction of A1-receptor-mediated potentiation of PLC activity, a property that was not found in these cultures before CBZ treatment. Thus, CBZ treatment for 10 days induces a new quality of adenosine A1-receptor-mediated signal transduction in cells that express low basal A1-receptor numbers.

Interleukin-6 enhances expression of adenosine A(1) receptor mRNA and signaling in cultured rat cortical astrocytes and brain slices.

The inhibitory neuromodulator adenosine is released in the brain in high concentrations under conditions of exaggerated neuronal activity such as ischemia and seizures, or electroconvulsive treatment. By inhibiting neural overactivity, adenosine counteracts seizure activity and promotes neuronal survival. Since stimulation of adenosine A(2b) receptors on astrocytes induces increased synthesis and release of interleukin-6, which also exerts neuroprotective effects, we hypothesized that the effects of interleukin-6 and of adenosine might be related. We report here that stimulation with interleukin-6 of cultured astrocytes, of cultured organotypic brain slices from newborn rat cortex, and of freshly prepared brain slices from rat cortex induces a concentration- and time-dependent upregulation of adenosine A(1) receptor mRNA. This increased adenosine A(1) receptor mRNA expression is accompanied in astrocytes by an increase in adenosine A(1) receptor-mediated signaling via the phosphoinositide-dependent pathway. Since upregulation of adenosine A(1) receptors leads to increased neuroprotective effects of adenosine, we suggest that the neuroprotective actions of interleukin-6 and adenosine are related and might be mediated at least in part through upregulation of adenosine A(1) receptors. These results may be of relevance for a better understanding of neuroprotection in brain damage but also point to a potential impact of neuroprotection in the mechanisms of the antidepressive effects of chronic carbamazepine, electroconvulsive therapy, and sleep deprivation, which are all accompanied by adenosine A(1) receptor upregulation.

Effects of caffeine and paracetamol alone or in combination with acetylsalicylic acid on prostaglandin E(2) synthesis in rat microglial cells.

Paracetamol has mild analgesic and antipyretic properties and is, along with acetylsalicylic acid, one of the most popular "over the counter" analgesic agents. However, the mechanism underlying its clinical effects is unknown. Another drug whose mechanism of action is unknown is caffeine, which is often used in combination with other analgesics, augmenting their effect. We investigated the inhibitory effect of paracetamol and caffeine on lipopolysaccharide (LPS)-induced cyclooxygenase (COX)- and prostaglandin (PG)E(2)-synthesis in primary rat microglial cells and compared it with the effect of acetylsalicylic acid, salicylic acid, and dipyrone. Furthermore, combinations of these drugs were used to investigate a possible synergistic inhibitory effect on PGE(2)-synthesis. Both paracetamol (IC(50)=7.45 microM) and caffeine (IC(50)=42.5 microM) dose-dependently inhibited microglial PGE(2) synthesis. In combination with acetylsalicylic acid (IC(50)=3.12 microM), both substances augmented the inhibitory effect of acetylsalicylic acid on LPS-induced PGE(2)-synthesis. Whereas paracetamol inhibited only COX enzyme activity, caffeine also inhibited COX-2 protein synthesis. These results are compatible with the view that the clinical activity of paracetamol and caffeine is due to inhibition of COX. Furthermore, these results may help explain the clinical experience of an adjuvant analgesic effect of caffeine and paracetamol when combined with acetylsalicylic acid.

The non-steroidal anti-inflammatory drug tepoxalin inhibits interleukin-6 and alpha1-anti-chymotrypsin synthesis in astrocytes by preventing degradation of IkappaB-alpha.

Tepoxalin is a structurally and functionally novel non-steroidal anti-inflammatory drug (NSAID) with potent anti-inflammatory and analgesic properties. Apart from its inhibitory effect on cyclooxygenase activity, tepoxalin is able to inhibit production of cytokines in peripheral cells outside the CNS. No data, however, are available concerning the effects of this drug in the CNS. Since cytokines such as interleukin-1 (IL-1) or interleukin-6 (IL-6) as well as acute-phase proteins such as alpha1-anti-chymotrypsin (ACT) participate in the etiopathology of Alzheimer's disease (AD), we were interested whether tepoxalin is able to inhibit the synthesis of these immunomodulators in primary rat microglia and astrocytes as well as in the human astrocytoma cell line U373 MG. We found that tepoxalin markedly inhibits IL-1beta-induced IL-6 and ACT synthesis in astrocytes and the synthesis of IL-1beta and IL-6 in lipopolysaccharide (LPS)-stimulated microglial cells. Electrophoretic mobility shift and reporter gene assays revealed that tepoxalin exerts its inhibitory effect through the inhibition of nuclear factor kappaB (NF-kappaB), a transcription factor involved in the induction of IL-1, IL-6 and ACT gene expression. We show that inhibition of NF-kappaB activation by tepoxalin is mediated by preventing IkappaB-alpha degradation. Based on this inhibitory effect of tepoxalin on cytokine and ACT synthesis and the documented therapeutic efficacy of NSAIDs in AD, we conclude that tepoxalin may be of therapeutic benefit for the treatment of AD patients and should therefore be tested in clinical trials.

Protein kinase C-mediated regulation of inducible nitric oxide synthase expression in cultured microglial cells.

Nitric oxide (NO) has been implicated in a number of important brain functions, such as long-term potentiation (LTP) and long-term depression (LTD), and in events associated with neurodegeneration and neuroprotection. In response to brain injury or disease NO production is increased by an inducible enzyme (iNOS), which is only expressed under these conditions. Activated microglia are a major cellular source of iNOS in brain. Due to the important role of iNOS in brain injury and disease, a detailed understanding of intracellular events triggering the expression of iNOS in microglia would facilitate pharmacotherapeutic approaches. It is shown here, that iNOS mRNA, protein and NO product are induced in cultured microglia by lipopolysaccharide (LPS). This induction is reduced by a number of substances elevating intracellular cyclic AMP levels. It is unabated, however, in the presence of substances inhibiting cyclooxygenase-1 and/or cyclooxygenase-2 (e.g., acetyl salicylic acid, SC 58125, L 745337), but is decreased by approx. 50% with PDTC, a scavenger of reactive oxygen intermediates (ROI) that inhibits nuclear factor kappaB (NF-kappaB) activation. Furthermore, inhibitors of protein kinase C (PKC) strongly inhibit iNOS mRNA and protein induction. PKC, therefore, constitutes a major second messenger component (besides NF-kappaB) in the signaling pathway regulating iNOS expression in microglia.

Stimulation of the sphingomyelin pathway induces interleukin-6 gene expression in human astrocytoma cells.

Interleukin-6 (IL-6) has previously been shown to participate in neurodegenerative processes including Alzheimer's disease. However, the mechanisms leading to increased IL-6 expression in the brain remain largely unknown. We have studied the effects of synthetic ceramides and sphingomyelinase as possible regulators of IL-6 gene expression in a human astrocytoma cell line. The synthetic ceramides C2- and C6-ceramide as well as the enzyme sphingomyelinase were able to induce IL-6 gene transcription and protein synthesis in a dose-dependent manner with maximal IL-6 mRNA levels being reached after 4 h of ceramide treatment. We propose that the sphingomyelin pathway is part of the signal transduction cascade leading to IL-6 gene expression in astrocytes, and that this pathway may be involved in IL-6-mediated neurodegenerative processes.

Effects of substance P and selected other neuropeptides on the synthesis of interleukin-1 beta and interleukin-6 in human monocytes: a re-examination.

Recent studies have suggested that substance P (SP) and some other neuropeptides are able to induce the synthesis of the proinflammatory cytokines interleukin-1 (IL-1) and interleukin-6 (IL-6) in peripheral blood mononuclear cells. In the present study, we re-examined these findings by using a completely endotoxin-free monocyte cultivation system. We demonstrate that the neuropeptides SP, vasoactive intestinal peptide, substance K. cholecytokinine, alpha-endorphin and beta-endorphin are consistently unable to induce the synthesis of IL-1 and IL-6 in human peripheral blood monocytes. However, low amounts of LPS (1 pg/ml) synergized with SP to induce IL-6 mRNA expression. In contrast to its lack of effect in monocytes, we were able to confirm the ability of SP to induce cytokine synthesis in astrocytic cells. Our results raise questions about previous results claiming a neuropeptide-induced synthesis of proinflammatory cytokines in human monocytes. In conjunction with other studies, we suggest that undetected levels of endotoxin/LPS in the culture medium may have been primarily responsible for results suggesting an inductive effect of neuropeptides on cytokine synthesis in monocytes.

Strategies to delay the onset of Alzheimer's disease.

Several processes are implicated in the neuropathology of Alzheimer's disease (AD), such as the deposition of amyloid, the formation of paired helical filaments and the proinflammatory activation of microglial and astroglial cells. Proinflammatory activation of glial cells has been a focus of research for a mere ten years now. However, the availability of and broad experience with anti-inflammatory drugs has led to several ongoing clinical trials to verify the capacity of anti-inflammatory drugs to ameliorate the deterioration in AD. The enzymatic cleavage of the amyloid-precursor-protein or the hyperphosphorylation of tau as well as the subsequent aggregation of the resulting products are further targets for drugs intended to delay the neuropathological destruction observed in AD.

Anti-inflammatory drugs: a hope for Alzheimer's disease?

Human brain cells are capable of initiating and amplifying a brain specific inflammatory response involving the synthesis of cytokines, acute-phase proteins, complement proteins, prostaglandins and oxygen radicals. In Alzheimer's disease (AD), all signs of an inflammatory microglial and astroglial activation are present inside and outside amyloid depositions and along axons of neurones with neurofibrillary tangles. Cell culture and animal models suggest a bidirectional relationship between inflammatory activation of glial cells and the deposition of amyloid. Although it remains unclear which of the different pathophysiological processes in AD may be the driving force in an individual case, the inflammatory activation may increase the speed of cognitive decline. Epidemiological studies point to a reduced risk of AD among users of anti-inflammatory drugs. Therefore, anti-inflammatory drugs have become the focus of several new treatment strategies. A clinical trial with the non-steroidal anti-inflammatory drug (NSAID) indomethacin showed promising results, while a clinical trial with steroids did not show a beneficial effect. Further trials with NSAIDs such as unselective cyclooxygenase (COX) and selective cyclooxygenase-2 (COX-2) inhibitors are on their way. COX inhibitors may not only act on microglial and astroglial cells but also reduce neuronal prostaglandin production. New data suggest that prostaglandins enhance neurotoxicity or induce pro-inflammatory cytokine synthesis in astroglial cells. Amongst these promising new strategies to reduce microglial or monocyte activation, interfering with intracellular pathways has been shown to be effective in various cell culture and animal models but clinical studies have not yet been performed.

Cytokine-induced transcription of protein-tyrosine-phosphatases in human astrocytoma cells.

Interleukin-1 (IL-1) and Tumor Necrosis Factor-a (TNFalpha) are potent mediators of inflammatory reactions in the brain. Although much is known about the effects of IL-1 on expression of secretory proteins, few studies have addressed the question of a selective, IL-1-dependent expression of genes involved in neuromodulatory effects of inflammation. Protein-tyrosine-phosphatases (PTP's) have been shown to regulate signal transduction and adhesion processes in the developing nervous system. They are candidates for inflammation-induced neuromodulation. Therefore, we investigated if IL-1 regulates expression of PTP's. We applied a DNA-fingerprinting method based on the PCR-amplification of conserved domains of gene families and observed IL-1-dependent induction of two PTP's, cytoplasmic PTPvarepsilon and receptor-PTPgamma, RPTPgamma, in human U373-MG astrocytoma cells. Using Northern blot analysis, we confirmed this result and also show that in addition to IL-1, TNFalpha but not IL-6 induces the transcription of cytoplasmic PTPvarepsilon and RPTPgamma in human astrocytoma cells. Given the important role for PTP's in neuromodulatory aspects such as axonal guidance and neurite outgrowth, cytokine-induced induction of PTP's may play an important pathenogenic role in the development of chronic inflammatory diseases in the brain.

IL-1 beta and TNF alpha, but not IL-6, induce alpha 1-antichymotrypsin expression in the human astrocytoma cell line U373 MG.

Cytokines such as interleukin-1 (IL-1) and interleukin-6 (IL-6) have previously been shown to participate in neurodegenerative processes including Alzheimer's disease. However, the molecular consequences of increased cytokine expression in the brain remain largely unknown. We have studied the effects of IL-1, IL-6 and TNF alpha on the expression of the acute-phase protein alpha 1-antichymotrypsin (ACT) in human astrocytoma cell lines. Both IL-1 and TNF alpha, but not IL-6, were able to induce ACT gene transcription and protein synthesis. The synthetic glucocorticoid dexamethasone enhanced cytokine-induced ACT mRNA expression and protein synthesis. We conclude that IL-1-induced expression of ACT may be part of the inflammatory response in the brain and may be involved in the pathology of Alzheimer's disease.

Effects of NSAIDs on IL-1 beta-induced IL-6 mRNA and protein synthesis in human astrocytoma cells.

Inflammatory processes contribute to the aetiopathology of Alzheimer's disease (AD). Interleukin-6 (IL-6), a proinflammatory cytokine, is found in the brains of AD patients, but not in brains of normal control persons. In the present study, the effects of seven non-steroidal anti-inflammatory drugs (NSAIDs) on interleukin-1 beta (IL-1 beta)-induced IL-6 mRNA expression and protein synthesis in a human astrocytoma cell line were investigated. Tenidap, naproxen and meloxicam inhibited the IL-1 beta-induced synthesis of IL-6, whereas ibuprofen, piroxicam, diclofenac and indomethacin had no effect. While the effects of naproxen and meloxicam were small and restricted to protein synthesis, tenidap strongly inhibited IL-6 protein synthesis and also affected IL-6 mRNA levels. It is concluded that NSAIDs, and particularly tenidap, may be useful for the treatment of inflammatory processes associated with AD.

Glutamate but not interleukin-6 influences the phosphorylation of tau in primary rat hippocampal neurons.

Alzheimer's disease (AD) is characterized by amyloid plaques, neuritic degenerations, disturbed glutamatergic neurotransmission and a peculiar inflammatory response. Diffuse plaques develop into neuritic plaques when neurites undergo degeneration in the plaque area. Hyperphosphorylation of tau proteins is a major step in neuritic pathology. Interleukin-6 (IL-6) has been found in diffuse and neuritic amyloid plaques in AD. Therefore the question arises whether IL-6 is involved in the transformation of diffuse into neuritic plaques by affecting tau phosphorylation. We investigated the influence of glutamate and IL-6 on tau phosphorylation in cultured primary rat hippocampal neurons. Glutamate but not IL-6 induced a dephosphorylation of tau. Furthermore IL-6 did not influence the glutamate-induced dephoshorylation of tau. We conclude that the role of IL-6 in AD is not related to the phosphorylation of tau.

Nerve growth factor (NGF) expression in rat microglia is induced by adenosine A2a-receptors.

Microglial response to stimuli is characterized by secretion of both neurotoxic and neurotrophic factors. Various adenosine receptor agonists stimulated the production of nerve growth factor (NGF) in microglia. Using reverse transcription-polymerase chain reaction (RT-PCR)- and ELISA-techniques, we show that the mixed A1- and A2-agonist 5'-(N-ethylcarboxamido)-adenosine (NECA) induces an increase in NGF mRNA expression and NGF protein release. Whereas the A1-specific agonist cyclopentyladenosine (CPA) only minimally affected NGF release, the A2a-specific agonist CGS-21680 triggered the greatest increase in microglial NGF synthesis. Analyzing the selective antagonist (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (EXIP), as well as modulators of the cyclic AMP (cAMP) pathway, we identified an adenosine A2a-receptor-sensitive, cAMP-mediated mechanism of microglial NGF synthesis. Our results indicate that A2a-adenosine receptors modulate microglial neurotrophin expression and release.

Vascular endothelial growth factor expression, vascular volume, and, capillary permeability in human brain tumors.

OBJECTIVE: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and a potent inducer of vascular permeability. In this study, we determined whether expression of VEGF is correlated with in vivo measurements of the capillary permeability and vascular volume of primary human brain tumors. METHODS: Tumor samples (seven glioblastomas, one anaplastic astrocytoma, two low-grade astrocytomas, one pilocytic astrocytoma, and three primary cerebral lymphomas) were stereotactically obtained from 14 patients. A semiquantitative polymerase chain reaction was used to quantify the relative expression of VEGF messenger ribonucleic acid in the tumors. VEGF protein was demonstrated in tissue sections by immunohistochemical techniques. A two-compartment dynamic computed tomographic method was used to quantitatively measure the aforementioned parameters in the regions from which the biopsies were obtained. RESULTS: In glial tumors, there was significant correlation of VEGF messenger ribonucleic acid levels with capillary permeability (P < 0.05) and vascular volume (P < 0.01). Although all primary cerebral lymphomas showed considerable increases in capillary permeability and vascular volume, VEGF expression was only slightly upregulated in these tumors. CONCLUSION: Our findings are consistent with the hypothesis that VEGF may be responsible for endothelial cell proliferation and vascular permeability in glial tumors. This relationship has implications for clinical applications, i.e., assessment of delivery of water-soluble drugs, treatment of edema, and antiangiogenesis therapy based on inhibition of VEGF function.