Search

Bloodstream infections (BSIs) are serious complications with high mortality and morbidity in patients with critical illness. This study was conducted to analyze the clinical and microbiological characteristics as well as outcomes in patients with intensive care unit (ICU)-acquired BSIs. Data from 1,545 patients admitted to the ICU were retrospectively collected from January 2005 to December 2010. ICU-acquired BSI was defined as a positive blood culture for a clinically significant bacterial or fungal pathogen obtained >72 h after admission to the ICU. Data on clinical and demographic characteristics, comorbid illness, causes of infections, causative pathogens, and clinical outcomes were analyzed. Among the 1,545 ICU patients analyzed, 129 ICU-acquired BSIs occurred in 124 patients. Catheter-related BSIs (CR-BSIs) and ventilator-associated pneumonia (VAP) were the most common causes (29.4 and 20.9 %, respectively). The most common isolates were Staphylococcus aureus in 35 (25.7 %) and Candida species in 32 (24.8 %) cases. Ninety-eight patients died (overall hospital mortality rate, 75.9 %). ICU-acquired BSI-related mortality occurred in 23 (63.8 %) and 7 (19.4 %) of the VAP and CR-BSIs cases, respectively. The most commonly isolated microorganisms from these fatalities were S. aureus (12, 25.7 %) and Acinetobacter species (12, 25.7 %). In 99 ICU-acquired BSI cases, patients did not receive adequate empirical antimicrobial treatment at the onset of BSIs, whereas the patients in 30 cases did. ICU-acquired BSIs may be associated with high mortality in patients with critical illness. Meticulous infection control and adequate treatment may reduce ICU-acquired BSI-related mortality.

White matter is frequently involved in ischemic stroke, and progressive ischemic white matter injuries are associated with various neurologic dysfunctions in the elderly population. Demyelination and oligodendrocyte (OL) loss are prominent features of ischemic white matter injury. Endothelin-1 injection into the internal capsule resulted in a localized demyelinating lesion in mice, where loss of OL lineage cells and inflammatory cell infiltration were observed accompanied by upregulation of toll-like receptor 2 (TLR2). Intriguingly, the extent of demyelinating pathology was markedly larger in TLR2 deficient mice than that of wild-type (WT) mice. TLR2 deficient mice showed enhanced OL death and decreased phosphorylation of ERK1/2 compared with WT animals. Cultured OLs from TLR2 deficient mice were more vulnerable to oxygen-glucose deprivation than WT OLs. Applying TLR2 agonists Pam3CSK4 or Zymosan after oxygen-glucose deprivation substantially rescued WT OL death with augmentation of ERK1/2 phosphorylation. Treatment with Pam3CSK4 also reduced the extent of endothelin-1 induced ischemic demyelination in vivo. Our data indicate TLR2 may provide endogenous protective effects on ischemic demyelination and OL degeneration.

Abnormal aggregation of alpha-synuclein and sustained microglial activation are important contributors to the pathogenic processes of Parkinson's disease. However, the relationship between disease-associated protein aggregation and microglia-mediated neuroinflammation remains unknown. Here, using a combination of in silico, in vitro and in vivo approaches, we show that extracellular alpha-synuclein released from neuronal cells is an endogenous agonist for Toll-like receptor 2 (TLR2), which activates inflammatory responses in microglia. The TLR2 ligand activity of alpha-synuclein is conformation-sensitive; only specific types of oligomer can interact with and activate TLR2. This paracrine interaction between neuron-released oligomeric alpha-synuclein and TLR2 in microglia suggests that both of these proteins are novel therapeutic targets for modification of neuroinflammation in Parkinson's disease and related neurological diseases.

OBJECTIVES To evaluate if L-arginine: NO pathway is activated in tumor tissues during IL-2 therapy and to evaluate whether IL-2 induced NO synthesis represents an antitumor effector mechanism or an inhibitory factor against therapeutic effects of IL-2. METHODS Four groups [untreated control, NG-monomethyl-L-arginine (MLA) therapy only, IL-2 therapy only, IL-2/MLA therapy groups] of BALB/c mice were injected intraperitoneally with 2 x 10(8) Meth A tumor cells on day 0. MLA was administered subcutaneously with Alzet continuous infusion pumps on day 2. IL-2 therapy (180,000 IU s.c. every 12h for 5 days) was started on day 3. NO production within ascites tumors was assessed by measuring nitrite concentrations in cultures of ascites cells harvested on day 8. Survival and the rate of body weight increment of the mice were measured to evaluate therapeutic responses. Daily urinary nitrate excretion was monitored to demonstrate the effectiveness of MLA in inhibiting NO synthesis. RESULTS Nitrite production in supernatants of Meth A ascites cell cultures was 63 +/- 14 microM in IL-2 treated mice and 3.2 +/- 1.5 microM in untreated controls (p < 0.001). MLA prevented the IL-2 therapy induced increase in nitrite production. IL-2 therapy did not decrease the rate of body weight increment and marginally prolonged mean survival to 18.2 days, compared to 16.6 days in control mice (p = 0.255). MLA administration decreased the rate of body weight increment and prolonged mean survival of IL-2 treated mice (21.8 days, p = 0.001 versus IL-2 alone). Interestingly, the MLA treatment increased the rate of body weight increment and diminished the survival of control mice to 11.6 days (p = 0.003). MLA administration via Alzet continuous infusion pumps achieved approximately 60% suppression of urinary nitrate excretion by control mice. Subcutaneous IL-2 treatment strongly induced nitric oxide synthesis (up to 3.5 mumoles of urinary nitrate/ mouse/day). MLA also effectively suppressed IL-2 induced NO production. CONCLUSION L-arginine: NO pathway can be activated in malignant ascites by IL-2 therapy and NO synthesis functions as an inhibitory mechanism against IL-2 induced anti-tumor effects.

Inflammatory responses are pathological hallmarks of infectious diseases of the nervous system such as bacterial meningitis and viral encephalitis. Noninfectious neurological disease or injury often accompanies neuroinflammation, although the underlying mechanisms of such “sterile neuroinflammation” are not completely understood. Studies conducted over the past 10 years on the function of Toll-like receptors (TLRs) in the nervous system have shed new light on the molecular and cellular mechanisms of neuroinflammation. TLRs belong to a class of pattern-recognition receptors that play important roles in host defense against pathogens and tissue injury/recovery by recognizing a wide variety of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). In the nervous system, different members of the TLR family are expressed on astrocytes, microglia, oligodendrocytes, and Schwann cells, implicating these glial cells in neuroinflammation in pathological contexts. In this chapter, we summarize recent studies of TLR expression in the cells of the nervous system and discuss its roles in neuroinflammation in the context of infectious diseases as well as noninfectious neurological disorders such as stroke, spinal cord injury, and peripheral nerve injury.

c-Jun N-terminal kinase (JNK), a member of the MAPK family, is an important regulatory factor of synaptic plasticity as well as neuronal differentiation and cell death. Recently, JNK has been reported to modulate synaptic plasticity by the direct phosphorylation of synaptic proteins. The specific role of c-Jun phosphorylation in JNK mediated synaptic plasticity, however, remains unclear. In this study, we investigated the effects of c-Jun phosphorylation on synaptic structure and function by using c-Jun mutant mice, c-JunAA, in which the active phosphorylation sites at serines 63 and 73 were replaced by alanines. The gross hippocampal anatomy and number of spines on hippocampal pyramidal neurons were normal in c-JunAA mice. Basal synaptic transmission, input–output ratios, and paired-pulse facilitation (PPF) were also no different in c-JunAA compared with wild-type mice. Notably, however, the induction of long-term potentiation (LTP) at hippocampal CA3–CA1 synapses in c-JunAA mice was impaired, whereas induction of long-term depression (LTD) was normal. These data suggest that phosphorylation of the c-Jun N-terminus is required for LTP formation in the hippocampus, and may help to better characterize JNK-mediated modulation of synaptic plasticity.

Toll-like receptors (TLRs) are type I transmembrane signaling molecules that are expressed in cells of the innate immune system. In these cells, TLRs function as pattern recognition receptors (PRR) that recognize specific molecular patterns derived from microorganisms. Upon activation, TLRs trigger a cascade of intracellular signaling pathways in innate immune cells, leading to the induction of inflammatory and innate immune responses, which in turn regulate adaptive immune responses. In the nervous system, different members of the TLR family are expressed on glial cells (astrocytes, microglia, oligodendrocytes, and Schwann cells) and neurons. Recently, increasing evidence has supported the idea that TLRs also recognize endogenous molecules that are released from damaged tissue, thereby regulating inflammatory responses and subsequent tissue repair. These findings imply that TLRs on glial cells may also be involved in the inflammatory response to tissue damage in the nervous system. In this review, we discuss recent studies on TLR expression in the cells of the nervous system and their roles in acute neurological disorders involving tissue damage such as strokes, traumatic spinal cord and brain injuries, and peripheral nerve injuries.

Recent studies show that necrotic neuronal cells (NNC) activate microglia, thereby leading to neuronal cell death. This suggests that chemicals that inhibit microglia activation may be used as neuroprotective drugs. In this context, we screened a chemical library for inhibitors of microglia activation. Using a screening system based on a nitrite assay, we isolated two chemicals that inhibit nitric oxide (NO) release from activated microglia: triamcinolone acetonide (TA) and amcinonide. The half-maximal inhibitory concentrations (IC50) of TA and amcinonide for NO release inhibition were 1.78 nM and 3.38 nM, respectively. These chemicals also inhibited NNC-induced expression of the proinflammatory genes iNOS, TNF-α, and IL-1β in glial cells. A study based on a luciferase assay revealed that TA attenuated NNC-induced microglia activation by blocking the NF-κB signaling pathway. In addition, TA protected cortical neurons in coculture with microglia from LPS/IFN-γ-induced neuronal cell death. In conclusion, TA may inhibit microglia activation and may protect neuronal cells from death induced by microglial activation.

Background: Imiquimod (IQ) is known as an agonist of Toll-like receptor 7 (TLR7) and is widely used to treat various infectious skin diseases. However, it causes severe itching sensation as its side effect. The precise mechanism of how IQ causes itching sensation is unknown. A recent report suggested a molecular target of IQ as TLR7 expressed in dorsal root ganglion (DRG) neurons. However, we recently proposed a TLR7-independent mechanism, in which the activation of TLR7 is not required for the action of IQ in DRG neurons. To resolve this controversy regarding the involvement of TLR7 and to address the exact molecular identity of itching sensation by IQ, we investigated the possible molecular target of IQ in DRG neurons. Findings: When IQ was applied to DRG neurons, we observed an increase in action potential (AP) duration and membrane resistance both in wild type and TLR7-deficient mice. Based on these results, we tested whether the treatment of IQ has an effect on the activity of K+ channels, Kv1.1 and Kv1.2 (voltage-gated K+ channels) and TREK1 and TRAAK (K2P channels). IQ effectively reduced the currents mediated by both K+ channels in a dose-dependent manner, acting as an antagonist at TREK1 and TRAAK and as a partial antagonist at Kv1.1 and Kv1.2. Conclusions: Our results demonstrate that IQ blocks the voltage-gated K+ channels to increase AP duration and K2P channels to increase membrane resistance, which are critical for the membrane excitability of DRG neurons. Therefore, we propose that IQ enhances the excitability of DRG neurons by blocking multiple potassium channels and causing pruritus.

The inflammasome is a multimolecular complex that orchestrates the activation of proinflammatory caspases and interleukin (IL)-1β, which is generally increased in the cerebrospinal fluids of patients with tuberculous meningitis. However, it has not been clarified whether mycobacteria can activate the inflammasome and induce IL-1β maturation in microglia. In this study, we found that the priming of primary murine microglial cells with conditioned media from cultures of macrophages infected with Mycobacterium tuberculosis (Mtb) led to robust activation of caspase-1 and IL-1β secretion after Mtb stimulation. Potassium efflux and the lysosomal proteases cathepsin B and cathepsin L were required for the Mtb-induced caspase-1 activation and maturation of IL-1β production in primed microglia. Mtb-induced IL-1β maturation was also found to depend on the nucleotide binding and oligomerization of domain-like receptor family pyrin domain containing 3 protein (NLRP3) and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), as well as the generation of mitochondrial reactive oxygen species (ROS). Notably, the priming of microglia with tumor necrosis factor-α or oncostatin M resulted in caspase-1 cleavage and IL-1β secretion in response to Mtb. Moreover, dexamethasone, as an adjunctive therapy for patients of tuberculous meningitis, significantly reduced the Mtb-induced maturation of IL-1β through inhibition of mitochondrial ROS generation. Collectively, these data suggest that Mtb stimulation induces activation of the microglial NLRP3 inflammasome (composed of NLRP3, ASC, and cysteine protease caspase-1) through microglia-leukocyte interactions as a priming signal, and that dexamethasone decreases inflammasome activation through inhibition of ROS of mitochondrial origin.

Background: We have previously reported that nerve injury-induced neuropathic pain is attenuated in toll-like receptor 2 (TLR2) knock-out mice. In these mice, inflammatory gene expression and spinal cord microglia actvation is compromised, whereas the effects in e dorsal root ganglia (DRG) have not been tested. In this study, we investigated the role of TLR2 in inflammatory responses in the DRG after peripheral nerve injury. Results: L5 spinal nerve transection injury induced the expression of macrophage-attracting chemokines such as CCL2/MCP-1 and CCL3/MIP-1 and subsequent macrophage infiltration in the DRG of wild-type mice. In TLR2 knockout mice, however, the induction of chemokine expression and macrophage infiltration following nerve injury were markedly reduced. Similarly, the induction of IL-1β and TNF-α expression in the DRG by spinal nerve injury was ameliorated in TLR2 knock-out mice. The reduced inflammatory response in the DRG was accompanied by attenuation of nerve injury-induced spontaneous pain hypersensitivity in TLR2 knock-out mice. Conclusions: Our data show that TLR2 contributes to nerve injury-induced proinflammatory chemokine/cytokine gene expression and macrophage infiltration in the DRG, which may have relevance in the reduced pain hypersensitivity in TLR2 knock-out mice after spinal nerve injury.

Dysfunction in immune surveillance during anticancer chemotherapy of patients often causes weakness of the host defense system and a subsequent increase in microbial infections. However, the deterioration of organ-specific function related to microbial challenges in cisplatin-treated patients has not yet been elucidated. In this study, we investigated cisplatin-induced TLR4 expression and its binding to LPS in mouse cochlear tissues and the effect of this interaction on hearing function. Cisplatin increased the transcriptional and translational expression of TLR4 in the cochlear tissues, organ of Corti explants, and HEI-OC1 cells. Furthermore, cisplatin increased the interaction between TLR4 and its microbial ligand LPS, thereby upregulating the production of proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, via NF-κB activation. In C57BL/6 mice, the combined injection of cisplatin and LPS caused severe hearing impairment compared with that in the control, cisplatin-alone, or LPS-alone groups, whereas this hearing dysfunction was completely suppressed in both TLR4 mutant and knockout mice. These results suggest that hearing function can be easily damaged by increased TLR expression and microbial infections due to the weakened host defense systems of cancer patients receiving therapy comprising three to six cycles of cisplatin alone or cisplatin combined with other chemotherapeutic agents. Moreover, such damage can occur even though patients may not experience ototoxic levels of cumulative cisplatin concentration.

Recent studies indicate that Toll-like receptors (TLRs), originally identified as infectious agent receptors, also mediate sterile inflammatory responses during tissue damage. In this study, we investigated the role of TLR2 in excitotoxic hippocampal cell death using TLR2 knock-out (KO) mice. TLR2 expression was up-regulated in microglia in the ipsilateral hippocampus of kainic acid (KA)-injected mice. KA-mediated hippocampal cell death was significantly reduced in TLR2 KO mice compared with wild-type (WT) mice. Similarly, KA-induced glial activation and proinflammatory gene expression in the hippocampus were compromised in TLR2 KO mice. In addition, neurons in organotypic hippocampal slice cultures (OHSCs) from TLR2 KO mouse brains were less susceptible to KA excitotoxicity than WT OHSCs. This protection is partly attributed to decreased expression of proinflammatory genes, such as TNF-α and IL-1β in TLR2 KO mice OHSCs. These data demonstrate conclusively that TLR2 signaling in microglia contributes to KA-mediated innate immune responses and hippocampal excitotoxicity.

Increasing evidence supports the notion that spinal cord microglia activation plays a causal role in the development of neuropathic pain after peripheral nerve injury; yet the mechanisms for microglia activation remain elusive. Here, we provide evidence that NADPH oxidase 2 (Nox2)-derived ROS production plays a critical role in nerve injury-induced spinal cord microglia activation and subsequent pain hypersensitivity. Nox2 expression was induced in dorsal horn microglia immediately after L5 spinal nerve transection (SNT). Studies using Nox2-deficient mice show that Nox2 is required for SNT-induced ROS generation, microglia activation, and proinflammatory cytokine expression in the spinal cord. SNT-induced mechanical allodynia and thermal hyperalgesia were similarly attenuated in Nox2-deficient mice. In addition, reducing microglial ROS level via intrathecal sulforaphane administration attenuated mechanical allodynia and thermal hyperalgesia in SNT-injured mice. Sulforaphane also inhibited SNT-induced proinflammatory gene expression in microglia, and studies using primary microglia indicate that ROS generation is required for proinflammatory gene expression in microglia. These studies delineate a pathway involving nerve damage leading to microglial Nox2-generated ROS, resulting in the expression of proinflammatory cytokines that are involved in the initiation of neuropathic pain.

Toll-like receptors (TLRs) functionally expressed in salivary epithelial cells, but their roles remain elusive. Among TLRs family, TLR3 is activated by dsRNA, a byproduct of viral infection. The aim of this study was to investigate the role of TLR3 in the inflammatory immune responses using HSG cells. Reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR and ELISA were performed to identify expression of TLRs and TLR3-mediated chemokine inductions. The chemotaxis assay of activated T lymphocytes was also performed. Treatment of HSG cells with polyinosinic: polycytidylic acid (poly(I:C)) significantly increased interferon-γ-inducible protein 10 (IP-10), interferoninducible T-cell α chemoattractant (I-TAC), and regulated on activation, normal T-cells expressed and secreted (RANTES) gene expressions in a concentration-dependent manner. Anti-TLR3 antibody blocked the increases of IP-10 and I-TAC genes. Poly(I:C)-induced increases of IP-10 and I-TAC were also confirmed at protein levels from cell lysates, but their release into extracellular medium was detected only in IP-10. We found that the culture media from HSG cells stimulated with poly(I:C) significantly increases T lymphocyte migration. Our results suggest that TLR3 plays an important role in chemokine induction, particularly IP-10, in salivary epithelial cells.

Traumatic spinal cord injury (SCI) is followed by massive infiltration and activation of myeloid cells such as neutrophils and macrophages, but the functions of these cells are controversial. In this study, our objective was to elucidate the in vivo role of a signaling pathway involved in activation of these innate immune cells in SCI using myeloid cell-specific IκB kinase (IKK)-β conditional knockout (ikkβΔmye) mice. In these mice, the ikkβ gene has been specifically deleted from myeloid cells, compromising their in vivo IKK/NF-κB-dependent activation. We found that ikkβΔmye mice had significantly reduced neutrophil and macrophage infiltrations after SCI compared to ikkβ(+/+) controls. SCI-induced proinflammatory gene expression was also reduced in ikkβΔmye mice. Reduced neuroinflammation in ikkβΔmye mice was accompanied by attenuated neuronal loss and behavioral deficits in motor activity. In addition, the SCI-induced expression of CXC ligand 1 was reduced in ikkβΔmye mice, which may be responsible for the reduced neutrophil infiltration in these mice. Our data demonstrate that IKK-β-dependent myeloid cell activation potentiates neuroinflammation and neuronal damage after SCI.

The sensory system is developed and optimized by experiences given in the early phase of life in association with other regions of the nervous system. To date, many studies have revealed that deprivation of specific sensory experiences can modify the structure and function of the central nervous system; however, the effects of sensory overload remains unclear. Here we studied the effect of overloading the taste sense in the early period of life on the synaptic plasticity of rat hippocampus and somatosensory cortex. We prepared male and female Sprague Dawley rats with ad libitum access to a 0.1% saccharin solution for 2 hrs per day for three weeks after weaning on postnatal day 22. Saccharin consumption was slightly increased in males compared with females; however, saccharin intake did not affect chow intake or weight gain either in male or in female rats. We examined the effect of saccharin-intake on long term potentiation (LTP) formation in hippocampal Schaffer collateral pathway and somatosensory cortex layer IV - II/III pathways in the 6-week old saccharin-fed rats. There was no significant difference in LTP formation in the hippocampus between the control group and saccharin-treated group in both male and female rats. Also in the somatosensory cortex, we did not see a significant difference in LTP among the groups. Therefore, we conclude that saccharin-intake during 3~6 weeks may not affect the development of physiological function of the cortical and hippocampal synapses in rats.

The skin of Vitis coignetiae Pulliat (meoru) grown wild in the Republic of Korea was analyzed for anthocyanins via HPLC coupled to ESI-MS/MS in positive ion mode. Chromatographic separation was conducted via RP HPLC using a C(18) column, with a 50-min gradient from 0 to 80% methanol in water containing 0.5% formic acid. A total of 18 anthocyanins were identified. Among them, nine compounds were newly determined by comparing the retention time (t(R)) and mass fragmentation patterns with those of the previously reported anthocyanins for other grape varieties: malvidin hexose, peonidin 3-galactoside, malvidin 3-galactoside, cyanidin, petunidin, petunidin 3-(6''-coumaroyl)-5-diglucoside, peonidin, malvidin, and malvidin 3-(6''-coumaroyl)-5-diglucoside. The antioxidant activity of the V. coignetiae Pulliat anthocyanins was determined via 2,2-diphenyl-2-picrylhydrazyl radical scavenging and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation assays in a range of concentration from 25 to 500 mg/L. The capacity increased with concentration. The IC(50) values, defined as the concentration of sample required to scavenge 50% of free radicals, were calculated as follows: 189.63+/-11.31 mg/L and 141.29+/-6.70 mg/L for 2,2-diphenyl-2-picrylhydrazyl and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation, respectively. The antioxidant activity of the V. coignetiae Pulliat anthocyanins is substantially higher than that of ascorbic acid and is similar to the effects of the extracts obtained from other grape varieties.

As watermelon is farmed primarily by protected and successive cultivation techniques, a number of pesticides are required for the control of pests and diseases. To evaluate the harmful effects of pesticides in watermelon and to guarantee consumers' safety, a rapid screening process for pesticides is required. A LC-MS/MS method was applied for the direct quantitation of 44 pesticide residues in watermelon. A Zorbax XDB-C(18) column was selected for analysis, with a mobile phase consisting of a gradient system of water and 5 mM methanolic ammonium formate. MS/MS experiments were performed in ESI positive ion and multiple reaction monitoring modes. The LOQs were in the range of 1-26 microg/kg, thereby indicating good sensitivity. Most of the recoveries ranged between 70-131% with RSDsor = 20%. We suggested that the amount of pesticide residues such as pyroquilon (pyn), boscalid (bd), and dimethomorph (di) in amides (AM) and cinosulfuron (ci) in ureas (UR) may have been overestimated for the pesticides owing to increased alpha-error risk, whereas the amounts of pesticide residues, such as imibenconazole (ie) in the triazoles (TR) and fenpyroximate (fee) in the imidazoles (IM), may have been underestimated as the result of increased beta-error risk. The current method allowed for the rapid quantitation and identification of low pesticide levels in the watermelon samples. No pesticide residues were detected in any of the surveyed watermelons obtained from eight local markets in the Republic of Korea. Statistical analysis of the recoveries classified the 44 pesticides into nine groups and three overall categories.

Ca2+ signaling is an important determining factor in many cellular processes, especially in cancer cell proliferation, motility and invasion. Glioblastoma is the deadliest brain cancer with its average survival time of less than a year, with the most prominent cellular feature being the ability of these cells to migrate to and invade the neighboring tissue. We hypothesized that disturbing the Ca2+ signaling pathway would decrease the propensity for these cells to migrate. Thus, we investigated the detailed Ca2+ signaling pathway of the glioblastoma cells in response to various receptor tyrosine kinases (RTK) and G-protein coupled receptor (GPCR) agonists. Here we report that caffeine, which is a well-known activator of ryanodine receptors (RyRs), paradoxically inhibits inositol-1, 4, 5-triphospate receptor(IP3R)-mediated Ca2+ increase by selectively targeting IP3R subtype 3(IP3R3), whose mRNA expression is significantly increased in glioblastoma cells. Consequently, by inhibiting IP3R3-mediated Ca2+ release, caffeine was found to inhibit the invasion and migration of various glioblastoma cell lines in scrape motility, Matrigel invasion, soft agar, and brain slice implantation assays. In a mouse xenograft model of glioblastoma, caffeine intake via drinking water greatly increased mean survival duration of subject animals. These findings propose IP3R3 as a novel target for glioblastoma treatment and that caffeine may be a useful adjunct therapy that slows glioblastoma invasion and migration by selectively targeting IP3R3.

We investigated anti-invasive effects of the anthocyanins from fruits of Vitis coignetiae Pulliat (known as meoru in Korea) on human hepatoma Hep3B cells. The anthocyanins inhibited cell invasion in a dose-dependent manner as measured by Matrigel (BD Biosciences, San Jose, CA, USA) invasion assays. They also inhibited expression of matrix metalloproteinase (MMP)-2 and MMP-9 and activation of nuclear factor kappaB (NF-kappaB) stimulated by tumor necrosis factor alpha. Taken together, the results of this study indicate that the anthocyanins isolated from fruits of V. coignetiae Pulliat have anti-invasive effects on human hepatoma Hep3B cells and inhibit MMP-2 and MMP-9 gene expression at least in part through the inhibition of NF-kappaB activation.

Hyperglycemia, abnormal lipid and antioxidant profiles are the most usual complications in diabetes mellitus. Thus, in this study, we investigated the anti-diabetic and anti-oxidative effects of anthocyanins (ANT) from black soybean seed coats in streptozotocin (STZ)-induced diabetic rats. The administration of ANT markedly decreased glucose levels and improved heart hemodynamic function (left ventricular end diastolic pressure, +/-dp/dt parameters). ANT not only enhanced STZ-mediated insulin level decreases, but also decreased the triglyceride levels induced by STZ injection in serum. Diabetic rats exhibited a lower expression of glucose transporter 4 proteins in the membrane fractions of heart and skeletal muscle tissues, which was enhanced by ANT. In addition, ANT activated insulin receptor phosphorylation, suggesting an increased utilization of glucose by tissues. Moreover, ANT protected pancreatic tissue from STZ-induced apoptosis through regulation of caspase-3, Bax, and Bcl-2 proteins. Furthermore, ANT significantly suppressed malondialdehyde levels and restored superoxide dismutase and catalase activities in diabetic rats. Interestingly, the observed effects of ANT were superior to those of glibenclamide. Taken together, ANT from black soybean seed coat have anti-diabetic effects that are due, in part, to the regulation of glucose transporter 4 and prevention of insulin resistance and pancreatic apoptosis, suggesting a possible use as a drug to regulate diabetes.

Autoantibodies specific for alpha-fodrin fragments are found in the tissues of persons afflicted with Sjögren’s syndrome (SS). However, the mechanism for alpha-fodrin degradation remains elusive. The following experiments utilized Par C5 cells to examine the role of P2X7 receptor (P2X7R) in apoptosis, particularly in the cleavage and release of alpha-fodrin, an apparent SS autoantigen. Five mM ATP stimulation induced apoptotic cell death with a sustained Ca2+ influx, which was mimicked in HEK cells transfected with P2X7R. ATP also induced cleavage of alpha-fodrin mediated by caspase-3 and calpain, releasing alpha-fodrin fragments through membrane blebs. However, both apoptotic cell death and alpha-fodrin cleavage were inhibited in the presence of 300 μM oxidized-ATP (ox-ATP), an irreversible blocker of P2X7R, or in Ca2+-free solution. We concluded that P2X7R plays an important role in apoptosis and alpha-fodrin degradation in salivary epithelial cells, providing an important clue elucidating the presence of alpha-fodrin fragments in SS tissues.

Anthocyanins belong to a class of flavonoids exhibiting antioxidant and anti-inflammatory actions as well as a variety of chemotherapeutic effects. However, little is known about the cellular and molecular mechanism of anticancer activity. In this study, we investigated if the anthocyanins (delphinidin-3,5-diglucoside: cyanidin-3,5-diglucoside: petunidin-3,5-diglucoside: delphinidin-3-glucoside: malvdin-3,5-diglucoside: peonidin-3,5-diglucoside: cyanidin-3-glucoside: petunidin-3-glucoside: peonidin-3- glucoside: malvidin-3- glucoside = 27:63:8.27:1:2.21:2.21:6.7:1.25:5.72:1.25) [corrected] isolated from meoru (Vitis coignetiae Pulliat) exerted antiproliferative and anti-invasive and apoptotic effects on human hepatoma Hep3B cells. It was found that the anthocyanins could inhibit cell growth by 75% at the concentration of 400 microg/mL for 48 h. Flow cytometric analysis showed that the anthocyanins increased the amount of DNA fragments (sub-G1 fraction) in a dose-dependent manner, which is closely related to mitochondrial dysfunction and reduction in antiapoptotic proteins (Bcl-2, xIAP, cIAP-1, and cIAP-2). The anthocyanins also significantly inhibited the migration and invasion of Hep3B cells through a matrigel-coated chamber. Taken together this study indicates that the anthocyanins from meoru have antiproliferative and anti-invasive effects and may induce apoptosis through the activation of the mitochondrial pathway and inhibition of antiapoptotic proteins. This study provides evidence that the anthocyanins isolated from meoru might be useful in the treatment of human hepatitis B-associated hepatoma.

Peripheral nerve injury triggers a series of responses in the injured nerve, such as the dissolution of distal axons, the activation of Schwann cells, the production of various proinflammatory mediators, and the infiltration of circulating immune cells. These orchestrated events regulate the degeneration and subsequent regeneration of the injured nerve. In addition, peripheral nerve injury often accompanies chronic pain. Studies in this field have revealed that spinal cord microglia activation plays a critical role in the development of pain hypersensitivity. Recent studies using genetically modified mice indicate that Toll-like receptors (TLRs) are involved in nerve degeneration (Wallerian degeneration) and chronic pain (neuropathic pain) development after nerve injury. Here, we review studies that have implicated TLRs in mediating nerve degeneration/regeneration and neuropathic pain following nerve injury. In addition, we discuss possible mechanisms underlying the roles of TLRs in these neurological disorders.

It would be preferable for pesticide residues substituted by hydrolyzable functionality to be analyzed after cooking because their structures are apt to degrade during boiling and/or heating. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of 44 pesticide residues with hydrolyzable functional group in five typical vegetable widely consumed in Republic of Korea is described. The sample clean-up was carried out according to the method of Food Code No. 83 established by the Korea Food and Drug Administration (KFDA). Zorbox XDB-C(18) column was selected for the analysis because of the best peak separation. The LC mobile phase consisted of water and 5 mm methanolic ammonium formate, which resulted in a peak shape with good symmetry at each run. Tandem mass spectroscopic (MS/MS) experiments were performed in ESI positive mode and the multiple reaction monitoring modes. A conventional matrix effect was modified to more comprehensive form 100gamma(ij) (%). A high matrix effect (

Peripheral nerve injury triggers a series of responses in the injured nerve, such as the dissolution of distal axons, the activation of Schwann cells, the production of various proinflammatory mediators, and the infiltration of circulating immune cells. These orchestrated events regulate the degeneration and subsequent regeneration of the injured nerve. In addition, peripheral nerve injury often accompanies chronic pain. Studies in this field have revealed that spinal cord microglia activation plays a critical role in the development of pain hypersensitivity. Recent studies using genetically modified mice indicate that Toll-like receptors (TLRs) are involved in nerve degeneration (Wallerian degeneration) and chronic pain (neuropathic pain) development after nerve injury. Here, we review studies that have implicated TLRs in mediating nerve degeneration/regeneration and neuropathic pain following nerve injury. In addition, we discuss possible mechanisms underlying the roles of TLRs in these neurological disorders.

Membrane lipid rafts are enriched in cholesterol and play an important role as signalling platforms. However, the roles of lipid rafts and associated signalling molecules in the innate immune responses to mycobacteria remain unknown. Here we show that stimulation with Mycobacterium tuberculosis 19 kDa lipoprotein, a TLR2/1 agonist, results in translocation of TLR2 to lipid rafts, coalescence of lipid rafts and production of reactive oxygen species (ROS) that drive pro-inflammatory responses. Disruption of lipid raft organization markedly reduced lipoprotein-induced ROS and inflammatory responses. Remarkably, the atypical protein kinase C (PKC) zeta was specifically recruited into detergent-resistant membrane fractions and associated with TLR2. PKCzeta activity was critical for lipoprotein-dependent ROS generation, raft coalescence and the pro-inflammatory responses by macrophages. Moreover, lipid raft organization was required for 19 kDa mediated PKCzeta activation. These results demonstrate that TLR2 trafficking and raft coalescence play an essential role for the initiation of lipoprotein-induced innate immune responses via TLR2 and ROS signalling. In addition, PKCzeta targets to lipid rafts and may act as a critical adaptor molecule to regulate lipid raft dynamics during TLR2 signalling.

Recently, autoantibodies directed against muscarinic type 3 receptor (M3R) have been reported in patients with primary SS. However, the precise epitope(s) of the M3R that interacts with SS autoantibodies remains unclear. The aim of this study was to identify the functional epitope of M3R which interacts with SS immunoglobulin G (IgG).Purified IgGs were obtained from the sera of seven SS patients (six primary and one secondary SS) and two normal persons. We examined whether SS IgG inhibits M3R function and identified the epitope using six synthetic peptides covering all the extracellular domains of M3R by microspectrofluorimetry and surface plasmon resonance-based optical biosensor system (BIAcore system).A volume of 0.5 mg/ml SS IgG inhibited carbachol (CCh)-induced [Ca(2+)](i) transient (CICT) in human submandibular gland (HSG) cells. However, co-incubation of SS IgG with the 6th peptide (514-527 amino acid region) corresponding to the third extracellular loop of M3R, recovered CICT. The result was further confirmed by BIAcore analysis. We found that the 6th peptide interacts with IgGs from three primary SS patients in a concentration-dependent manner. The synthetic peptide which consists of amino acids 228-237 corresponding to the COOH-terminus of the second extracellular loop of M3R also bound to SS IgG. However, normal IgGs did not interact with the 6th peptide.The results suggest that the third extracellular loop of M3R represents a functional epitope bound by SS IgG, and thereby partly inhibits M3R function.

Mycobacterium abscessus (Mab) is an emerging and rapidly growing non-tuberculous mycobacterium (NTM). Compared with M. tuberculosis, which is responsible for tuberculosis, much less is known about NTM-induced innate immune mechanisms. Here we investigated the involvement of pattern-recognition receptors and associated signalling in Mab-mediated innate immune responses. Mab activated the extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinases (MAPKs), and induced the secretion of tumour necrosis factor-alpha, interleukin (IL)-6 and IL-12p40 in murine macrophages via Toll-like receptor (TLR) 2. Notably, the activation of ERK1/2, but not p38, was crucial for Mab-induced pro-inflammatory cytokine production. The ITAM-like motif of dectin-1 critically contributed to Mab internalization and cytokine secretion by macrophages. In addition, dectin-1, in cooperation with TLR2, was required for the efficient phagocytosis of Mab, ERK1/2 activation and pro-inflammatory cytokine secretion. Co-immunoprecipitation and confocal analysis showed the physical interaction and colocalization of dectin-1 with TLR2 following Mab stimulation. Moreover, dectin-1-induced Syk activation was essential for the production of inflammatory cytokines and the release of reactive oxygen species by Mab-infected macrophages. Collectively, these data demonstrate that Mab actively internalizes into and robustly activates innate immune responses in macrophages through a physical and functional interaction between TLR2 and dectin-1.

BackgroundActivated microglia elicits a robust amount of pro-inflammatory cytokines, which are implicated in the pathogenesis of tuberculosis in the central nervous system (CNS). However, little is known about the intracellular signaling mechanisms governing these inflammatory responses in microglia in response toMycobacterium tuberculosis(Mtb).MethodsMurine microglial BV-2 cells and primary mixed glial cells were stimulated with sonicated Mtb (s-Mtb). Intracellular ROS levels were measured by staining with oxidative fluorescent dyes [2',7'-Dichlorodihydrofluorescein diacetate (H2DCFDA) and dihydroethidium (DHE)]. NADPH oxidase activities were measured by lucigenin chemiluminescence assay. S-Mtb-induced MAPK activation and pro-inflammatory cytokine release in microglial cells were measured using by Western blot analysis and enzyme-linked immunosorbent assay, respectively.ResultsWe demonstrate that s-Mtb promotes the up-regulation of reactive oxygen species (ROS) and the rapid activation of mitogen-activated protein kinases (MAPKs), including p38 and extracellular signal-regulated kinase (ERK) 1/2, as well as the secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-12p40 in murine microglial BV-2 cells and primary mixed glial cells. Both NADPH oxidase and mitochondrial electron transfer chain subunit I play an indispensable role in s-Mtb-induced MAPK activation and pro-inflammatory cytokine production in BV-2 cells and mixed glial cells. Furthermore, the activation of cytosolic NADPH oxidase p47phox and MAPKs (p38 and ERK1/2) is mutually dependent on s-Mtb-induced inflammatory signaling in murine microglia. Neither TLR2 nor dectin-1 was involved in s-Mtb-induced inflammatory responses in murine microglia.ConclusionThese data collectively demonstrate that s-Mtb actively induces the pro-inflammatory response in microglia through NADPH oxidase-dependent ROS generation, although the specific pattern-recognition receptors involved in these responses remain to be identified.

The activation of spinal cord glial cells has been implicated in the development of neuropathic pain upon peripheral nerve injury. The molecular mechanisms underlying glial cell activation, however, have not been clearly elucidated. In this study, we found that damaged sensory neurons induce the expression of tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and inducible nitric-oxide synthase genes in spinal cord glial cells, which is implicated in the development of neuropathic pain. Studies using primary glial cells isolated from toll-like receptor 2 knock-out mice indicate that damaged sensory neurons activate glial cells via toll-like receptor 2. In addition, behavioral studies using toll-like receptor 2 knock-out mice demonstrate that the expression of toll-like receptor 2 is required for the induction of mechanical allodynia and thermal hyperalgesia due to spinal nerve axotomy. The nerve injury-induced spinal cord microglia and astrocyte activation is reduced in the toll-like receptor 2 knock-out mice. Similarly, the nerve injury-induced pro-inflammatory gene expression in the spinal cord is also reduced in the toll-like receptor 2 knock-out mice. These data demonstrate that toll-like receptor 2 contributes to the nerve injury-induced spinal cord glial cell activation and subsequent pain hypersensitivity.

Inflammation in the peripheral nervous system (PNS) is one of the characteristics of virus-induced peripheral neuropathy. In this inflammatory response, Schwann cells are actively involved. Previously, toll-like receptor 3 (TLR3) was reported as a receptor for double-stranded RNA (dsRNA) that induces antiviral and inflammatory responses in cells of the innate immune system. In this study, we investigated the expression and putative role of TLR3 in Schwann cells. TLR3 was constitutively expressed in Schwann cells. Stimulation with polyinosinic-polycytidylic acid, a synthetic dsRNA analogue, induced the expression of inducible nitric oxide synthase (iNOS) gene in Schwann cells. Studies on the intracellular signal transduction pathways using iSC, an immortalized Schwann cell line, revealed that dsRNA induces the activation of NF-kappaB, p38, and c-Jun N-terminal kinase (JNK). The activation of NF-kappaB, p38, JNK, and dsRNA-dependent protein kinase is required for dsRNA-mediated iNOS gene expression. However, the activation of PI3 kinase and GSK-3beta inhibited iNOS gene induction, a process mediated by their inhibitory effects on NF-kappaB and p38 activation. dsRNA-induced NO production caused neuronal cell death in cultured dorsal root ganglion. Finally, the introduction of dsRNA into the rat sciatic nerve induced iNOS gene expression and peripheral nerve demyelination in vivo. Taken together, these data suggest that viral RNA may induce inflammatory Schwann cell activation via TLR3 and peripheral nerve damage in the PNS.

Retinoic acid-inducible gene-I (RIG-I) mediates part of the cell signaling in response to viral infection. Polyinosinic-polycytidilic acid (poly IC) is a synthetic double-stranded RNA (dsRNA) and mimics viral infection when applied to cell cultures. The CC chemokine, RANTES (regulated on activation, normal T-cell expressed and secreted), is a potent attractant for inflammatory cells such as memory T-lymphocytes, monocytes and eosinophils. In the present study, we demonstrated that poly IC enhances the expression of RIG-I in U373MG human astrocytoma cells. The RNA interference of RIG-I resulted in the suppression of the poly IC-induced RANTES expression. Pretreatment of the cells with SB203580, an inhibitor of p38 mitogen-activated protein kinase, and dexamethasone inhibited the poly IC-induced expression of RIG-I. Furthermore, poly IC upregulated RIG-I in normal human astrocytes in culture and the in vivo injection of poly IC into the striatum of the mouse brain induced the expression of RIG-I in astrocytes. We conclude that RIG-I may be involved in immune reactions against viral infection, at least in part, through the regulation of RANTES expression in astrocytes.

Although eugenol is widely used in dentistry, little is known about the molecular mechanisms responsible for its anesthetic properties. In addition to calcium channels, recently demonstrated by our group, there could be another molecular target for eugenol. Using a whole-cell patch-clamp technique, we investigated the effect of eugenol on voltage-gated sodium channel currents ( I Na) in rat dental primary afferent neurons identified by retrograde labeling with a fluorescent dye in maxillary molars. Eugenol inhibited action potentials and I Na in both capsaicin-sensitive and capsaicin-insensitive neurons. The pre-treatment with capsazepine, a competitive antagonist of transient receptor potential vanilloid 1 (TRPV1), failed to block the inhibitory effect of eugenol on I Na, suggesting no involvement of TRPV1. Two types of I Na, tetrodotoxin (TTX)-resistant and TTX-sensitive I Na, were inhibited by eugenol. Our results demonstrated that eugenol inhibits I Na in a TRPV1-independent manner. We suggest that I Na inhibition by eugenol contributes to its analgesic effect.

Schwann cells play an important role in peripheral nerve regeneration. Upon nerve injury, Schwann cells are activated and produce various proinflammatory cytokines and chemokines, resulting in the recruitment of macrophages and the phagocytosis of myelin debris. However, it is unclear how nerve injury induces Schwann cell activation. Recently, it was reported that necrotic cells induce immune cell activation via toll-like receptors (TLRs). This suggests that the TLRs expressed on Schwann cells may recognize nerve injury by binding to the endogenous ligands secreted by the damaged nerve, thereby inducing Schwann cell activation. To explore such a possibility, we stimulated rat Schwann cells with necrotic neuronal cells (NNC). The stimulation of Schwann cells with NNC induced the expression of various inflammatory mediators, including TNF-alpha and iNOS. Studies on the NNC-mediated intracellular signaling pathways revealed that p38 and JNK are involved in the NNC-mediated Schwann cell activation. In addition, NNC-induced proinflammatory gene expression was reduced in mouse Schwann cells derived from TLR2 or TLR3 knockout mice. In summary, these results suggest that necrotic neuronal cells induce inflammatory Schwann cell activation via TLR2 and TLR3, which might be involved in Wallerian degeneration upon peripheral nerve injury.

This study characterized the upstream signalling molecules involved in extracellular signal-regulated kinase (ERK) 1/2 activation and determined their effects on differential tumour necrosis factor (TNF)-alpha expression by monocytes/macrophages infected with virulent or avirulent mycobacteria. The avirulent Mycobacterium tuberculosis (MTB) strain H37Ra (MTBRa) induced higher levels of activation of ERK 1/2 and the upstream MAPK kinase (MEK)1 and, subsequently, higher levels of TNF-alpha expression in human primary monocytes and monocyte-derived macrophages, as compared with MTB strain H37Rv (MTBRv). The MTB-induced activation of ERK 1/2 was not dependent on Ras or Raf. However, inhibition of the activity of atypical protein kinase C (PKC) zeta decreased the in vitro phosphorylation of MEK, ERK 1/2 activation and subsequent TNF-alpha induction caused by MTBRv or MTBRa. Toll-like receptor (TLR) 2 was found to play a major role in MTB-induced TNF-alpha expression and PKCzeta phosphorylation. Co-immunoprecipitation experiments showed that PKCzeta interacts physically with TLR2 after MTB stimulation. Moreover, PKCzeta phosphorylation was increased more in macrophages following MTBRa, versus MTBRv, infection. This is the first demonstration that PKCzeta interacts with TLR2 to play an essential role in MTB-induced ERK 1/2 activation and subsequent TNF-alpha expression in monocytes/macrophages.

Patterns of salivary HCO3−secretion vary and depend on species and gland types. However, the identities of the transporters involved in HCO3−transport and the underlying mechanism of intracellular pH (pHi) regulation in salivary glands still remain unclear. In this study, we examined the expression of the Na+-HCO3−cotransporter (NBC) and its role in pHiregulation in guinea pig salivary glands, which can serve as an experimental model to study HCO3−transport in human salivary glands. RT-PCR, immunohistochemistry, and pHimeasurements from BCECF-AM-loaded cells were performed. The amiloride-sensitive Na+/H+exchanger (NHE) played a putative role in pHiregulation in salivary acinar cells and also appeared to be involved in regulation in salivary ducts. In addition to NHE, NBC also played a role in pHiregulation in both acini and ducts. In the parotid gland, NBC1 was functionally expressed in the basolateral membrane (BLM) of acinar cells and the luminal membrane (LM) of ducts. In the submandibular gland, NBC1 was expressed only in the BLM of ducts. NBC1 expressed in these two types of salivary glands takes up HCO3−and is involved in pHiregulation. Although NBC3 immunoreactivity was also detected in submandibular gland acinar cells and in the ducts of both glands, it is unlikely that NBC3 plays any role in pHiregulation. We conclude that NBC1 is functionally expressed and plays a role in pHiregulation in guinea pig salivary glands but that its localization and role are different depending on the type of salivary glands.

We recently found that the concentration of HCO 3 - in guinea-pig saliva is very similar to that of human saliva; however, the entity that regulates HCO 3 - transport has not yet been fully characterized. In order to investigate the mechanism of HCO 3 - transport, we identified, cloned, and characterized a sodium bicarbonate ( Na + / HCO 3 - cotransporter found in guinea-pig parotid glands (gpNBC1). The gpNBC1 gene encodes a 1079-amino acid protein that has 95% and 96% homology with human and mouse parotid NBC1, respectively. Oocytes expressing gpNBC1 were exposed to HCO 3 - or Na+-free solutions, which resulted in a marked change in membrane potentials (Vm), suggesting that gpNBC1 is electrogenic. Likewise, a gpNBC1-mediated pH recovery was observed in gpNBC1 transfected human hepatoma cells; however, in the presence of 4, 4-diisothiocyanostilbene-2,2-disulfonic acid, a specific NBC1 inhibitor, such changes in Vm and pHi were not observed. Together, the data show that the cloned guinea-pig gene is a functional, as well as sequence homologue of human NBC1.

Pilocarpine has been used as a choice of drugs for treatment of impaired salivary flow. Although considerable data are available as to the stimulatory effect of pilocarpine on the salivary secretion in human, its underlying mechanism, at the cellular level, has not been rigorously studied. In this experiment, we studied the effect of pilocarpine on the ion channel activity, cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) and aquaporin (AQP)-5 expression, which play key roles in the secretary process and determine the capacity of fluid secretion. In human submandibular gland (SMG) acinar cells, 10(-5) M pilocarpine activated the outward rectifying-current, which was predominantly K(+) selective in the whole cell patch clamp study. The pilocarpine increased [Ca(2+)](i) in a concentration-dependent manner in the range of 10(-6) M to 10(-4) M. We found that both increases of [Ca(2+)](i) and outward rectifying- K(+) current were inhibited by 10(-5) M U-73122, a specific phospholipase C inhibitor. The magnitudes of pilocarpine-induced [Ca(2+)](i) transients were approximately 55% lower than those with the same concentration of carbachol (CCh). Pilocarpine also increased the amount of AQP-5 protein in the apical membrane (APM) in human SMG acinar cells. Our results suggest that pilocarpine induce salivary secretions in human by activating K(+) channels, increasing [Ca(2+)](i) via phospholipase C dependent pathway, and increasing AQP-5 protein expression in the APM of SMG acinar cells.

It has been demonstrated that spinal microglial activation is involved in formalin-induced pain and that minocycline, an inhibitor of microglial activation, attenuate behavioral hypersensitivity in neuropathic pain models. We investigated whether minocycline could have any anti-nociceptive effect on inflammatory pain, after intraperitonial administration of minocycline, 1 h before formalin (5%, 50 microl) injection into the plantar surface of rat hindpaw. Minocycline (15, 30, and 45 mg/kg) significantly decreased formalin-induced nociceptive behavior during phase II, but not during phase I. The enhancement in the number of c-Fos-positive cells in the L4-5 spinal dorsal horn (DH) and the magnitude of paw edema induced by formalin injection during phase II were significantly reduced by minocycline. Minocycline inhibited synaptic currents of substantia gelatinosa (SG) neurons in the spinal DH, whereas membrane electrical properties of dorsal root ganglion neurons were not affected by minocycline. Analysis with OX-42 antibody revealed the inhibitory effect of minocycline on microglial activation 3 days after formalin injection. These results demonstrate the anti-nociceptive effect of minocycline on formalin-induced inflammatory pain. In addition to the well-known inhibitory action of minocycline on microglial activation, the anti-edematous action in peripheral tissue, as well as the inhibition of synaptic transmission in SG neurons, is likely to be associated with the anti-nociceptive effect of minocycline.

Local anesthetics have been generally accepted as being safe. However, recent clinical trials and basic studies have provided strong evidence for the neurotoxicity of local anesthetics, especially through apoptosis. We hypothesized that local anesthetics cause neural complications through Schwann cell apoptosis. Among local anesthetics tested on the Schwann cell line, RT4-D6P2T, bupivacaine significantly induced cell death, measured by the methyl tetrazolium (MTT) assay, in a dose- (LD50= 476 μM) and time-dependent manner. The bupivacaine-induced generation of reactive oxygen species (ROS), which was initiated within 5 hrs and preceded the activation of caspase-3 and poly ADP-ribose polymerase (PARP) degradation, was suggested to trigger apoptosis, exhibited by Hoechst 33258 nuclear staining and DNA fragmentation. Furthermore, concomitant block of ROS by anti-oxidants significantly inhibited bupivacaine-induced apoptosis. Among the local anesthetics for peripheral neural blocks, bupivacaine induced apoptosis in the Schwann cell line, which may be associated with ROS production.

Glial activation is known to contribute to pain hypersensitivity following spinal sensory nerve injury. In this study, we investigated mechanisms by which glial cell activation in medullary dorsal horn (MDH) would contribute to tactile hypersensitivity following inferior alveolar nerve and mental nerve transection (IAMNT). Activation of microglia and astrocytes was monitored at 2 h, 1, 3, 7, 14, 28, and 60 days using immunohistochemical analysis with OX-42 and GFAP antibodies, respectively. Tactile hypersensitivity was significantly increased at 1 day, and this lasted for 28 days after IAMNT. Microglial activation, primarily observed in the superficial laminae of MDH, was initiated at 1 day, maximal at 3 days, and maintained until 14 days after IAMNT. Astrocytic activation was delayed compared to that of microglia, being more profound at 7 and 14 days than at 3 days after IAMNT. Both tactile hypersensitivity and glial activation appeared to gradually reduce and then return to the basal level by 60 days after IAMNT. There was no significant loss of trigeminal ganglion neurons by 28 days following IAMNT, suggesting that degenerative changes in central terminals of primary afferents might not contribute to glial activation. Minocycline, an inhibitor of microglial activation, reduced microglial activation, inhibited p38 mitogen-activated protein kinase (MAPK) activation in microglia, and significantly attenuated the development of pain hypersensitivity in this model. These results suggest that glial activation in MDH plays an important role in the development of neuropathic pain and activation of p38 MAPK in hyperactive microglia contributes to pain hypersensitivity in IAMNT model.

The transcription factor NF-κB in human intestinal epithelial cells plays a central role in regulating genes that govern the onset of mucosal inflammatory responses following intestinal microbial infection. Nod1 is a cytosolic pattern recognition receptor in mammalian cells that senses components of microbial peptidoglycans and signals the activation of NF-κB. The aim of these studies was to assess the functional importance of Nod1 in activating NF-κB and NF-κB proinflammatory target genes in human intestinal epithelium. Human colon epithelial cells that constitutively express Nod1 were used as a model intestinal epithelium. These cells do not signal through Toll-like receptor 4 (TLR4) or respond to bacterial lipopolysaccharide, but they express functional TLR5 and interleukin 1 (IL-1) receptors that signal the activation of NF-κB in response to bacterial flagellin or IL-1 stimulation. Stable expression of dominant negative (DN) Nod1 in colon epithelial cells prevented IκB kinase and NF-κB activation in response to infection with enteroinvasiveEscherichia coli. In contrast, DN Nod1 did not eliminate IL-1 or flagellin-stimulated NF-κB activation. Inhibition of NF-κB was accompanied by inhibition of NF-κB target genes that provide signals for the mucosal influx of neutrophils during intestinal infection. We conclude that signaling through Nod1 is required for activating NF-κB in human intestinal epithelial cells infected with gram-negative enteric bacteria that can bypass TLR activation. Signaling through Nod1 provides the intestinal epithelium with a backup mechanism for rapidly activating innate immunity during infection with a group of highly invasive pathogenic gram-negative bacteria.

Although the spontaneous rupture of adrenal pheochromocytoma is rare, it can be lethal because it can induce serious changes in the circulation. We describe a 32 year old man with bilateral pheochromocyroma presenting as abdominal pain. In the emergency room, an abdominal MRI showed an aneurysmal vessel in the right adrenal mass and accompanying hemorrhage around the tumor capsule. The bleeding site was found by transfemoral abdominal angiography. Coil embolization was done in the bleeding vessels, specifically branches of the right adrenal artery. The hemorrhage was successfully controlled and vital signs of the patient were restored. Following emergency care, biochemical and imaging studies showed compatible findings of a bilateral adrenal pheochromocytoma. Postoperative histologic findings confirmed these observations. A ruptured pheochromocytoma should be considered as a cause of acute abdomen in cases of a concomitant adrenal mass. Intratumoral aneurysmal bleeding may be a cause of ruptured tumor, and careful angiographic intervention will help to ensure safe control of bleeding in such an emergency situation, even in cases of bilateral tumor.

Microglia, the resident macrophages in the central nervous system, can rapidly respond to pathological insults. Toll-like receptor 2 (TLR2) is a pattern recognition receptor that plays a fundamental role in pathogen recognition and activation of innate immunity. Although many previous studies have suggested that TLR2 contributes to microglial activation and subsequent pathogenesis following brain tissue injury, it is still unclear whether TLR2 has a role in microglia dynamics in the resting state or in immediate-early reaction to the injury in vivo. By using in vivo two-photon microscopy imaging and Cx3cr1 (GFP/+) mouse line, we first monitored the motility of microglial processes (i.e. the rate of extension and retraction) in the somatosensory cortex of living TLR2-KO and WT mice; Microglial processes in TLR2-KO mice show the similar motility to that of WT mice. We further found that microglia rapidly extend their processes to the site of local tissue injury induced by a two-photon laser ablation and that such microglial response to the brain injury was similar between WT and TLR2-KO mice. These results indicate that there are no differences in the behavior of microglial processes between TLR2-KO mice and WT mice when microglia is in the resting state or encounters local injury. Thus, TLR2 might not be essential for immediate-early microglial response to brain tissue injury in vivo.

Fas/Apo-1 is a member of the TNF receptor superfamily that signals apoptotic cell death in susceptible target cells. Fas or Fas ligand (FasL)-deficient mice are relatively resistant to the induction of experimental allergic encephalomyelitis, implying the involvement of Fas/FasL in this disease process. We have examined the regulation and function of Fas expression in glial cells (astrocytes and microglia). Fas is constitutively expressed by primary murine microglia at a low level and significantly up-regulated by TNF-alpha or IFN-gamma stimulation. Primary astrocytes express high constitutive levels of Fas, which are not further affected by cytokine treatment. In microglia, Fas expression is regulated at the level of mRNA expression; TNF-alpha and IFN-gamma induced Fas mRNA by approximately 20-fold. STAT-1alpha and NF-kappaB activation are involved in IFN-gamma- or TNF-alpha-mediated Fas up-regulation in microglia, respectively. The cytokine TGF-beta inhibits basal expression of Fas as well as cytokine-mediated Fas expression by microglia. Upon incubation of microglial cells with FasL-expressing cells, approximately 20% of cells underwent Fas-mediated cell death, which increased to approximately 60% when cells were pretreated with either TNF-alpha or IFN-gamma. TGF-beta treatment inhibited Fas-mediated cell death of TNF-alpha- or IFN-gamma-stimulated microglial cells. In contrast, astrocytes are resistant to Fas-mediated cell death, however, ligation of Fas induces expression of the chemokines macrophage inflammatory protein-1beta (MIP-1beta), MIP-1alpha, and MIP-2. These data demonstrate that Fas transmits different signals in the two glial cell populations: a cytotoxic signal in microglia and an inflammatory signal in the astrocyte.