Levocetirizine, a second generation non-sedating antihistamine that blocks the H₁ histamine receptor, may exhibit immunoregulatory properties that augment its primary pharmacological mechanism. To investigate this possibility, 13 Kuwaiti seasonal allergic rhinitis (SAR) patients were treated with levocetirizine for four weeks in comparison with a 7-member placebo-treated control group, followed by clinical evaluation and flow cytometric analysis of peripheral venous blood for inflammatory cell and lymphocyte subpopulation profiles. Relative to the controls, levocetirizine-treated patients exhibited an expected reduction in early phase allergic symptoms, including sneezing (P<0.001), nasal itching (P<0.01), nasal congestion, and running nose (P<0.001); reduced percentages of eosinophils (P<0.05); and three subpopulations of activated T lymphocytes: CD4+CD29+, CD4+CD212+, and CD4+CD54+ (P<0.05). Levocetirizine treatment also correlated with a significant increase in the percentage of CD4+CD25+ T cells (P<0.001). The ability of levocetirizine to reduce percentage representation of cell phenotypes known to contribute to inflammatory tissue damage (eosinophils, CD4+CD29+, CD4+CD212+, and CD4+CD54+) and expand percentages of CD4+CD25+, which may include protective immunoregulatory (Treg) cells, indicates that the drug has pharmacological potential beyond the immediate effects of H₁ histamine–receptor inhibition. Although the present data does not define a therapeutic mechanism, the results reported here establish important trends that may be used to guide future mechanistic examination of immunoregulatory capacity of H₁ inhibitors.

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Cytochrome P450 (CYP) functional expression is reduced in uremia and normalized after restoration of kidney function via transplantation. The aim of this study was to evaluate the effect of conventional hemodialysis on the functional expression of CYP1A, 2C, and 3A. We also investigated the role of nuclear factor-κB (NF-κB) in CYP regulation during uremia. Primary cultures of normal rat hepatocytes were incubated with serum obtained from end-stage renal disease patients pre- and post-hemodialysis and healthy control subjects, in the presence and absence of the NF-κB inhibitor andrographolide. Uremic pre-hemodialysis serum caused significant reductions (P<0.01) in CYP1A (44%), 2C (27%), and 3A (35%) protein expression compared to control serum, while dialyzed serum (i.e., obtained immediately post-hemodialysis) had no effect. CYP1A2, 2C11, and 3A2 mRNA expression, as well as CYP3A activity, were similarly impacted by uremic serum and were improved to >80% of control values after hemodialysis. NF-κB inhibition nearly eliminated the effect of uremic serum on CYP functional expression. This is the first study to demonstrate that conventional hemodialysis acutely improves altered CYP functional expression observed in rat hepatocytes incubated with uremic human serum.

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Enhanced levels of intracellular stresses such as oxidative stress and endoplasmic reticulum (ER) stress are implicated in various neuropathological conditions including brain ischemia and neurodegeneration. During a search for compounds that regulate ER stress and ER stress-induced cell death, we identified a carbazole derivative 16-14 [9-(3-cyanobenzyl)-1,4-dimethylcarbazole] that protected against both ER stress and glutathione depletion. 16-14 suppressed tunicamycin (Tm)-induced cell death in both F9 Herp KO cells and PC12 cells, and its regulation of ER stress was associated with reduced levels of unfolded protein response (UPR) signaling. ER stress caused by overexpression of a fluorescent ER-resident protein, GFP-KDEL, was also attenuated by 16-14 without altering the expression levels of GFP-KDEL. 16-14 also prevented glutathione depletion-induced cell death caused by buthionine sulfoximine (BSO), but not likely via its anti-oxidative activity. Further analysis revealed that 16-14 suppressed increases in intracellular Ca²⁺ in response to thapsigargin (Tg). These results suggest that 16-14 may protect cells against different stresses via the maintenance of intracellular Ca²⁺ homeostasis.
[Supplementary Fig. 1: available only at http://dx.doi.org/10.1254/jphs.08136FP]

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To measure separately electromyogram (EMG) activity of A-delta and C fiber–mediated responses and study the characteristics of drug effects on them in mice, we modified our original method designed to measure these responses associated with hindlimb withdrawal movements. Single electrical stimulation applied to the toe elicited biphasic EMG activity in the ipsilateral femoris biceps muscle and withdrawal movement. Times to peak of the short- and long-latency response of EMG activity were almost consistent with those obtained from the hindlimb movement. As the short- and the long-latency responses of EMG activity were selectively inhibited by tetrodotoxin (TTX) (3 and 10 μM) and capsaicin (0.98 and 3.27 mM) applied to the sciatic nerve, the responses were considered to occur via A-delta and C fibers, respectively. A 0.5-Hz repetitive conditioning stimulus (CS) elicited significant wind-up in both the A-delta and C fiber–mediated responses. The wind-up of the A-delta fiber–mediated response disappeared during the CS and that of the C fiber–mediated response continued throughout the CS. This method is simple but useful for studying the effect of analgesic agents on A-delta and C fiber–mediated nociceptive responses and clarifying the roles of these afferent fibers in chronic pain in mice.

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In this study, we provide evidence of critical changes in the expression of non-selective cation currents (NSCC) during culture in rat aortic myocytes. A selective TRPV4 agonist, 4α-phorbol 12,13-didecanoate (4αPDD), had little effect on membrane currents and intracellular Ca²⁺ (Ca²⁺_i) in freshly isolated cells from the aorta. In contrast, in cultured aortic myocytes with and without serum, 4αPDD at a concentration range between 0.3 and 3 μM effectively elevated Ca²⁺_i, which was abolished in the absence of external Ca²⁺. Application of 4αPDD to cultured aortic myocytes also activated NSCC, which had a reversal potential of +3 mV. Both of these signals were blocked by ruthenium red (RuR), an effective blocker of TRPVs. Although the expression of TRPV4 mRNA transcript was found in cultured as well as non-cultured aortic myocytes, significant immunoreactivity to TRPV4 protein was only detected in cultured rat aortic myocytes. Moreover, cultured human pulmonary arterial smooth muscle cells (hPASM) had a substantial response to 4αPDD, which was susceptible to the removal of external Ca²⁺ and application of RuR. These results provide a strong basis for our proposal that endogenous TRPV4 functions as an important regulator of Ca²⁺_i in vascular myocytes under some physiological and pathophysiological conditions.

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This study was conducted to determine whether Na_v1.8 contributes to the release and/or synthesis of substance P (SP) in adult mice dorsal root ganglion (DRG) neurons. The SP released from cultured DRG neurons of Na_v1.8 knock-out mice exposed to either capsaicin or KCl was significantly lower than that from wild-type (C57BL/6) mice based on a radioimmunoassay. The SP level of L6 DRG in Na_v1.8 knock-out mice was also lower than that in wild-type mice. After chronic constriction injury (CCI) of the sciatic nerve, the level of SP decreased in the L6 ipsilateral DRG of wild-type but not Na_v1.8 knock-out mice. The preprotachykinin-A (PPT-A) mRNAs in L4 – 6 DRGs of Na_v1.8 knock-out mice also fell to half their normally abundant levels of expression. There were significant increases in Na_v1.8 expression of the L6 contralateral DRG from wild-type mice and in the percentage of neurons expressing neurokinin-1 receptor in the cytosol of L6 DRGs from wild-type or Na_v1.8 knock-out mice. These findings suggest that Na_v1.8 is involved in the regulation of the release and synthesis of SP in the DRG neurons of wild-type mice.

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Ruscogenin (RUS), first isolated from Ruscus aculeatus, also a major steroidal sapogenin of traditional Chinese herb Radix Ophiopogon japonicus, has been found to exert significant anti-inflammatory and anti-thrombotic activities. Our previous studies suggested that ruscogenin remarkably inhibited adhesion of leukocytes to a human umbilical vein endothelial cell line (ECV304) injured by tumor necrosis factor-α (TNF-α) in a concentration-dependent manner. Yet the underlying mechanisms remain unclear. In this study, the in vivo effects of ruscogenin on leukocyte migration and celiac prostaglandin E₂ (PGE₂) level induced by zymosan A were studied in mice. Furthermore, the effects of ruscogenin on TNF-α–induced intercellular adhesion molecule-1 (ICAM-1) expression and nuclear factor-κB (NF-κB) activation were also investigated under consideration of their key roles in leukocyte recruitment. The results showed that ruscogenin significantly suppressed zymosan A–evoked peritoneal total leukocyte migration in mice in a dose-dependent manner, while it had no obvious effect on PGE₂ content in peritoneal exudant. Ruscogenin also inhibited TNF-α–induced over expression of ICAM-1 both at the mRNA and protein levels and suppressed NF-κB activation considerably by decreasing NF-κB p65 translocation and DNA binding activity. These findings provide some new insights that may explain the possible molecular mechanism of ruscogenin and Radix Ophiopogon japonicus for the inhibition of endothelial responses to cytokines during inflammatory and vascular disorders.

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The present study was performed to study the participation of histamine H₃ receptors in nasal symptoms using Sch 50971, a potent and selective agonist of the H₃ receptor. Repeated topical application of antigen caused an increase in sneezing and nasal rubbing in sensitized mice. Oral administration of Sch 50971 and imetit, specific H₃-receptor agonists, resulted in an inhibition of nasal symptoms induced by an antigen similar to an H₁-receptor antagonist, cetirizine. Furthermore, simultaneous use of H₃-receptor agonists, Sch 50971 or imetit, and an H₁-receptor antagonist, cetirizine, caused a significant inhibitory effect on nasal symptoms at doses that showed no effect when used separately. The number of eosinophils in the nasal mucosa of mice sensitized with antigen was significantly decreased by cetirizine; however, Sch 50971 and imetit had no effect on eosinophil infiltration. These results clearly indicate that H₃ receptors are involved in the etiology of nasal allergy, and the stimulation of H₃ receptors may be useful as a novel therapeutic approach in nasal allergy.

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The effect of ganoderic acid Me (GA-Me), which was purified from the fermentation mycelia of the traditional Chinese medicinal mushroom Ganoderma lucidum as reported (Tang W, Gu TY, Zhong JJ. Biochem Eng J. 2006;32:205–210), on anti-invasion was investigated. Wound healing assay indicated that GA-Me inhibited cell migration of 95-D, a human highly metastatic lung tumor cell line, in dose- and time-dependent manners. Results of cell aggregation and adhesion assays showed that GA-Me promoted cell homotypic aggregation and inhibited cell adherence to extracellular matrix (ECM). In addition, GA-Me suppressed matrix metalloproteinases 2/9 (MMP2/9) gene expressions at both mRNA and protein levels in 95-D cells according to qRT-PCR and Western blotting, respectively. The results demonstrated that GA-Me effectively inhibited tumor invasion, and it might act as a new MMP2/9 inhibitor for anti-metastasis treatment of carcinoma cells.

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To obtain compounds that inhibit the interaction of von Willebrand factor (vWF) and glycoprotein (GP) Ib, a novel binding assay was established. The binding of fixed platelets to vWF-R497 mutant was quantified by a solid phase assay. In this assay, fixed platelets bound to the vWF-R497 mutant, carrying the deletion of Glu497-Tyr508 and the missense mutation of Arg545 to Ala, without binding modulators such as ristocetin. The K_d value of the binding was 2.8 nM, which was consistent with the result from liquid binding assay. The binding was inhibited by aurin tricarboxylic acid (ATA) and an anti GPIb antibody, AK2. Using this binding assay, we screened our library compounds and obtained D74-3736. This compound also inhibited ristocetin-induced platelet aggregation in the human platelet-rich plasma.

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To explore the cellular effects of mild electrical stimulation (MES), we treated A549 cells with low-intensity direct current at 5 V applied for 10 min. MES did not induce cell cytotoxicity or the unfolded protein response (UPR). Interestingly, the expression of ubiquitinated proteins and heat shock protein (Hsp) 72 was increased but not that of other Hsps. MES attenuated the degradation of Hsp72, which is a substrate of the proteasome-ubiquitin system. These results, along with the observed increase in expression of ubiquitinated proteins, imply that MES may affect the proteasome system, which regulates the fate of many proteins.

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