Methyl 4-(β-D-glucopyranosyloxy)-3-hydroxy-5- methoxybenzoate, isolated from Sanguisorba officinalis, inhibits CpG-DNA-induced inflammation

Purpose: To evaluate the anti-inflammatory effect of methyl-4-(β-D-glucopyranosyloxy)-3-hydroxy-5-methoxybenzoate (comp-1) on immune cells.Methods: Comp-1 was isolated from Sanguisorba officinalis. After treating with comp-1, cell viability and levels of pro-inflammatory cytokines were assessed utilizing MTT assay and ELISA, respectively. Besides, the effects of comp-1 on nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and iNOS were determined using western blotting. Moreover, nitric oxide production was assessed using the Griess reagent.Results: Treatment of dendritic cells (DCs) with CpG DNA upregulated cytokine expression. Comp-1 markedly downregulated the expressions of IL-12 p40, IL-6, and TNF-α, with 50% inhibitory concentrations (IC50) of 1.077 ± 0.04 (p < 0.01), 0.28 ± 0.01 (p < 0.01), and 0.79 ± 0.02 μM (p < 0.01),respectively. Treatment of DCs with CpG DNA upregulated NF-κB and MAPK activation. However, pretreatment of the cells with Comp-1 suppressed CpG DNA-induced NF-κB and MAPK activation. Moreover, comp-1 exhibited a strong anti-inflammatory effect by inhibiting nitric oxide production and iNOS expression.Conclusion: These results reveal that comp-1 has significant anti-inflammatory effect on immune cells. Keywords: Natural compound, Inflammation, Pro-inflammatory cytokine, Toll-like receptor

Chemical Modification of the Human Ether-a-go-go-related gene(HERG) K* Current by the Amino-Group Reagent Trinitrobenzene Sulfonic Acid

We investigated the effects of trinitrobenzene sulfonic acid (TNBS), an amino-group reagent, on the humanether-a-go-go-related gene (HERG) K+ channels expressed inXenopus oocytes. TNBS neutralizes the positively charged amino-agroups of peptideN-terminal and lysine residues. External application of TNBS at 10 mM for 5 min irreversibly shifted the curves for currents at the end of the pulse and tail currents of HERG to a more negative potential and decreased the maximal amplitude of the Itail curve (Itail, max). TNBS had little effect on either the activated current-voltage relationship or the reversal potential of HERG current, indicating that TNBS did not change ion selectivity properties. TNBS shifted the time constant curves of both activation and deactivation of the HERG current to a more hyperpolarized potential; TNBS's effect was greater on channel opening than channel closing. External H+ is known to inhibit HERG current by shifting V1/2 to the right and decreasing Itail, max. TNBS enhanced the blockade of external H+ by exaggerating the effect of H+ on Itail, max, not on V1/2. Our data provide evidence for the presence of essential amino-groups that are associated with the normal functioning of the HERG channel and evidence that these groups modify the blocking effect of external H+ on the current.We are grateful to Dr. Jokubas Ziburkus for reading and editing this manuscript and to Hee-Kyung Hong for technical support. Ji-Hyun Yun was the recipient of the BK21 fellowship for graduate students in 2006. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (R04- 2003-000-10007-0)

Clarithromycin Susceptibility Testing of Mycobacterium avium Complex Using 2,3-Diphenyl-5-thienyl-(2)-tetrazolium Chloride Microplate Assay with Middlebrook 7H9 Broth

A series of 119 Mycobacterium avium complex isolates were subjected to clarithromycin susceptibility testing using microplates containing 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC). Among 119 isolates, 114 (95.8%) were susceptible to clarithromycin and 5 were resistant according to the new and the standard method. STC counts the low cost and reduces the number of procedures needed for susceptibility testing

MicroRNA-150 modulates intracellular Ca2+ levels in naïve CD8+ T cells by targeting TMEM20

Regulation of intracellular Ca2+ signaling is a major determinant of CD8+ T cell responsiveness, but the mechanisms underlying this regulation of Ca2+ levels, especially in naïve CD8+ T cells, are not fully defined. Here, we showed that microRNA-150 (miR-150) controls intracellular Ca2+ levels in naïve CD8+ T cells required for activation by suppressing TMEM20, a negative regulator of Ca2+ extrusion. miR-150 deficiency increased TMEM20 expression, which resulted in increased intracellular Ca2+ levels in naïve CD8+ T cells. The subsequent increase in Ca2+ levels induced expression of anergy-inducing genes, such as Cbl-b, Egr2, and p27, through activation of NFAT1, as well as reduced cell proliferation, cytokine production, and the antitumor activity of CD8+ T cells upon antigenic stimulation. The anergy-promoting molecular milieu and function induced by miR-150 deficiency were rescued by reinstatement of miR-150. Additionally, knockdown of TMEM20 in miR-150-deficient naïve CD8+ T cells reduced intracellular Ca2+ levels. Our findings revealed that miR-150 play essential roles in controlling intracellular Ca2+ level and activation in naïve CD8+ T cells, which suggest a mechanism to overcome anergy induction by the regulation of intracellular Ca2+ levels115Ysciescopu

Development of EKC after Eximer Laser Photorefractive Surgery and Subsequent Recurrence of EKC-like Keratitis

This research focuses on four cases of patients having undergone eximer laser photorefractive surgery who were diagnosed with adenoviral keratoconjunctivitis during the postoperative period and who later developed epidemic keratoconjunctivitis (EKC)-like keratitis. Two of the patients had undergone laser-assisted subepithelial keratectomy (LASEK), one had undergone laser in situ keratomileusis and one had photorefractive keratectomy. After the surgery adenoviral keratoconjunctivitis and recurrent late-developing EKC-like keratitis were observed in the patients. Recurrent late-developing EKC-like keratitis occurred in one of the patients, who had received LASEK as many as three times. The others had only one or two episodes.The corneal infiltrates of keratitis mainly occurred in the central cornea. Successful resolution of recurrent late-developing EKC-like keratitis was achieved through the use of topical steroids without sequelae and the final best-corrected visual acuity was as good as the base line. These keratitis infiltrates have been presumed to represent an immune response to the suspected adenoviral antigens deposited in corneal stroma during the primary adenoviral infection. Previous reports argued that patients with a history of adenoviral ketatoconjunctivitis were succeptible to adenoviral keratoconjunctivitis becoming reactivated; however, in our research, our patients had their first adenoviral infections after the eximer laser photorefractive surgery and reactivation was confirmed. We recommend that attention be paid to adenoviral infection after laser refractive operations, because these patients seem to have more frequent recurrences

Anti-inflammatory Role of Mesenchymal Stem Cells in an Acute Lung Injury Mouse Model

Background Mesenchymal stem cells (MSCs) attenuate injury in various lung injury models through paracrine effects. We hypothesized that intratracheal transplantation of allogenic MSCs could attenuate lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice, mediated by anti-inflammatory responses. Methods Six-week-old male mice were randomized to either the control or the ALI group. ALI was induced by intratracheal LPS instillation. Four hours after LPS instillation, MSCs or phosphate-buffered saline was randomly intratracheally administered. Neutrophil count and protein concentration in bronchoalveolar lavage fluid (BALF); lung histology; levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and macrophage inflammatory protein-2; and the expression of proliferation cell nuclear antigen (PCNA), caspase-3, and caspase-9 were evaluated at 48 hours after injury. Results Treatment with MSCs attenuated lung injury in ALI mice by decreasing protein level and neutrophil recruitment into the BALF and improving the histologic change. MSCs also decreased the protein levels of proinflammatory cytokines including IL-1β, IL-6, and TNF-α, but had little effect on the protein expression of PCNA, caspase-3, and caspase-9. Conclusions Intratracheal injection of bone marrow-derived allogenic MSCs attenuates LPS-induced ALI via immunomodulatory effects