Doo Jin Paik

Bacillus subtilis-specific poly-γ-glutamic acid regulates development pathways of naive CD4+ T cells through antigen-presenting cell-dependent and -independent mechanisms

Peripheral naive CD4(+) T cells selectively differentiate to type 1 T(h), type 2 T(h) and IL-17-producing T(h) (T(h)17) cells, depending on the priming conditions. Since these subsets develop antagonistically to each other to elicit subset-specific adaptive immune responses, balance between these subsets can regulate the susceptibility to diverse immune diseases. The present study was undertaken to determine whether poly-gamma-glutamic acid (gamma-PGA), an edible and safe exopolymer that is generated by microorganisms such as Bacillus subtilis, could modulate the development pathways of T(h) subsets. The presence of gamma-PGA during priming promoted the development of T(h)1 and T(h)17 cells but inhibited development of T(h)2 cells. gamma-PGA up-regulated the expression of T-bet and ROR-gammat, the master genes of T(h)1 and T(h)17 cells, respectively, whereas down-regulating the level of GATA-3, the master gene of T(h)2 cells. gamma-PGA induced the expression of IL-12p40, CD80 and CD86 in dendritic cells (DC) and macrophages in a Toll-like receptor-4-dependent manner, and the effect of gamma-PGA on T(h)1/T(h)2 development was dependent on the presence of antigen-presenting cells (APC). Furthermore, gamma-PGA-stimulated DC favored the polarization of naive CD4(+) T cells toward T(h)1 cells rather than T(h)2 cells. In contrast, gamma-PGA affected T(h)17 cell development, regardless of the presence or absence of APC. Thus, these data demonstrate that gamma-PGA has the potential to regulate the development pathways of naive CD4(+) T cells through APC-dependent and -independent mechanisms and to be applicable to treating T(h)2-dominated diseases.

Modification of cardiovascular response of adenosine A1 receptor agonist by cyclic AMP in the spinal cord of the rats.

This study was performed to investigate the influence of the spinal adenosine A1 receptors on the central regulation of blood pressure (BP) and heart rate (HR), and to define whether its mechanism is mediated by cyclic AMP (cAMP) or cyclic GMP (cGMP). Intrathecal (i.t.) administration of drugs at the thoracic level were performed in anesthetized, artificially ventilated male Sprague-Dawley rats. Injection (i.t.) of adenosine A1 receptor agonist, N6-cyclohexyladenosine (CHA; 1, 5 and 10 nmol) produced dose dependent decrease of BP and HR. Pretreatment with a cAMP analogue, 8-bromo-cAMP, attenuated the depressor and bradycardiac effects of CHA (10 nmol), but not with cGMP analogue, 8-bromo-cGMP. These results suggest that adenosine A1 receptor in the spinal cord plays an inhibitory role in the central cardiovascular regulation and that this depressor and bradycardiac actions are mediated by cAMP.

Combined Therapy of Cilazapril and Losartan Has No Additive Effects in Ameliorating Adriamycin-Induced Glomerulopathy

Aims: Effects of the blockade of renin-angiotensin system (RAS), by angiotensin-converting enzyme inhibitor (ACEi), type 1 angiotensin II receptor blocker (ARB), or a combination of both, were evaluated in Adriamycin (ADR)-induced glomerulopathy. Methods: Male Sprague-Dawley rats (180–250 g) were induced of glomerulopathy by treatment with ADR (2 mg/kg, i.v.). Six weeks later, they were treated with cilazapril (1 mg/kg/day) and/or losartan (10 mg/kg/day) for an additional 6 weeks. Results: The urinary excretion of protein progressively increased following the treatment with ADR, which was prevented by ACEi, ARB, and a combination of both. Similarly, the glomerulopathy assessed by glomerulosclerosis index was also ameliorated by ACEi or ARB. However, combined therapy of both ACEi and ARB was without an additional effect (Control 1.4 ± 0.4%, ADR 10.7 ± 2.7%**, ACEi 0.8 ± 0.4%, ARB 2.6 ± 1.0%, ACEi+ARB 1.7 ± 1.5%, ** p < 0.01 vs. Control). The expression of transforming growth factor-β1 was increased following the treatment with ADR (1.4 ± 0.07-fold, p < 0.05 vs. Control), however, the degree of which was similarly blunted by either ACEi, ARB, or the combination of both. The expression of type 1 angiotensin II receptor mRNA increased following the treatment with ADR, the degree of which was further upregulated by ACEi and decreased by ARB to the control level (ADR 1.3 ± 0.06-fold*, ACEi 1.8 ± 0.05-fold***, ARB 1.0 ± 0.04-fold, * p < 0.05 and *** p < 0.001 vs. Control). The combined therapy of ACEi and ARB still showed an upregulation of type 1 angiotensin II receptor mRNA, however, of which degree was mitigated compared with that induced by ACEi alone (ACEi+ARB 1.5 ± 0.04-fold, ** p < 0.01 vs. Control). On the contrary, the expression of type 2 angiotensin II receptor mRNA was downregulated following the treatment with ADR, which was similarly restored to the control level by ACEi, ARB, and a combination of both (ADR 0.5 ± 0.08-fold**, ACEi 1.0 ± 0.06-fold, ARB 1.0 ± 0.05-fold, ACEi+ARB 1.0 ± 0.05-fold, ** p < 0.01 vs. Control). Conclusion: It is suggested that combined therapy of ACEi and ARB with relatively high or maximal doses of each drug has no additive or synergistic benefits on the progression of ADR-induced glomerulopathy. Effects of RAS blockade may in part be related to differential regulation of type 1 and type 2 angiotensin II receptors.

A Cadaveric Anatomical Study of the Levator Aponeurosis and Whitnall's Ligament

Purpose To identify the anatomy of the levator aponeurosis (LA) and Whitnalls ligament (WL) in Korean subjects using cadavers. Methods Orbital exenteration was performed in ten cadavers (20 eyeballs) that had no history of trauma near the eyeball. We observed characteristics of WL (tension, density, and shape) and the relationship between the superior rectus muscle (SR) and the levator palpebrae superioris. We measured the distance from both the eyelid margin and the upper border of the tarsal plate to the insertion of the LA medially, centrally, and laterally. Results The WLs we observed showed several shapes. In 12 eyes, we saw clear, white fibrotic bands, while in four others, we found thin, less taut bands. In four eyes, we were unable to identify the precise shape of the band. The insertions of the LA showed nasal dehiscence in 13 eyes and parallel attachment in seven eyes. The distances from the eyelid margin to the insertion of the LA medially, centrally, and laterally were 8.31 mm, 5.57 mm, and 5.15 mm, respectively. The distances from the upper border of the tarsal plate to the insertion of the LA medially, centrally, and laterally were 2.75 mm, 4.82 mm, and 4.29 mm, respectively. Conclusions This study examined the anatomy of WL and the LA in Korean subjects and may be helpful as a reference in levator muscle surgery.

Mediation of the cardiovascular response of adenosine A1 receptor through a GABAB receptor in the spinal cord of the rats

Cardiovascular inhibitory effects induced by intrathecal (i.t.) administration of adenosine A1 receptor agonist and its modulation by cyclic AMP was suggested by our previous report. In this experiment, we examined the mediation of cardiovascular effects of adenosine A1 receptor by gamma-aminobutyric acid receptors A and B [GABA(A) and GABA(B)] in the spinal cord. I.t. administration of 10 nmol of N6-cyclohexyladenosine (CHA), an adenosine A1 receptor agonist, and pretreatment with bicuculline (10 nmol, i.t), a GABA(A) receptor antagonist, and 5-aminovaleric acid (50 nmol, i.t.), a GABA(B) receptor antagonist, prior to injection of CHA were performed in anesthetized, artificially ventilated Sprague-Dawley rats. I.t. injection of 50 nmol of 5-aminovaleric acid significantly attenuated the inhibitory cardiovascular effects of CHA but 10 nmol of bicuculline did not alter CHA-induced cardiovascular actions. It is suggested that cardiovascular responses of adenosine A1 receptor is mediated by GABA(B) receptor in the spinal cord.

Requirement of de novo protein synthesis for aminopterin-induced apoptosis in a mouse myeloma cell line.

Cells synthesize nucleotides through de novo and salvage pathways that require the activities of dihydrofolate reductase (DHFR) and hypoxanthine-guanine phosphoribosyltransfease (HGPRT), respectively. Aminopterin, an inhibitor of dihydrofolate reductase, has been demonstrated to allow HGPRT(-) cells to be negatively selected. However, the pathway by which aminopterin leads to cell death remains to be clarified. In this study, we characterized features of cellular responses induced by aminopterin treatment in P3-X63-Ag8.653, a mouse HGPRT(-) myeloma cell line. Upon treatment with aminopterin, the cells readily underwent an apoptotic process, as assessed by DNA fragmentation assay and electron microscopic analysis. Aminopterin-induced apoptosis was drastically reduced by addition of actinomycin D and cycloheximide, indicating that active RNA and protein synthesis is required for the apoptotic effect of aminopterin. Interestingly, the induction of c-myc gene expression preceded the activity of DNA fragmentation in aminopterin-treated cells. Taken together, these results suggest that cells deficient in the salvage pathway of purine biosynthesis are susceptible to aminopterin-induced apoptosis that requires de novo synthesis of proapoptotic factors, including Myc oncoprotein.

Effect of dietary legumes on bone-specific gene expression in ovariectomized rats

In previous studies, we found that the consumption of legumes decreased bone turnover in ovariectomized rats. The purpose of the present study is to determine whether the protective effects on bone mineral density (BMD) and the microarchitecture of a diet containing legumes are comparable. In addition, we aim to determine their protective actions in bones by studying bone specific gene expression. Forty-two Sprague-Dawley rats are being divided into six groups during the 12 week study: 1) rats that underwent sham operations (Sham), 2) ovariectomized rats fed an AIN-93M diet (OVX), 3) ovariectomized rats fed an AIN-93M diet with soybeans (OVX-S), 4) ovariectomized rats fed an AIN-93M diet with mung beans (OVX-M), 5) ovariectomized rats fed an AIN-93M diet with cowpeas (OVX-C), and 6) ovariectomized rats fed an AIN-93M diet with azuki beans (OVX-A). Consumption of legumes significantly increased BMD of the spine and femur and bone volume of the femur compared to the OVX. Serum calcium and phosphate ratio, osteocalcin, expression of osteoprotegerin (OPG), and the receptor activator of nuclear factor κB ligand (RANKL) ratio increased significantly, while urinary excretion of calcium and deoxypyridinoline and expression of TNF-α and IL-6 were significantly reduced in OVX rats fed legumes, compared to OVX rats that were not fed legumes. This study demonstrates that consumption of legumes has a beneficial effect on bone through modulation of OPG and RANKL expression in ovariectomized rats and that legume consumption can help compensate for an estrogen-deficiency by preventing bone loss induced by ovarian hormone deficiency.

Comparative analyses of influenza virus receptor distribution in the human and mouse brains

Accumulating evidence suggests a potential link between influenza A virus infection and the occurrence of influenza-associated neurological disorders. As influenza infection is mediated by specific receptors on the host cell surface, it is important to understand the distribution patterns of influenza receptors in target organs. We carried out comprehensive experiments to localize influenza receptors in the brains of two different mouse strains and the human brain for comparison using lectin histochemistry. We further compared the brain regions in which influenza receptors were expressed and the regions in which experimental influenza infection was observed. Our results show that the expression patterns for influenza receptors in mouse and human brains are different. In the mouse brain, human influenza virus receptors (HuIV-R) were expressed in part of brainstem and cerebellar white matter while avian influenza virus receptors (AIV-R) were expressed in the cerebellar Purkinje neurons. In contrast, in the human brain, many neurons and glia in widespread regions, including the cerebral cortex, hippocampus, brainstem, and cerebellum, express both AIV-R and HuIV-R. Importantly, vascular endothelial cells, choroid plexus epithelial cells and ependymal cells in both mouse and human brains express high levels of HuIV-R and AIV-R. The regional reciprocity was not observed when comparing regions with influenza receptor expression and the regions of influenza infection within the mouse brain. Our results demonstrate a differential influenza receptor expression pattern in mouse and human brains, and a disparity between influenza receptor distribution and regions with actual influenza infection.

Agrobacterium sp.-derived β-1,3-glucan enhances natural killer cell activity in healthy adults: a randomized, double-blind, placebo-controlled, parallel-group study

BACKGROUND/OBJECTIVES The present study investigated the hypothesis that a highly pure linear β-1,3-glucan produced by Agrobacterium sp. R259 enhances human natural killer (NK) cell activity and suppresses pro-inflammatory cytokines. SUBJECTS/METHODS In an eight-week, double-blind, randomized, placebo-controlled clinical trial, 83 healthy adults with white blood cell counts of 4,000-8,000 cells/µL were participated and randomly assigned to take two capsules per day containing either 350 mg β-1,3-glucan or placebo. Six participants withdrew their study consent or were excluded due to NK cell activity levels outside the normal range. NK cell activity and serum levels of immunoglobulin G (IgG) and cytokines, such as interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12 and tumor necrosis factor (TNF)-α were measured. RESULTS NK cell activity and the serum levels of IL-10 were significantly higher from baseline to week 8 in the β-glucan group compared with the placebo group (P = 0.048, P = 0.029). Consumption of β-1,3-glucan also significantly increased NK cell activity compared with placebo after adjusting for smoking and stress status (P = 0.009). In particular, the effect of β-1,3-glucan on NK cell activity was greater in participants with severe stress than in those experiencing mild stress. However, the administration β-1,3-glucan did not significantly modulate the levels of IFN-γ, IL-2, IL-4, IL-6, IL-12, TNF-α and IgG compared with the placebo. CONCLUSION The results showed that supplementation with bacterial β-1,3-glucan significantly increased NK cell activity without causing any adverse effects. Additionally, the beneficial effect of β-1,3-glucan on NK cell activity was greater in participants experiencing severe stress.

Effects of maternal hyperthermia on myogenesis‐related factors in developing upper limb

BACKGROUND Maternal hyperthermia is one causative factor in various congenital anomalies in experimental animals and humans. In the present study, we assessed the effects of high temperature on limb myogenesis in mice. METHODS Pregnant mice, C57BL/6 strain, were exposed to hyperthermia (43 degrees C, 5 minutes) on embryonic day (ED) 8. Fetuses on ED 11, 13, 15, and 17 and neonates on postnatal day (PD) 1 were collected. To characterize the effects of hyperthermia on myogenesis-related factors Pax3, MyoD, myogenin, and myosin heavy chain (MyHC) during skeletal muscle development, we performed RT-PCR, western blotting, immunohistochemistry, and transmission electron microscopy. RESULTS Pax3 gene expression was still detected on ED 13 in hyperthermia-exposed fetuses. The expression of MyoD protein was down-regulated in fetuses exposed to hyperthermia. In contrast, myogenin and MyHC protein expression were up-regulated on PD 1 and ED 17, respectively, in the group exposed to hyperthermia. Immunohistochemical analysis confirmed the findings from western blot analysis. Compared with control neonates, a TEM study revealed immature muscle fibers in PD 1 hyperthermia neonates. Thus, our studies showed that maternal hyperthermia induced delayed expression of Pax3 and inhibited expression of MyoD proteins, which are known to play important roles in migration of myogenic progenitor cells, and in myoblast proliferation. In addition, maternal hyperthermia also delayed the expression of myogenin protein for the formation of myotubes, and MyHC protein, which is one of the final muscle differentiation factors. CONCLUSION Our data suggest that maternal hyperthermia delays limb myogenesis in part by disregulating the expression of key myogenesis-related factors.