Improvement of indirect enzyme-linked immunosorbent assay for detection of Japanese encephalitis virus antibodies in swine sera

Japanese encephalitis (JE) is an important zoonosis caused by the mosquito-transmitted JE virus (JEV), which is a causative agent of reproductive failure in pregnant sows. Detection of JEV antibodies in swine is performed by hemagglutination inhibition (HI), virus neutralization (VN), and the plaque reduction neutralization test (PRNT). The most stringent PRNT is the 90% endpoint PRNT (PRNT₉₀). These conventional assays are difficult to carry out in diagnostic laboratories with insufficient instruments or cell culture systems. An alternative assay that is easily conducted and time efficient is required. In this study, we improved the indirect enzyme-linked immunosorbent assay (I-ELISA) with clarified antigen for the detection of JEV antibodies. The I-ELISA results obtained from 175 swine serum samples were compared with HI, VN, and PRNT₉₀ results. The sensitivity of I-ELISA was 91.8%, 95.0%, and 94.7% compared with HI, VN, and PRNT₉₀ results, respectively. The specificity of I-ELISA was 92.2%, 94.7%, and 94.7% compared with HI, VN, and PRNT₉₀ results, respectively. Moreover, the I-ELISA results were significantly correlated with the HI (r = 0.93), VN (r = 0.95), and PRNT₉₀ (r = 0.92) results. These results suggest that the improved I-ELISA is useful for serosurveillance of JEV in swine.

Effect of chicken egg yolk antibody on canine parvoviral enteritis in pups / 대한수의학회지

Preventive and therapeutic effects of egg yolk antibody, immunoglobulin Y (IgY), against canine parvovirus (CPV) was evaluated in 25 pups orally challenged with CPV-2a. Oral administration of IgY using powder, paste and coated paste delivery systems was compared. Each type of IgY was administered orally for 17 days from 3 days before challenge. The group of pups administered coated IgY showed mild symptoms such as a moderate decrease in total white blood cell count, no depression, vomiting and diarrhea when compared with other groups. The overall clinical score of the group of pups administered coated IgY was significantly lower than that of the challenge control group. However, mortality did not differ among groups because not all pups received symptomatic treatment. These results implied that oral treatment of coated IgY could improve therapeutic effects against CPV challenge if pups received symptomatic treatment.

Differential regulation of inducible nitric oxide synthase and cyclooxygenase-2 expression by superoxide dismutase in lipopolysaccharide stimulated RAW 264.7 cells

Inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) have been known to be involved in various pathophysiological processes such as inflammation. This study was performed to determine the regulatory function of superoxide dismutase (SOD) on the LPS-induced expression of iNOS, and COX-2 in RAW 264.7 cells. When a cell-permeable SOD, Tat-SOD, was added to the culture medium of RAW 264.7 cells, it rapidly entered the cells in a dose-dependent manner. Treatment of RAW 264.7 cells with Tat-SOD led to decrease in LPS-induced ROS generation. Pretreatment with Tat-SOD significantly inhibited LPS-induced expression of iNOS and NO production but had no effect on the expression of COX-2 and PGE2 production in RAW 264.7 cells. Tat-SOD inhibited LPS-induced NF-kappaB DNA binding activity, IkappaBalpha degradation and activation of MAP kinases. These data suggest that SOD differentially regulate expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells.

Inhibition of LPS-induced cyclooxygenase 2 and nitric oxide production by transduced PEP-1-PTEN fusion protein in Raw 264.7 macrophage cells

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor. Although it is well known to have various physiological roles in cancer, its inhibitory effect on inflammation remains poorly understood. In the present study, a human PTEN gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-PTEN fusion protein. The expressed and purified PEP-1-PTEN fusion protein were transduced efficiently into macrophage Raw 264.7 cells in a time- and dose- dependent manner when added exogenously in culture media. Once inside the cells, the transduced PEP-1-PTEN protein was stable for 24 h. Transduced PEP-1-PTEN fusion protein inhibited the LPS-induced cyclooxygenase 2 (COX-2) and iNOS expression levels in a dose-dependent manner. Furthermore, transduced PEP-1-PTEN fusion protein inhibited the activation of NF-kappa B induced by LPS. These results suggest that the PEP-1-PTEN fusion protein can be used in protein therapy for inflammatory disorders.

Suppression of HIV-1 Tat-induced monocyte adhesiveness by a cell-permeable superoxide dismutase in astrocytes

HIV-1 Tat is considered to be one of key players to facilitate monocyte entry into the CNS, which is characteristic feature of AIDS-related encephalitis and dementia. This study was performed to determine the regulatory function of superoxide dismutase (SOD) on the HIV-1 Tat-induced signaling pathways leading to NF-kappaB activation, expression of adhesion molecules, and monocyte adhesion in CRT-MG human astroglioma cells by using cell-permeable SOD. When cell-permeable SOD was added to the culture medium of CRT-MG cells, it rapidly entered the cells in dose- and time-dependent manners. Treatment of astrocytes with cell-permeable SOD led to decrease in Tat-induced ROS generation as well as NF-kappaB activation. Cell-permeable SOD inhibited the activation of MAP kinases including ERK, JNK and p38 by HIV-1 Tat. Treatment of CRT-MG cells with cell-permeable SOD significantly inhibited protein and mRNA levels of ICAM-1 and VCAM-1 up-regulated by HIV-1 Tat, as measured by Western blot analysis and RT-PCR. Furthermore, enhanced adhesiveness of monocyte to astrocyte by HIV-1 Tat was significantly abrogated by pretreatment with cell-permeable SOD fusion proteins. These data indicate that SOD has a regulatory function for HIV-1 Tat-induced NF-kappaB activation in astrocytes and suggest that cell-permeable SOD can be used as a feasible therapeutic agent for regulation of ROS-related neurological diseases.

Human glutamate dehydrogenase is immunologically distinct from other mammalian orthologues

Five monoclonal antibodies (mAbs) that recognize human glutamate dehydrogenase (GDH) have been selected and designated as monoclonal antibodies hGDH60-6, hGDH60-8, hGDH63-10, hGDH63-11, and hGDH91-14. A total of five mAbs recognizing different epitopes of the enzyme were obtained, two of which inhibited human GDH activity. When total proteins of human homogenate separated by SDS- PAGE, were probed with mAbs, a single reactive protein band of 55 kDa, which co-migrated with purified recombinant human GDH was detected. When the purified GDH was incubated with each of the mAbs, its enzyme activity was inhibited by up to 58%. Epitope mapping analysis identified, two subgroups of mAbs recognizing different peptide fragments. Using the individual anti-GDH antibodies as probes, the cross reactivities of brain GDH obtained from human and other animal brain tissues were investigated. For the human and animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 55 kDa when hGHD60-6, hGHD60-8, or hGHD91-14 mAbs were used as probes. However, the anti-human GDH mAbs immunoreactive to bands on Western blots reacted differently on the immunoblots of the other animal brains tested, i.e., the two monoclonal antibodies hGDH63-10 and hGDH63-11 only produced positive results for human. These results suggest that human brain GDH is immunologically distinct from those of other mammalian brains. Thorough characterization of these anti-human GDH mAbs could provide potentially valuable tool as immunodiagnostic reagents for the detection, identification and characterization of the various neurological diseases related to the GDH enzyme.

The effect of cytokines and endotoxin on the nitric oxide production and its relation to mitochondrial aconitase activity in cultured rat lung microvascular endothelial cells / 대한내과학회지

OBJECTIVE: Both constitutive and inducible forms of nitric oxide synthase exist in endothelial cells. Disorders that produce acute lung injury frequently release endotoxin and cytoknes, such as interferon(IFNgamma) and tumor necrosis factor (TNFalpha). Endotoxin and these cytokines likely act as important mediators of cell injury. Because nitric oxide (NO) avidly reacts with iron, it may affect the activity of key enzymes, such as mitochondrial aconitase, which contain an iron-sulfur structure as a prosthetic group. METHOD: We studied the effect of IFNgamma, TNFalpha and E. coli lipopolysaccharide(LPS) on NO production and mitochondrial aconitase activity in cultured rat lung microvascular endothelial cells(RLMVC). RESULT: Exposing RLMVC for 24 hours to IFNgamma(500 U/mL), TNFalpha(300 U/mL) and LPS(5 microgram/mL) significantly increases nitrite production to 20+/-1 micrometer compared to 0.07 micrometer in control cells(P<0.05, n=4). Cytokine treatment also reduced mitochondrial aconitase activity from 196+/-8 to 102+/-34 nmole/min/mg of cell protein(P<0.05, n=4). Treatment with the inhibitor of nitric oxide synthase N-monomethyl-L-arginine(NMMA) (0.5 mM) not only significantly blunted the cytokine-mediated increase in nitrite formation (3+/-0.5 micrometer vs 20+/-1 micrometer with cytokines, P<0.05, n=4), but also prevented the cytokine-mediated drop in aconitase activity (161+/- 24 vs. 196+/-8 nmole/min/mg of cell protein, NS). CONCLUSION: Exposing RLMVC to IFNgamma, TNFalpha and E. coli LPS substantially decreases mitochondrial aconitase activity. Nitric oxide appears to mediate this effect. Our results suggest that the excessive production of NO by endothelial cells, in response to cytokines and endotoxin, may inhibit the function of the endothelial cell itself.

The Changes of c-fos and c-jun after Capsaicine Treatment in the Rat Brain / 대한해부학회지

The expression of c-fos and c-jun in the brain of the rat after capsaicin treatment was investigated by in situ hybridization, dot blot hybridization and immunocytochemical methods. Adult male Sprague-Dawley rats[200g] were used for this study. The first set of rats received a single subcutaneous injection of capsaicin[50mg/Kg] dissolved in 10% Tween-80 and 10% ethanol in saline. The rats were decapitated 1, 3, 5, 10, 24, 72 hours and 1 week after capsaicin treatment. The control set of rats were treated with saline instead of capsaicin. In situ hybridization and dot blot hybridization were carried out. O1igonucleotide probe complimentary to c-fos mRNA sequences were used for this study and labeling of oligonucleotides was accomplished using the DNA tailing kit. The expression of c-fos mRNA on the nucleus of neurons in in situ hybridization was observed throughout the brain, and was especially abundant in the olfactory cortex, nucleus of diagonal band of Broca, habenular nuclei, periaqueductal gray, parabrachial nucleus, entopeduncular nucleus, ventral posterolateral nucleus of the thalamus and cerebellum. Compared to the control rats, c-fos mRNA were increased 24 hours after capsaicin injection and gradually decreased after 72 hours, returning to the normal control level 1 week after capsaicin injection. c-fos mRNA was detected only 1 week after capsaicin injection in the various areas of the brain. The fos protein-like immunoreactivity was initially somewhat decreased at 24 hours, but increased at 72 hours and reactions was maximally observed at 1 week after capsaicin treatment. But Jun protein immunoreactivity was not increased, on the contrary, it was even decreased both in numbers of reactive cells and immunoreactivity 1 week after capsaicin injection. From the above results, c-fos gene expression was pronounced in the nucleus concerned with pain, olfaction and taste such as VPL nucleus of the thalamus, olfactory cortex and parabrachial nucleus, in the limbic system concerned with stress and emotion such as nucleus of diagonal band of Broca, periaqueductal gray and habenular nucleus, in the structure concerned with somatic motor function such as entopeduncular nucleus and cerebellum. Also, the c-fos gene was activated by the capsaicin early in the course of effects, then the fos protein increased as a results of c-fos activation. On the other hand, c-jun did not respond to capsaicin treatment early in the course, but Jun protein decreased late in the course of capsaicin effects.

Clinical significance of the combined assay of pleural fluid ADA activity and CEA level in the various pleural effusion / 결핵

No abstract available.

Natural killer activity and antibody-dependent cellular cytotoxicity in patients with primary lung cancer

The NK activity and ADCC of peripheral blood mononuclear cell were examined to evaluate the contribution of ADCC and NK activity to host immune response against lung cancer. The NK activity and ADCC were examined in 58 patients with primary lung cancer and 40 healthy volunteers as normal controls. The NK activity of patients with lung cancer was significantly subnormal, but ADCC was at a normal level. The NK activity was decreased in non-small cell lung cancer (NSCLC), but not in small cell lung cancer (SCLC) compared to normal controls. According to stage, the NK activity in stage II, III-M0 and III-M1 NSCLC showed low levels compared to that of stage I NSCLC, but there was no difference of NK activity in patients with SCLC. The NK activity was not affected by performance status. There was no significant difference of ADCC in patients with lung cancer according to cell type, stage and performance compared with that of normal controls. The NK activity and ADCC were not changed after chemotherapy and operation respectively.

The bronchoalveolar lavage fluid cell analysis with normal lung and unaffected side lung of patients with minor symptoms or radiologic abnormalities / 결핵

No abstract available.

Adenosine deaminase activity in bronchoalveolar lavage fluid in patients with pulmonary tuberculosis / 결핵

No abstract available.