Ischemic preconditioning (IPC), i.e., a preliminary brief episode of ischemia and reperfusion, has been shown to reduce the cell damage induced by long ischemia and reperfusion. Superoxide radical which is produced during reperfusion after ischemia was recongnized as a factor of the ischemic injury and it is dismutated into H2O2 and O2 by two types of intracellular superoxide dismutase (SOD), Cu,Zn-SOD in cytoplasm and Mn-SOD in mitochondria. Recently oxygen free radicals are suggested to induce the apoptosis, however mechanism of the reduced apoptosis by ischemic precondition was unknown, while many studies performed in mammalian heart indicated that ATP-sensitive K+(K atp) channel activation related with the protective effects. The aim of present study is toinvestigate 1)whether IP upregulate the Cu,Zn-SOD and Mn-SOD activities, and 2)whether ischemic preconditioning decreases apoptosis via K atp channel activation in timely reperfused skeletal muscle after long ishemia. The experimental animals, Sprague-Dawley rats weighing 250~300g, were divided into 8 group; 1)control group, 2)ischemic preconditioning only groups, 3)pinacidil, a K atp channel opener, treatment only groups, 4)glibenclamide, a K atp channel blocker, treatment only groups, 5)ischemia groups, 6)ischemia after IPC groups, 7)ischemia and pinacidil treatment groups, and 8)IP and ischemia after glibenclamide pretreatment groups. Animals of the control group were administered with the vehicle (DMSO) alone. Pinacidil (1mg/kg) was administred intravenously 5 minutes after initiation of ischemia, and glibenclamide(0.5mg/kg) was injected intravenously 20 minutes before IPC. In rats that were ischemic preconditioned, the left common iliac artery was occluded for 5 minutes followed by 5 minutes of reperfusion by three times usign vascular clamp. Ischemia was done by occlusion of the same artery for 4 hours. The specimens of left rectus femoris muscle were obtained immediately (0 hours), 12 hours, 24 horus after drug administrations, IP or ischemia and reperfusion. The immunoreactivities of SOD and its alterations were observed by use of sheep antihuman Cu,Zn-SOD and Mn-SOD antibodies onthe 10 micro meter cryosections. The incidencies of apoptosis were observed by TUNEL methods with in situ apoptosis detection kit on 6 micro meter paraffine section. The results obtained were as follows: 1. After IPC, immunoreactivities of Cu,Zn-SOD mainly in the small-sized fibers were increased by 24 hours, that of Mn-SOD at 0 hour and 24 hours. 2. No significant changes in immunoreactivities of SDO was observed in the pinacidil and in the glibenclamide treatment only groups, and in the ischemia only groups. 3. The immunoreactivities of the Cu,Zn-SOD were incresed in the ischemia after IPC groups and the ischemia and pinacidil treatment groups. 4. The immunoreactivities of the Cu,Zn-SOD in the IPC and ischemia after glibenclamide pretreantment groups were not increased except for the 12 hours reperfusion group. But, Mn-SOD immunoreactivities were incresed in to 0 hours, 12 hours and 24 hours after reperfusion. 5. In the control group, the IPC only groups, and the pinacidil treatment only groups, negative or trace apoptotic reactions were observed, but the positive apoptotic reaction occured in the glibenclamide treatment groups. 6. Moderate or many number of apoptosis were revealed in the ischemia groups, and also the IPC and ischemia after glibenclamide pretreatment group except for 12 hours and 24 hours after reperfusion. However, the incidence of apoptosis was decreased in the ischemia after IPC groups and in theischemia and pinacidil treatment groups. 7. There is a coincidence between the increase of Cu,Zn-SOD immunoreactivities and the decrease of apoptosis in thepresence of ischemia and reperfusion. These results suggest that the protective effects of ishemic preconditioing may related to the SOD activation, and the ischemic preconditioning decreases the apoptosis partially via K atp channel activation in timely reperfused rat skeletal muscle. It is also suggested that inhibition of apoptosis by IPC may related with the SOD activation.

Galectin-3 plays an important role in cell development, differentiation and cancer metastasis, including cell-cell/extracellular matrix (ECM) interactions and is supposed to have an effect of apoptosis on T-cells in thymic clonal selection. In this study, to know the effect of galectin-3 on thymocyte development, we used recombinant human galectin-3 (rHgal-3) from Escherichia coli, JM105, which was inserted with human gal-3 gene-transformed plasmid vector (prGal-3) to express human galectin-3. Expressed rHgal-3 was confirmed by western blot using the culture supernant of hybridoma (M3/38) producing monoclonal antibody to human galectin-3. Sepharose gel affinity chromatography was used to purify the expressed rHgal-3. Thymocytes and hepatocytes from 6-week-old male BALB/c mice were incubated with rHgal-3 and showed marked increase of apoptotic population on analysis using flow cytometry with 7-AAD in a dosedependent manner. However, rHgal-3 failed to induce apoptosis on hepatocytes. Interestingly, this apoptotic effect was not inhibited by lactose, a specific lectin domain inhibitor. From these results, we concluded that extrinsic galectin-3 induces apoptosis on mouse thymocytes, and galectin-3 may have an apoptotic effect on T-cells in thymic clonal selection.

Test vaccines comprised of inactivated water-in-oil emulsions containing various antigen levels were prepared using a non-pathogenic H5N1 avian influenza (AI) virus, A/duck/Hokkaido/Vac-1/04 (H5N1). The potencies of these test vaccines were evaluated by two experiments. In the first experiment, the triangular relationship among the antigen levels of test vaccines, the hemagglutination inhibition (HI) antibody response, and the protective effect against challenge with a highly pathogenic avian influenza (HPAI) virus, A/chicken/Yamaguchi/7/04 (H5N1), was confirmed. Then lasting immunity of chickens after a single-shot vaccination was confirmed in the second experiment. As a result, complete protection after the challenge was observed in chickens immunized by test vaccines with an antigen level of 160 HA units/dose or higher. Thus, it was ascertained that the minimum antigen level in the AI vaccine was 160 HA units/dose, and the minimum HI antibody titer that could protect chickens from HPAI virus infection-related death was considered to be 1:16. Dose-dependent HI antibody responses were observed in chickens after the vaccination. Thus, 640 HA units/dose were thought to be similar to the optimal antigen level. Alternatively, the HI antibody titers of chickens, injected with the vaccine containing 640 HA units/dose, were maintained at 1:181 or higher for 100 weeks after the single-shot vaccination.

In this study ELISA was standardized for detecting antibodies against Hydropericardium Syndrome Virus. Factors like Antigen, Conjugate and incubation time were studied. Standardization was carried out by checker board titration method. Optimum incubation time for colour development was found to be 15 minutes while 50 mug/ml of antigen gave better results than 100 mug/ml. The conjugate dilution of 1:500 was observed to give satisfactory results as determined by regression analysis. Cut off value was determined by adding 2SDs and 3SDs in the mean optical density (OD) values of negative serum samples. It was found that mean OD+2SD (0.0684) resulted in better diagnostic sensitivity) and diagnostic specificity than mean OD+3SDs (0.0741).

The quick diagnosis of Newcastle disease requires known serum against the disease. In this study, an attempt was made to raise anti- Newcastle disease virus hyperimmune serum in rabbits. Three different inoculum were prepared to inoculate in the rabbits; (i) Fresh harvested allantoic fluid containing Newcastle disease virus (NDV) Mukteswar vaccine strain; (ii) Freshly harvested ND virus pelletted through centrifuging at 40,000 rpm for two hours and resuspended in normal saline and (iii) Pelletted virus (centrifuged and suspended as in ii) with addition of incomplete Freund's adjuvant. It was observed during the monitoring of haemagglutination inhibition (HI) titre that the serum collected after series of inoculation of 1st and 2nd inoculum provided maximum titre upto 1:256. However, the serum collected after series of injection of 3rd inoculum gave maximum HI titre 1:1024. This study suggested that antigen containing incomplete Freund's adjuvant provided better immune response against Newcastle disease virus in rabbits.