A novel culture method of canine peripheral blood lymphocytes with concanavalin a and recombinant human interleukin-2 for adoptive immunotherapy

This study was designed to develop a novel culture method for the efficient proliferation of canine peripheral blood lymphocytes (cPBL) for adoptive immunotherapy. When cPBL were cultured in the presence of concanavalin A (Con A), proliferation of cPBL was induced and expression of interleukin-2 receptor (IL-2R) which enables to respond to exogenously added IL-2 was upregulated. And then, when cPBL were cultured with recombinant human interleukin-2 (rhIL-2) in addition to Con A, proliferation was accelerated and increased to about 10-fold after 1 week. The phenotypic analysis showed that the main population of the cultured cPBL was consisted of CD8+ positive lymphocytes. Among them, CD4+CD8+ double positive (DP) lymphocytes had significantly increased, and the ratio of CD4+ single positive (SP) lymphocytes to CD8+ SP lymphocytes (CD4+SP/CD8+SP) was decreased as compared to before culturing. To evaluate the cytotoxic activity of cPBL cultured with Con A and rhIL-2, furthermore, cytotoxic assay was carried out against xenogeneic melanoma cell line (MeWo), which resulted in MHC-unrestricted cytokilling. These results suggest that the culture method of cPBL by the use of Con A and rhIL-2 may be useful for generating lymphokine activated killer cells, and also this may be beneficial for adoptive immunotherapy of tumor-bearing dogs.     

Silicon content in rice seedlings to protect rice blast fungus at the nursery stage

To control rice blast effectively at the nursery stage, the absolute SiO₂ content necessary for rice plants to resist blast disease was investigated using various rice cultivars and soils. Nine rice cultivars with different complete resistance genes and different degrees of partial resistance were grown on nursery soils amended with silica gel at different rates to change the SiO₂ content of rice plant. The rice seedlings were then inoculated 28 days after sowing with Pyricularia grisea to estimate their blast resistance. In all rice cultivars, the number of lesions was significantly reduced when SiO₂ content increased in the rice seedling; lesions were reduced to 5%–20% of the number on the seedlings grown in soil without silica gel when the seedling SiO₂ content reached 5%. Additionally, the susceptibility to blast disease of rice seedlings grown on eight soils collected from different districts, with varying amounts of silica gel, was compared. The number of lesions decreased significantly when the SiO₂ content in the seedlings reached 5%. These results suggest that SiO₂ content of at least 5% in the rice plant can control this disease at the nursery stage under any conditions.     

Trophinin is expressed in the porcine endometrium during the estrous cycle

We investigated endometrial expression of trophinin mRNA and protein, homophilic cell adhesion molecules, during the estrous cycle of gilts. An immunopositive reaction for trophinin was observed in the luminal and glandular epithelia of the endometrium at all stages of the estrous cycle, but not in endometrial stromal cells or the myometrium. A partial coding sequence of porcine trophinin was similar to sequences in humans and mice, with homologies of 75% and 70%, respectively. As in humans and mice, the trophinin gene is expressed in the endometrium. Trophinin, however, is expressed in the endometrium of the pig throughout the estrous cycle, higher expression levels were observed at some points of the luteal phase, as in humans. These findings suggest that regulation of trophinin gene expression in the pig is different from that in mice, but similar to that in humans. Furthermore, the present results suggest that the pig might be a suitable model for studying the physiological importance of trophinin in early pregnancy in humans.     

Percutaneous ultrasound-guided over-the-wire catheterization of the portal and hepatic vessels in cattle

A new method for catheterization of the portal and hepatic veins in cattle by means of the over-the-wire system was investigated to maintain more reliable long-term patency of catheters. Four cattle were used to evaluate the success rate, patency and safety of the procedure. The catheters, coated by urokinase were patent as long as they were in situ. In addition, the introducer was useful to prevent the catheter from being broken. No complications developed during the10 days after the procedure. Two cows were then euthanized. Post mortem findings were minimal. The results of the study reported here are promising, the benefits are significant and there is no apparent disadvantage to its use.     

Identification of endotrypanum species from a sloth,a squirrel and Lutzomyia sandflies in ecuador by PCR amplification and sequencing of the mini-exon gene

PCR amplification and nucleotide sequencing of the mini-exon gene revealed that four strains isolated from a sloth (Choloepus hoffmanni), a squirrel (Sciurus granatensis) and two sandflies (Lutzomyia hartmanni) in Ecuador were indistinguishable from Endotrypanum monterogeii. Another strain isolated from Lu. hartmanni showed the high sequence similarity to E. schaudinni. Since three of these strains have been previously identified as Leishmania (Viannia) equatorensis, the results demonstrate that L. (V.) equatorensis is genetically closely related to the genus Endotrypanum. The present study also indicates that Endotrypanum species are distributed in arboreal animals and sandflies in Ecuador, and that mini-exon gene amplification is useful for epidemiological studies of Leishmania and Endotrypanum in the New World.     

Effects of propofol-sevoflurane anesthesia on the maternal and fetal hemodynamics blood gases, and uterine activity in pregnant goats

To determine the effects of propofol and sevoflurane on hemodynamics, acid-base balance and uterine activity in pregnant animals, a prospective experimental study was designed by use of ten pregnant goats. Propofol was intravenously administered at a bolus dose of 5 mg/kg and then infused a rate of 0.3 mg/kg/min for 5 min. Following the induction, the animals were incrementally inhaled 2.7 and 4.1% of end-tidal concentration of sevoflurane each for 30 min, and then recovered. The maternal and fetal heart rate (HR), arterial blood pressure (BP) and acid-base balance, the intrauterine pressure (IUP), and the uterine blood flow (UBF) were measured. Following the pre-anesthetic data, the parameters were measured 7 times throughout the anesthetic and recovering periods. The propofol infusion induced 1.37 times of HR increase and produced decrease in PO(2) and a relevant metabolic acidemia in the mother, with no effect in the fetus. Sevoflurane reduced BP in the fetus from 30 (2.7%) to 60 (4.1%) min of inhalation. The uterine contractions disappeared throughout sevoflurane inhalation, and then recurred within 15 min after the cessation of sevoflurane. Propofol injection increases HR, and induces a moderate hypoxemia and metabolic acidemia associated with the suppressed ventilation for pregnant goats, with less effect on the fetal hemodinamics. Sevoflurane causes minimal change in maternal hemodynamics, but induces significant hypotension in the fetus and reduction of uterine activity. These data may be useful in making anesthetic choices combined with analgesia for Caesarian section in goats.     

Growth of recombinant equine herpesvirus 1 (EHV-1) replaced with passage-induced mutant gene 1 and gene 71 derived from an attenuated EHV-1 in cell cultures and in the lungs of mice

The relationship of passage-induced mutant genes 1 and 71 of an attenuated equine herpesvirus 1 (EHV-1) with virulence was analysed by constructing nine recombinant EHV-1 viruses by homologous recombination. Gene 1 or/and gene 71 of a virulent EHV-1 strain, HH1, was replaced by a mutant gene 1 or/and 71 of an attenuated HH1 strain, BK343, respectively. The beta-galactosidase gene of Escherichia coli was inserted within the gene 1 or 71 coding sequence of HH1 to inactivate the genes. Virus replications of these recombinant viruses in cell cultures were similar, but release of the gene 71-inactivated virus from infected cells was delayed compared to that of the other viruses. Plaque sizes of the recombinant viruses were similar to those of HH1, but those of BK343 were significantly smaller, indicating an effect of some unknown factor(s) on viral cell-to-cell spread. The growth abilities of the recombinant viruses with a mutant gene 1 or/and 71 in lungs of mice were similar to those of HH1, but those of gene 71-inactivated viruses were reduced to the level of BK343 and the titers were about 100-times lower than those of the other recombinant viruses. These results indicate that the mutant genes 1 and 71 of BK343 might not confer an attenuated nature to EHV-1.