Glutathione content of in vivo and in vitro matured canine oocytes collected from different reproductive stages

Glutathione (GSH) concentrations of oocytes are considered as an important marker of the cytoplasmic maturation. The present study was designed to compare GSH concentrations of in vivo and in vitro matured canine oocytes. In vivo matured oocytes were collected 72 hr after ovulation by flushing fallopian tubes after laparotomy. Ovaries were collected from bitches with different reproductive stages, and collected oocytes were divided into 2 groups according to the size viz. < 120 microm and > 120 microm in diameter and cultured for 72 hr in Tissue Culture Medium-199 supplemented with 10% FBS, 2.2 mg/ml sodium bicarbonate, 2.0 microg/ml estrogen, 0.5 microg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin-streptomycin solution in the presence or absence of 50 microM beta-mercaptoethanol. GSH concentrations were determined by the dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. GSH concentrations of immature canine oocytes were 2.9 and 3.8, 3.5 and 6.8, and 3.1 and 6.5 pM/oocyte for < 120 microm and > 120 microm in diameter oocyte groups at anestrous, follicular and luteal stage, respectively (P<0.05). In vivo matured oocytes had significantly higher GSH concentrations compared with in vitro matured oocytes. The GSH content was 19.2 pM/oocyte for in vivo matured oocytes, while 4.1 to 8.1 and 5.7 to 13.2 pM/oocyte for in vitro matured oocytes cultured in the absence or presence of beta-mercaptoethanol, respectively (P<0.05). Presence of beta-mercaptoethanol increased GSH synthesis in canine oocytes cultured in vitro, and oocytes collected from follicular and luteal stage was superior to anestrus oocytes.     

The Effects of Salivary Gland Extracts from <Emphasis Type="Italic">Boophilus microplus</Emphasis> Ticks on Mitogen-stimulated Bovine Lymphocytes

The effect of salivary gland extract (SGE) from the tick Boophilus microplus was examined in mitogen-stimulated lymphocytes in vitro. SGE was added to lymphocytes of seven cattle together with the mitogens concanavalin A (ConA), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). Semi-purified B cells from another seven cattle were stimulated with the mitogen lipopolysaccharide (LPS). PHA and ConA stimulated proliferation of lymphocytes to the same extent, but the inhibition due to SGE of Boophilus microplus on the proliferative response stimulated by PHA (39.0% ± 9.3%) was less than the inhibition of proliferative response stimulated by ConA (75.4% ± 6.9%). In contrast, SGE of B. microplus stimulated the proliferation of B cells in the presence of LPS in a dose-dependent manner. Enhanced stimulation of B cells by SGE at >4 μg in culture was greater than twice that observed when B cells were stimulated by LPS alone. SGE does not have a direct suppressive effect on bovine B cell proliferation; however, in vivo the effectiveness of B cell responses might be influenced by other immune factors, such as cytokine profiles.     

Decrease in intestinal endocrine cells in Balb/c mice with CT-26 carcinoma cells

The density of intestinal endocrine cells, in Balb/c mice with colon 26 (CT-26) carcinoma cells, were examined immunohistochemically at 28 days after implantation. After CT-26 cell administration there was a significant decrease in most of the intestinal endocrine cells (p < 0.01) compared with the control group. The significant quantitative changes in the intestinal endocrine cell density might contribute to the development of the gastrointestinal symptoms commonly encountered in cancer patients.     

Retrospective assessment of tolerability and efficacy of zoledronate in the palliative treatment of cancer-bearing dogs

Zoledronate is a bisphosphonate frequently used for the treatment of hypercalcaemia of malignancy and tumour-associated bone pain in dogs, however, there is a paucity of information regarding its use in veterinary medicine. The aim of this retrospective study was to report the tolerability of zoledronate in the palliative treatment of cancer-bearing dogs and secondarily to to assess the efficacy of zoledronate for the treatment of hypercalcaemia of malignancy. Thirty-seven dogs (22 with tumour-associated bone pain and 15 with hypercalcaemia of malignancy) that received 114 zoledronate infusions were included. Tolerability was assessed by the absence of post-zoledronate hypocalcaemia or other adverse events as defined by Veterinary Cooperative Oncology Group-Common Terminology Criteria for Adverse Events criteria. Efficacy was assessed by comparison of available ionized calcium levels before and after zoledronate administration in hypercalcaemic dogs. In 79% of zoledronate infusions, no adverse events were reported. The majority of adverse events which occurred in the other 21% of infusions could be attributed to concurrent chemotherapy or the underlying neoplastic disease. There was a small but significant increase in creatinine following treatment with zoledronate, however, none of the dogs developed clinically significant renal disease. In eight hypercalcaemic dogs with available ionized calcium following zoledronate administration, ionized calcium decreased rapidly within 7 days following treatment with zoledronate. Zoledronate is well-tolerated with few recorded adverse events, however, monitoring of serum creatinine is advised. Zoledronate seems to be effective in the treatment of hypercalcaemia of malignancy.     

Tumour necrosis factor stimulated gene 6 intrinsically regulates PD-L1 expressions in breast cancer cells,leading to modulation of tumour microenvironment

Recent studies have shown that tumour cells express tumour necrosis factor-inducible gene 6 (TSG-6) and its protein, which is known to play a key role in regulating excessive immune responses and proliferation and growth of mesenchymal stem cells (MSCs). It has not been confirmed whether the inhibition of TSG-6 for tumour cells can suppress tumour cell growth and regulate the activation of immune cells in the tumour microenvironment (TME). TSG-6-specific small interfering RNA was transfected into canine and human breast cancer cells (CIPp, CIPm and BT-20). TSG-6-down-regulated (siTSG-6) cells showed decreased cell proliferation, migration, and invasion abilities. Decreased mRNA expressions of NF-κB, STAT3 and Sox2, confirming that TSG-6 is an upper factor governing tumour growth and metastasis. Notably, siTSG-6 cells showed significantly decreased expression levels of CD44 and PD-L1. Direct and indirect co-culture of canine peripheral blood mononuclear cells (cPBMCs) and the siTSG-6 cells showed significant activation in M1 type macrophages and cytotoxic T cells. They also showed a tendency to decrease in the expression of CTLA-4 and increase in the expression of PD-1. In conclusion, this study suggests that the down-regulation of TSG-6 in breast cancer cells could not only suppress tumour growth and metastasis, and but also regulate TME. Since modulation of immune checkpoint proteins occurs in both tumour cells and immune cells, inhibiting TSG-6 and its protein within the TME could be novel therapeutic target for anticancer treatment.     

Effect of dopamine on excretory urographic image quality and the prevention of contrast-induced nephropathy in dogs

The effect of low dose dopamine on the excretory urographic image quality and contrast media-induced nephropathy in normal dogs (experiment 1) and the dogs with decreased renal function (experiment 2) were assessed. In experiment 2, decreased renal function was induced by gentamicin overdose. In each experiment, animals were divided into 3 groups. In group 1, only contrast medium (iohexol) was administered. In group 2, contrast medium plus intravenous fluid (0.9% saline) were administered. And in group 3, contrast medium plus intravenous fluid and low dose dopamine were administered. Investigated parameters included intrarenal resistive index (RI), serum BUN and creatinine concentrations, contrast medium elimination time and radiographic image quality. In experiment 1, RI of group I increased at 80 min after contrast medium administration (p<0.05), but RI of group 3 decreased at 48 and 72 hr (p<0.05). Serum BUN concentration of group 1 was higher than that of group 2 and 3 (p<0.05); in radiographic examination, contrast medium elimination time decreased in group 2 and 3, but image quality of group 2 was inferior to that of group 3. In experiment 2, image quality of group 3 only provided adequate visualization of renal structures. The formula of contrast medium plus low dose dopamine was found to provide good nephrogram and pyelogram image quality without supplemental contrast medium, and to protect renal tubules from prolonged exposure to concentrated contrast medium.     

Enzyme-linked immunosorbent assay for screening the plasma residues of tetracycline antibiotics in pigs.

The recommended therapeutic doses of three kinds of tetracyclines, oxytetracycline (OTC, withdrawal period, 10 days), chlortetracycline (CTC, withdrawal period, 5 days) and tetracycline (TC, withdrawal period, 5 days), were each administered to a group of 15 pigs. Blood was sampled before drug administration and during the withdrawal period. The concentration of tetracyclines in plasma, determined by semi-quantitative ELISA, was compared with that of internal standard (10 ppb as oxytetracycline). The absorbance ratio of internal standard to sample (B/Bs) was employed as an index to determine the tissue residues in pigs. All 45 plasma samples from nontreated pigs showed negative in the residue of any of three tetracycline antibiotics. OTC was detected in plasma of pigs treated until the 8th day, CTC until the 4th day, and TC was detected until the 3rd day of its withdrawal period. The present study showed that the semi-quantitative ELISA easily be adopted in predicting tissue residues for tetracycline antibiotics in live pigs.