Cytotoxic T lymphocyte mediated recognition of human pancreatic cancer cells

T lymphocytes play an important role in tumor rejection and their response to human malignant melanoma has been well documented. In contrast, the existence of cytotoxic T lymphocytes (CTL) to pancreatic cancer remains unclear. Tumor-associated lymphocytes (TAL) and peripheral blood monocytes (PBMC) were isolated from pancreatic cancer patients. Tumor-specific CTL were generated from TAL and PBMC using solid-phase anti-CD3, low-dose IL-2 (50 IU/ml) and repetitive autologous tumor stimulation. The specificity of CTL was tested in standard cytotoxicity assays using autologous tumor cells, autologous fibroblasts when available, several allogeneic pancreatic tumor cells and the NK-sensitive cell line K562. Anti-HLA-Class I MAb, W6/32, was used to demonstrate that tumor-specific CTL were HLA-Class I restricted. HLA-molecules of human pancreatic cancer cells were washed out using acid elution. Eight consecutive, histologically confirmed pancreatic cancer specimen as well as peripheral blood mononuclear cells were analyzed. CTL were capable of lysing autologous tumor cells significantly after 3 stimulations with autologous tumor cells. T cell mediated recognition was HLA-Class I restricted as shown by incubation with MAb anti-HLA-Class I. In case of HLA-A2 positivity, incubation of tumor cells in cytotoxicity assays resulted in significant inhibition. Autologous fibroblasts or K562 cells were lysed significantly less. HLA-Class I molecule elution resulted in significantly lower recognition of these cells by CTL. These results show for the first time in a larger series the possibility of generating CTL in human pancreatic cancer. The identification of new tumor associated antigens or tumor antigens will be crucial for establishing new treatment strategies.     

Influence of ETR-p1/f1 antisense peptide on endothelin-induced constriction in rat renal arcuate arteries

1. This study set out to examine the endothelin receptor subtypes mediating vasoconstriction in the rat renal arcuate artery. This was done in isolated vessels 120–200 μm in diameter, incubated with a selective agonist and the novel ‘antisense'' peptide to part of the human endothelin_A receptor.
2. Groups of vessels (n=6) were incubated with increasing concentrations of endothelin-1 (ET-1), from 1 to 100 nM, which caused a 65% maximal contraction at the highest dose with an pEC₅₀ of 8.16±0.11 M. By contrast, in six other vessels sarafotoxin 6c over the same dose range gave a minimal contraction (around 5% of maximum).
3. Preincubation of six vessels with the antisense peptide ETR p₁/f1 at 1 μM had no effect on the ET-1 induced vasoconstriction, in terms of displacement of the concentration-response curve or the maximal tension achieved by the agonist. In the six vessels exposed to 4 μM ETR p₁/f1, there was a significant shift of the concentration-response curve and a lower pEC₅₀ at 7.78±0.09 M (P<0.05). At the highest concentrations of ETR p₁/f1, there was a marked suppression of all responses to ET-1, which at the maximal concentrations tested, 0.1 μM, only reached some 10% of the maximal achievable contraction.
4. Increasing ET-1 concentrations up to 2 μM in vessels incubated with 40 μM ETR-p₁/f1 showed that the blockade could be overcome and that the relationship was shifted to the right (P<0.001) by approximately one log unit with a pEC₅₀ of 7.13±0.11 M. A Schild plot of the data indicated the antagonist to be acting competitively at a single population of receptors.
5. At the highest concentrations tested, 40 μM, ETR-p₁/f1 had no effect on noradrenaline-induced contractions, indicating a lack of non-specific actions.
6. Together, these data suggest that at the rat renal arcuate artery the endothelin_A receptor is the predominant functional receptor mediating contraction. Furthermore, this study has shown the potential usefulness of this novel type of ‘antisense'' peptide in blocking receptor activation.

     

Effects of naftidrofuryl oxalate on microsphere embolism-induced decrease in regional blood flow of rat brain.

1. The purpose of the present study was to determine whether naftidrofuryl oxalate (naftidrofuryl), a vasodilator, is capable of improving brain regional blood flow of animals in sustained ischaemia. 2. Cerebral ischaemia was induced by injecting 900 microspheres (48 microns in diameter) into the right internal carotid artery of rats. Cerebral blood flow of brain regions was measured by a hydrogen clearance method on the 3rd, 7th and 28th days after the onset of ischaemia. Ischaemic animals were treated with naftidrofuryl, 15 mg kg-1 day-1 i.p., from the first to 28th day. 3. Microsphere-embolism caused a sustained decrease in cortical and striatal blood flow over a period of 28 days, whereas hippocampal blood flow was decreased on the 3rd day but not on the 7th or 28th day. On the 3rd day, the striatal and hippocampal but not cortical blood flow of naftidrofuryl-treated, microsphere-embolized rats was higher than untreated rats. On the 7th and 28th days, the cortical and striatal blood flow of the treated and untreated animals did not differ. 4. Brain slices from microsphere-embolized rats contained areas, which were not stained with triphenyltetrazolium chloride (TTC), to a similar degree on the 3rd, 7th and 28th days, indicating the genesis of cerebral infarction. TTC-unstained areas of microsphere-embolized rats that had received naftidrofuryl treatment were smaller than those of untreated rats on the 3rd and 7th days, but not on the 28th day.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of microsphere embolism-induced impairment of learning and memory function and the cholinergic system

The impairments of learning and memory function and of the cholinergic system were examined in rats with microsphere embolism. Microsphere embolism was induced by injection of 900 microspheres with a diameter of 48 microm into the right internal carotid artery. The retention latency of a passive avoidance test was shortened and the escape latency of a water maze test was prolonged, when the animals were tested on the 5th to 10th day after the embolism, suggesting learning and memory dysfunction. Cholinergic parameters of the striatum and hippocampus, such as acetylcholine (ACh) content (67 and 60% decrease, respectively), choline acetyltransferase (ChAT) activity (45 and 56% decrease, respectively), and Bmax of muscarinic acetylcholine M1-receptor (43 and 37% decrease, respectively), were reduced on the 11th day after the embolism, suggesting attenuation of ACh synthesis and a decrease in the number of muscarinic acetylcholine M1-receptors mainly in the striatum and hippocampus. Areas not stained with triphenyltetrazolium chloride, an indication of infarction, were detected mainly in the striatum and hippocampus and partly in the frontal cortex on the 11th day after the embolism. The results suggest that an animal with microsphere embolism may be a good ischemic model with relatively sustained impairments of learning and memory function and of the striatal and hippocampal cholinergic system.     

Beneficial effect of amosulalol and phentolamine on post-hypoxic recovery of contractile force and energy metabolism in rabbit hearts.

1. The effects of phentolamine, an alpha-adrenoceptor blocking agent and amousulalol, an alpha 1 and beta-adrenoceptor antagonist on hypoxia-induced impairment in cardiac function and metabolism were examined using the isolated heart Langendorff preparation of the rabbit. 2. Hypoxia induced cessation of cardiac contractile force, a rise in resting tension, a decrease in myocardial high-energy phosphates, an increase in tissue calcium content and the release of ATP metabolites from the heart. Subsequent reoxygenation resulted in little recovery of cardiac contractile force, and there were further increases in tissue calcium content and in the release of creatine kinase from the heart. 3. Treatment of hypoxic hearts with either 83 microM phentolamine or 45 microM amosulalol resulted in a suppression of the rise in resting tension, the tissue calcium accumulation and the release of creatine kinase and ATP metabolites during hypoxia. This treatment also elicited significant recovery of cardiac contractile force, restoration of myocardial high-energy phosphates, suppression of the release of creatine kinase and the accumulation of tissue calcium during reoxygenation. Both 83 microM phentolamine and 45 microM amosulalol a significant prolongation of the effective refractory period of rabbit isolated atria. 4. Lower concentrations of phentolamine (16 microM) and amosulalol) (9 microM), which are sufficient to exert an alpha-adrenoceptor blocking action, did not elicit an appreciable effect on the post-hypoxic recovery of cardiac contractile force. 5. These results suggest that phentolamine and amosulalol are capable of protecting the myocardium from hypoxia-induced derangements in cardiac function and metabolism. This effect is probably attributable to their membrane stabilizing effect, rather than to their alpha-adrenoceptor blocking action.     

Protective action of YM-12617, an alpha 1-adrenoceptor antagonist, on the hypoxic and reoxygenated myocardium

The present study was designed to determine whether the 1-form of YM-12617, which was developed recently as an alpha 1-adrenoceptor blocker, is capable of protecting the myocardium from hypoxia-induced disturbances of cardiac function and metabolism. Isolated rabbit hearts were perfused for 25 min under hypoxic conditions in the absence or the presence of 28 microM YM-12617, followed by 45 min with oxygenated perfusion medium, and functional and metabolic changes of the heart were examined. Hypoxia induced several pathophysiological changes. Upon subsequent reoxygenation, there was less than 10% recovery of the contractile force and an approximately 40% recovery of the myocardial high-energy phosphates. Treatment with YM-12617 during the hypoxic periods resulted in approximately 90% recovery of the cardiac contractile function upon subsequent reoxygenation. Treatment with YM-12617 restored the myocardial high-energy phosphates, such as ATP and creatine phosphate, to approximately 90 and 80% of the initial value, respectively, during the subsequent reoxygenation. These results suggest that YM-12617 is capable of protecting the myocardium from hypoxia-induced disturbances of cardiac function and metabolism.     

Beneficial effect of tan-shen, an extract from the root of Salvia, on post-hypoxic recovery of cardiac contractile force

The present study was undertaken to elucidate the possible effects of tanshinone VI, one of the extracts from the root of Salvia, on post-hypoxic recovery of cardiac contractile force. For this purpose, rat hearts were perfused for 45 min under reoxygenated conditions following 20-min hypoxic perfusion, and changes in tissue high-energy phosphates and calcium contents, and release of ATP metabolites and creatine kinase were examined. Post-hypoxic recovery of cardiac contractile force was augmented when hearts were treated with 42 nM tanshinone VI during hypoxia. This beneficial recovery was accompanied by enhanced restoration of myocardial high-energy phosphates, depression of hypoxia- and reoxygenation-induced increase in tissue calcium content, and suppression of release of ATP metabolites such as adenosine, inosine and hypoxanthine from the perfused heart. The results suggest that tanshinone VI is beneficial for the recovery of cardiac contractility after a certain period of oxygen-deficiency, possibly through mechanisms involving improvement of myocardial energy production upon oxygen-replenishment and/or inhibition of calcium accumulation in the cardiac cell.     

Role of the cyclooxygenase 2-thromboxane pathway in 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced decrease in mesencephalic vein blood flow in the zebrafish embryo

Previously, we reported that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) evoked developmental toxicity required activation of aryl hydrocarbon receptor type 2 (AHR2), using zebrafish embryos. However, the downstream molecular targets of AHR2 activation are largely unknown and are the focus of the present investigation. TCDD induces cyclooxygenase 2 (COX2), a rate-limiting enzyme for prostaglandin synthesis in certain cells. In the present study, we investigated the role of the COX2-thromboxane pathway in causing a specific endpoint of TCDD developmental toxicity in the zebrafish embryo, namely, a decrease in regional blood flow in the dorsal midbrain. It was found that the TCDD-induced reduction in mesencephalic vein blood flow was markedly inhibited by selective COX2 inhibitors, NS-398 and SC-236, and by a general COX inhibitor, indomethacin, but not by a selective COX1 inhibitor, SC-560. Gene knock-down of COX2 by two different types of morpholino antisense oligonucleotides, but not by their negative homologs, also protected the zebrafish embryos from mesencephalic vein circulation failure caused by TCDD. This inhibitory effect of TCDD on regional blood flow in the dorsal midbrain was also blocked by selective antagonists of the thromboxane receptor (TP). Treatment of control zebrafish embryos with a TP agonist also caused a reduction in mesencephalic vein blood flow and it too was blocked by a TP antagonist, without any effect on trunk circulation. Finally, gene knock-down of thromboxane A synthase 1 (TBXS) with morpholinos but not by the morpholinos' negative homologs provided significant protection against TCDD-induced mesencephalic circulation failure. Taken together, these results point to a role of the prostanoid synthesis pathway via COX2-TBXS-TP in the local circulation failure induced by TCDD in the dorsal midbrain of the zebrafish embryo.     

An electron microscopic study of apoptosis in the granulosa layer of ovarian follicles in rats treated with continuous illumination

Polycystic ovarian syndrome was induced in adult rats by treatment with continuous illumination to study follicular atresia. Granulosa cells of regressing antral follicles showed characteristic features of apoptosis ultrastructurally: condensed nuclei, apoptotic bodies, and phagocytosis of the apoptotic cells and their fragments by neighboring cells. In this study, moreover, it was observed that an intact granulosa cell was able to ingest another granulosa cell of normal appearance, presumably indicating an early stage of follicular atresia. The phagocytosing granulosa cell surrounded another granulosa cell to be phagocytosed by elongated cell processes. Gap junctions developed between the phagocytic cell and the phagocytosed cell. The phagocytic cell possessed abundant free ribosomes, well-developed rough endoplasmic reticulum and mitochondria, but few lysosomes. The ingested cell was similar in appearance to the phagocytic cell. In addition, granulosa cells were also observed ingesting karyopyknotic cells and degenerative organelles, both displayed in large vacuoles filled with fragments of nuclei and organelles. These findings suggest a process of apoptosis in the granulosa cells during follicular atresia. This study was presented at the 20th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Fukuoka, September 1–3, 1988.