A new tumor-rejection antigen recognized by cytotoxic T lymphocytes infiltrating into a lung adenocarcinoma.

Lung cancer is the most commonly occurring malignancy worldwide and one of the few that continues to show an increasing incidence. To understand the molecular basis of host immunity against lung cancer, we investigated tumor antigens recognized by HLA-A24-restricted CTLs established from T cells infiltrating into lung adenocarcinoma and report a new gene coding for antigens recognized by the CTLs. The mRNA of this gene was expressed at different levels in all of the malignant cells tested (high in adenocarcinomas and gliomas and low in esophageal cancers and malignant hematological disease). It was also expressed at the different levels in each of a panel of normal tissues (high in the thymus, low in peripheral blood mononuclear cells, and lowest in the stomach, small intestine, and skeletal muscle). This gene encodes a Mr 60,000 nuclear protein with 414 deduced amino acids. The three peptides at positions 158-165, 170-179, and 188-196 were recognized by the CTLs. One peptide at position 188-196 had the ability to induce HLA-A24-restricted and tumor-specific CTLs in peripheral blood mononuclear cells of lung cancer patients. These CTLs, however, did not lyse HLA-A24+ PHA-activated T cells in which the mRNA of this gene was highly expressed, even in the presence of excess amounts of a corresponding peptide in culture. These results suggest that this gene product and peptide could be applicable to specific immunotherapy of lung cancer patients.

Identification of a new endoplasmic reticulum-resident protein recognized by HLA-A24-restricted tumor-infiltrating lymphocytes of lung cancer.

To help clarify the molecular basis of tumor immunology in lung cancer, we have investigated antigens recognized by HLA-A24-restricted CTLs established from T cells infiltrating into lung adenocarcinoma and report a new gene encoding tumor epitopes recognized by the CTLs. This gene was located on chromosome 4q31.22 and encoded an unreported endoplasmic reticulum-resident protein with 412 deduced amino acids. This protein had a molecular mass of 46 kDa and was expressed in the majority of malignant cells and tissues tested, with the exception of T-cell leukemia cells, but was not expressed in a panel of normal cells and tissues, except in those of the testis, placenta, and fetal liver. Two peptides at positions 13-20 and 75-84 were recognized by the CTLs and had an ability to induce HLA-A24-restricted and tumor-specific CTLs in peripheral blood mononuclear cells of lung cancer patients. Thus, these peptides might be appropriate molecules for use in the specific immunotherapy of HLA-A24+ patients with lung and other cancers.

Complex molecular genetic abnormalities involving three or more genetic mutations are important prognostic factors for acute myeloid leukemia.

We conducted a comprehensive analysis of 28 recurrently mutated genes in acute myeloid leukemia (AML) in 271 patients with de novo AML. Co-mutations were frequently detected in the intermediate cytogenetic risk group, at an average of 2.76 co-mutations per patient. When assessing the prognostic impact of these co-mutations in the intermediate cytogenetic risk group, overall survival (OS) was found to be significantly shorter (P=0.0006) and cumulative incidence of relapse (CIR) significantly higher (P=0.0052) in patients with complex molecular genetic abnormalities (CMGAs) involving three or more mutations. This trend was marked even among patients aged ⩽65 years who were also FLT3-ITD (FMS-like tyrosine kinase 3 internal tandem duplications)-negative (OS: P=0.0010; CIR: P=0.1800). Moreover, the multivariate analysis revealed that CMGA positivity was an independent prognostic factor associated with OS (P=0.0007). In stratification based on FLT3-ITD and CEBPA status and 'simplified analysis of co-mutations' using seven genes that featured frequently in CMGAs, CMGA positivity retained its prognostic value in transplantation-aged patients of the intermediate cytogenetic risk group (OS: P=0.0002. CIR: P<0.0001). In conclusion, CMGAs in AML were found to be strong independent adverse prognostic factors and simplified co-mutation analysis to have clinical usefulness and applicability.

Three stages of differentiation in mouse megakaryocyte progenitor cells (CFU-Meg).

Mouse megakaryocyte colonies developed in a fibrin clot culture system were morphologically classified into three types: immature homogeneous (IH), heterogeneous (H), and mature homogeneous (MH) colonies. The colony size (number of megakaryocytes per colony), the ploidy distribution of megakaryocytes in colonies, and the ratios of their progenitors (megakaryocyte colony-forming units, CFU-Meg) in the S-phase of the cell cycle were compared among the three types. Also, morphological changes in colonies were examined in time sequence. The mode of the number of megakaryocytes per colony on day 6 of culture was greater than 64, between 17 and 32, and between 4 and 8, in IH, H, and MH colonies, respectively. The mean DNA content of megakaryocytes was 3.1N, 4.2N, and 7.2N on day 6 of incubation, in IH, H, and MH colonies, respectively, these values being significantly different from each other (p less than 0.001). The mean proportion of CFU-Meg synthesizing DNA was 21.9%, 26.0%, and 48.6% for CFU-Meg forming IH, H, and MH colonies, respectively (p less than 0.01; IH- versus MH-CFU-Meg and H- versus MH-CFU-Meg). Successive observation of the colony morphology from days 5 to 11 of culture suggested a transformation of the colony types from IH colony to MH colony through H colony. These observations indicate that the three types of megakaryocyte colonies classified on the basis of their morphological features reflect various stages of differentiation of mouse CFU-Meg.

Autoradiographic analysis on second-messenger systems and local cerebral blood flow in ischemic gerbil brain.

Alterations of the second-messenger systems, adenylate cyclase (AC) and protein kinase C (PKC), and local cerebral blood flow (lCBF) were evaluated during experimental cerebral ischemia in gerbils employing a quantitative autoradiographic method, which permitted these three parameters to be measured in the same brain. Ischemia was induced by occlusion of the right common carotid artery for 6 h. Animals attaining more than 5 in their ischemic scores were utilized for further experiments. At the end of ischemia, lCBF was measured by the [14C]iodoantipyrine method. The AC and PKC activities were estimated by the autoradiographic technique developed in our laboratory using [3H]forskolin (FK) and [3H]phorbol-12,13-dibutyrate (PDBu), respectively. The lCBF fell below 10 ml/100 g/min in most cerebral regions on the ligated side. The greatest reduction in FK binding was noted in the olfactory tubercle, caudate-putamen, and globus pallidus, followed by the hippocampus and cerebral cortices. The FK binding tended to be low at lCBF less than 20 ml/100 g/min in the cerebral cortices. However, the PDBu binding was relatively well preserved in each cerebral structure, and no significant correlation between lCBF and PDBu binding was noted in the cerebral cortices. The AC system may thus be vulnerable to ischemic insult over extensive brain regions, while the PKC system may be relatively resistant to ischemia.

Effects of lesioning of the substantia innominata on autoregulation of local cerebral blood flow in rats.

Recently, accumulated data have suggested that the nucleus basalis magnocellularis, i.e., the substantia innominata (SI), may represent the primary source of central cholinergic innervation in the rat cortical vasculature. We therefore examined the effects of unilateral lesion of the SI on the autoregulation of local CBF (lCBF) during induced hypotension in rats. Male Wistar rats were divided into three groups. The animals of groups 1 and 2 received an injection of 5 micrograms of ibotenate into the right SI stereotaxically. At 7 days after the injection, the lCBF was measured by the [14C]iodoantipyrine technique in the awake state. Group 1 was used as the normotensive group (MABP = 113.1 +/- 12.2 mm Hg). Group 2 formed the hypotensive group, and the lCBF was measured during hypotension (MABP = 80.0 +/- 5.5 mm Hg) induced by hemorrhage. Group 3, the sham-operated normotensive group, received vehicle injection into the right SI at 7 days prior to the lCBF measurement. In group 1, lCBF was significantly lower in the frontal, parietal, temporal, and striate cortices on the lesioned side compared to that on the contralateral side. In group 2, lCBF was significantly decreased in the cortices on the lesioned side, but there was no significant difference in magnitude of the lCBF reduction between groups 1 and 2. Group 3 exhibited no hemispheric asymmetries in lCBF. These findings suggest that the SI exerts an influence on cortical lCBF, but does not play a role in the autoregulation of lCBF during hypotension.

Flow threshold for reduction of cyclic AMP binding in the hippocampus CA1 and other brain regions during stroke development in gerbils.

The flow threshold for alterations of the in vitro [3H]cyclic AMP (cAMP) binding, an indicator of the total amount of particulate cAMP-dependent protein kinase, was evaluated in the gerbil brain after 30 min, 2 h, and 6 h of unilateral common carotid artery occlusion, respectively. The autoradiographic method developed in our laboratory enabled us to measure the [3H]cAMP binding and local CBF in each region of the same brain. The ischemic flow thresholds for reduction of the cAMP binding in the hippocampus CA1 were 18, 34, and 49 ml 100 g-1 min-1 after 30-min, 2-h, and 6-h ischemia, respectively. These values were higher than those in other regions such as the hippocampus CA, and temporal cerebral cortex in each duration of ischemia. These findings indicate that (a) the ischemic flow threshold for perturbation of the cAMP system may be higher in the hippocampus CA1 than in other brain regions, suggesting that the hippocampus CA1 could be especially vulnerable to acute ischemic stress; and (b) the level of the aforementioned threshold may increase progressively during the time course of ischemia in particular regions such as the hippocampus CA1 and CA3, suggesting that the duration of ischemia exerts a definite influence on the viability of the ischemic neuronal cells in these regions.

Noradrenergic nervous activity in migraine.

The autonomic nervous function in patients with migraine was studied during headache-free intervals. The following observations were made: (1) a decrease in overshoot in Valsalva's maneuver; (2) orthostatic hypotension; (3) low levels of plasma norepinephrine in the steady state; (4) failure in elevation of the plasma norepinephrine level after head-up tilting; (5) dilatation of the pupils after instillation in the eye of 1.25% epinephrine; and (6) a long recovery time in tests by bolus injection of 0.1 microgram of norepinephrine bitartrate per kilogram. The above findings suggest that patients with migraine show sympathetic hypofunction together with denervation hypersensitivity of the iris and the arteries, and that a defective noradrenergic nervous system may play a role in the pathogenesis of migraine.

Alteration of inositol 1,4,5-trisphosphate receptor after six-hour hemispheric ischemia in the gerbil brain.

In order to evaluate the influence of cerebral ischemia on the inositol 1,4,5-trisphosphate receptor, the alterations of inositol 1,4,5-trisphosphate receptor binding sites and local cerebral blood flow were examined 6 h after occlusion of the right common carotid artery in the gerbil brain. The autoradiographic method developed in our laboratory enabled us to measure both parameters within the same brain. Animals attaining ischemic scores of more than 5, as assessed 1 h after occlusion, were utilized. The local cerebral blood flow was measured 6 h after occlusion by the [14C]iodoantipyrine method. The inositol 1,4,5-trisphosphate binding sites were evaluated in vitro using [3H]inositol 1,4,5-trisphosphate as a specific ligand. The local cerebral blood flow fell below 15 ml/100 g per min in most of the cerebral regions on the occluded side. In contrast, a significant reduction in inositol 1,4,5-trisphosphate binding sites was noted only in the hippocampus CA1 on the occluded side. Inositol 1,4,5-trisphosphate binding tended to decrease when the values of local cerebral blood flow were below 20 ml/100 g per min in this region. On the other hand, the inositol 1,4,5-trisphosphate receptor immunoreactivity in the brain examined with a monoclonal antibody against inositol 1,4,5-trisphosphate receptor protein did not reveal any differences between the ischemia and sham groups on both sides, suggesting that the inositol 1,4,5-trisphosphate receptors may not undergo significant morphological degradation. These findings indicate that the suppression of inositol 1,4,5-trisphosphate binding in the hippocampus CA1 may be attributable to a regionally specific perturbation of the inositol 1,4,5-trisphosphate metabolism in this region.(ABSTRACT TRUNCATED AT 250 WORDS)

Variable expression on lung cancer cell lines of HLA-A2-binding MAGE-3 peptide recognized by cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) specific for HLA-A2-binding MAGE-3 peptide (FLWGPRALV) were generated by repetitive stimulation of PBMC with the peptide in the presence of EBV-transformed B blasts and IL-2. Using these CTL, we investigated the expression of the HLA-A2-binding MAGE-3 peptide on lung cancer cell lines. Of 14 cell lines investigated, 1-87, PC-9, OU-LC-KI, 11-18 and LK87 were derived from HLA-A2 positive patients. But cytofluorometry analysis showed that 1-87, PC-9 and OU-LC-KI, but not 11-18 or LK87 expressed the HLA-A2 antigen. All five cell lines expressed MAGE-3 gene mRNA. Twelve of thirteen CTL lines from two HLA-A2 positive donors showed no cytotoxicity against any of the 14 lung cancer cell lines. CTL line TI-1 showed cytotoxicity against 1-87 but not against any of the other cell lines. Treatment of 1-87 with IFN-gamma greatly augmented the cytotoxicity of TI-1 and induced it in the other 12 CTL lines, confirming the expression of the peptide on 1-87. No cytotoxicity was induced by IFN-gamma treatment of PC-9 or OU-LC-KI. However, PC-9 and OU-LC-KI pulsed with the peptide were killed efficiently by all of the CTL lines, suggesting no expression of the peptide on those cells. A low level of cytotoxicity was induced on 11-18 but not LK87 by IFN-gamma treatment, although expression of the HLA-A2 antigen was not observed by cytofluorometry. These findings showed that expression of the HLA-A2-binding MAGE-3 peptide recognized by CTL was variable on lung cancer cell lines.

Inhibition of aspartate chemotaxis of Escherichia coli by site-directed sulfhydryl modification of the receptor.

Thr-154 of the chemoreceptor Tar in Escherichia coli is important for aspartate sensing. Taking advantage of the fact that Tar has no Cys residues, we have further investigated the role of Thr-154 by replacing it with Cys in order to subject it to SH modification. Tar-T154C retained the abilities of aspartate sensing and repellent sensing. However, when cells with Tar-T154C were treated with an SH-modifying reagent, 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), they specifically lost the ability to sense aspartate; the ability was restored by the reducing reagent, 1,4-dithiothreitol. DTNB showed no detectable effect on the function of wild-type Tar or serine-replaced Tar, Tar-T154S. Thus, DTNB modifies Cys-154 of Tar-T154C in intact cells and causes a specific defect in the aspartate-sensing ability of Tar. The addition of 1 mM or higher concentrations of aspartate resulted in protection of Cys-154 from the modification; serine had no effect in this regard. These results that not only is Thr-154 important for aspartate sensing but also, it may be located at the actual aspartate-binding site.

Inhibition of nitric oxide synthesis impairs autoregulation of local cerebral blood flow in the rat.

The effect of intravenous administration of NG-monomethyl-L-arginine (L-NMMA, 30 mg kg-1), a specific inhibitor of nitric oxide (NO) synthesis, on the autoregulation of local cerebral blood flow (LCBF) was examined in the rat using the [14C]iodoantipyrine autoradiographic method. LCBF was significantly lower in various superficial regions such as the cerebral cortices and cerebellar cortex and in several deep brain regions in animals with haemorrhagic hypotension induced after L-NMMA infusion (the L-NMMA + HEM group) compared with animals without haemorrhagic hypotension after L-NMMA infusion (the L-NMMA group). The present findings suggest that NO synthesis may play a crucial role in the autoregulation of LCBF in response to a reduction in blood pressure in the cerebral cortices, cerebellar cortex and several deep brain regions.

Microvascular damage in the cortex of cat brain from middle cerebral artery occlusion and reperfusion.

Oxygen-dependent quenching of phosphorescence provides an extraordinarily powerful method for examining the effects of ischemia/hypoxia on the cortex of the brain. Video camera technology has permitted imaging, through a window in the skull, of the phosphorescence of an oxygen probe, Pd meso-tetra-(4-carboxyphenyl)-porphine, bound to albumin in the blood of anesthetized animals. Images of the phosphorescence taken at different times after the flash of excitation light were used to generate high-resolution two-dimensional maps of the oxygen pressure. These maps show that cortical oxygenation is spatially heterogeneous and that there is dynamic time-dependent modulation of regional oxygen pressures. When the middle cerebral artery was occluded, the region for which it supplied blood became hypoxic, the severity of the hypoxia varying among animals. Release after 60 min of occlusion resulted in a rapid rise of the oxygen pressure to above-normal levels followed by onset of a delayed period of hypoxia. This period is characterized by generally low tissue oxygen pressures with local regions of more severe hypoxia. The delayed period of hypoxia appears to result from damage to the microvasculature, and this microvascular damage is proposed to be an important determinant of the extent of irreversible brain damage.

Enhanced maximal binding capacity (Bmax) of second messenger ligand in the acute phase of cerebral ischemia--direct visualization by digital image analysis.

Autoradiographic visualization of the Bmax (maximal binding capacity) and Kd (dissociation constant) of [3H]phorbol 12,13-dibutyrate (PDBu) and [3H]forskolin (FK) was performed after 30-min unilateral carotid artery occlusion in the gerbil brain. These parameters and the local cerebral blood flow (CBF) were measured at the level of the caudate-putamen in the same brain using a digital image processing technique developed in our laboratory. The local CBF was measured at the end of the experiment. [3H]PDBu and [3H]FK were utilized as specific ligands to assess the activities of protein kinase C (PKC) and adenylate cyclase (AC), respectively. The local CBF on the occluded side was severely reduced and ranged from 0.2 to 9.0 ml/100 g/min, whereas the local CBF on the non-occluded side exhibited a moderate reduction except in the midline regions. The Bmax values of PDBu and FK were significantly increased not only on the occluded side but also on the non-occluded side in the ischemia group as compared to the corresponding values in the sham group. In contrast, the Kd value of each ligand remained unchanged in the ischemia group. These findings suggest that both the adenylate cyclase and protein kinase C systems may be significantly and diffusely activated in the initial stage of brain ischemia. Thus, severe hemispheric cerebral ischemia in the acute phase may induce severe perturbation of the second messenger systems in extensive bilateral regions.

Immobilization stress induces alterations of second-messenger systems in the gerbil brain.

The effects of immobilization stress on the cerebral second messenger (adenylate cyclase and phosphoinositide) were investigated autoradiographically in mongolian gerbils. After 10 min (10-min stress group, n = 7), or after 6 h (6-h stress group, n = 7) of fixation on a flat board while supine, in vitro autoradiography was performed using [3H]forskolin (3H-FK) and [3H]phorbol-12,13-dibutyrate (3H-PDBu) as specific ligands to identify the distribution of adenylate cyclase and protein kinase C, respectively. In another group of 7 gerbils (control group), the same autoradiographic procedure was performed immediately after the animals were removed from the cage. In the 10-min stress group, FK binding was significantly decreased in the hypothalamus and amygdala, but significantly increased in the basal ganglia including the caudate-putamen and globus pallidus. FK binding in the 6-h stress group tended to increase throughout the brain, rising significantly in the basal ganglia. PDBu binding in either stress group did not change significantly compared to the control group in any region except the hippocampal CA3 region of the 6-h stress group. Under immobilization stress, the adenylate cyclase system may undergo time-dependent and regionally specific changes, while the phosphoinositide system remains relatively stable.

A multicenter prospective study on the treatment of chronic idiopathic thrombocytopenic purpura.

A multicenter prospective study on the treatment of chronic idiopathic thrombocytopenic purpura (ITP) was conducted by the Idiopathic Disorders of Hematopoietic Organs Research Committee (IDHORC), the Ministry of Health and Welfare of Japan. The aim of the study was to establish an improved therapeutic guide for chronic ITP. Of the 247 eligible patients 175 have been followed up to the present time, 16 patients have died, and 56 have been lost to follow-up. The median follow-up time was 55 months (range: 1 to 91 months). Of the 206 patients treated with corticosteroids, 13.1% achieved sustained complete remission. Splenectomy produced remissions in 52.5% of 72 patients evaluated 12 months after operation. Eighty-five patients were treated with immunosuppressive agents, but the response rates were low and the effect was transient in most cases. In the patients followed up without any specific treatment, the mean platelet counts showed slight improvement. Of the 16 patients who died during the observation period, only one died of bleeding; the remaining 15 died of causes unrelated to thrombocytopenia. It is noteworthy that five patients who were treated with corticosteroids or immunosuppressive agents died of infection.

Carotid and aortic screening for coronary artery bypass grafting.

BACKGROUND: To identify risk factors for preexisting carotid and aortic disease in coronary artery bypass grafting (CABG), preoperative parameters were analyzed. METHODS: Three-hundred eight consecutive patients undergoing elective isolated CABG were investigated through preoperative duplex scanning of the carotid artery, computed tomography of the chest, and intraoperative ultrasonography of the ascending aorta. RESULTS: Prevalence of carotid stenosis and ascending aortic atherosclerosis was 14.3% (44 of 308) and 30.2% (93 of 308), respectively. Multivariate analysis indicated that significant independent risk factors for carotid stenosis were atherosclerosis of the ascending aorta (p = 0.028, odds ratio [OR] = 2.16), peripheral vascular disease (p = 0.008, OR = 4.08), and history of stroke (p = 0.0004, OR = 3.73). Significant independent risk factors for ascending aortic atherosclerosis were peripheral vascular disease (p = 0.029, OR = 3.05), age older than 60 years (p = 0.009, OR = 2.94), and carotid stenosis (p = 0.018, OR = 2.27). Modifications on the operative procedure for aortic atherosclerosis were carried out in 49 patients. Overall hospital mortality and morbidity for stroke were 0.97% and 0.65%, respectively. CONCLUSIONS: Prevalence of carotid and aortic disease was not low among candidates for CABG. Carotid and aortic screening may help to modify the operative strategy to reduce morbidity of stroke.

Percutaneous vertebral angioplasty before coronary artery bypass grafting.

We report a case of a 63-year-old male with three-vessel coronary heart disease complicated by stenosis of the bilateral vertebral arteries. Triple coronary bypass grafting, using arterial conduits, was successfully performed after percutaneous balloon angioplasty of the left vertebral artery. Precedent angioplasty of a stenotic vertebral artery is safe and protects the brain from ischemia during extracorporeal circulation.

Inhibition of nitric oxide synthesis induces a significant reduction in local cerebral blood flow in the rat.

The effect of intravenous administration of NG-monomethyl-L-arginine (L-NMMA, 30 mg/kg), a specific inhibitor of nitric oxide synthesis, on local cerebral blood flow (lCBF) was examined in the rat using the [14C]iodoantipyrine autoradiographic method. L-NMMA induced a statistically significant reduction in lCBF in the cerebral cortices as well as in various deep structures of the brain. This reduction in lCBF was accompanied by a clear increase in mean arterial blood pressure, suggesting that the cerebral resistance vessels constricted significantly beyond the autoregulatory response following L-NMMA administration. These findings indicate that the basal cerebral circulation may be closely related to nitric oxide production.

Dual effect of naloxone on blood platelet aggregation and cerebral blood flow in gerbils.

The effect of naloxone on blood platelet aggregation and cerebral blood flow in gerbils was studied. Administration of naloxone in dose 1 mg/kg to intact gerbils resulted in a marked increase in platelet aggregability accompanied by 27% reduction in cerebral blood flow. Focal cerebral ischemic injury significantly enhanced platelet aggregatory response and treatment with naloxone was without any additional effect on platelet aggregation. Cerebral blood flow in ischemic hemisphere, however, increased following naloxone injection by 46%. In vitro naloxone in millimolar concentrations inhibited platelet aggregation in a dose-dependent way. Apparent decrease in fluorescence of platelet membranes tagged with fluorescence probe due to naloxone suggests conformational changes in platelet membrane as a primary mechanism for the antiaggregatory effect of naloxone in vitro.