Identification of a naturally processed HLA-Cw7-binding peptide that cross-reacts with HLA-A24-restricted ovarian cancer-specific CTLs.

Here, we describe an human leukocyte antigen (HLA)-A*24:02-restricted cytotoxic T-lymphocyte (CTL) clone, 1G3, established from naïve CD8(+) T-lymphocytes obtained from a healthy donor stimulated with HLA-modified TOV21G, an ovarian cancer cell line. The 1G3 clone responds not only to ovarian cancer cells in the context of HLA-A*24:02 but also to allogeneic HLA-Cw*07:02 molecules through cross-reactive T-cell receptor recognition. Expression screening using a complementary DNA library constructed from TOV21G messenger RNA revealed that this alloreactivity was mediated through the nine-mer peptide VRTPYTMSY, derived from RNA-binding motif protein 4. To our knowledge, this study presents the first example of the allorecognition of an HLA-Cw molecule by HLA-A-restricted T-cells, thereby revealing a naturally processed epitope peptide. These findings provide the structural bases for the allorecognition of human T-cells. In addition, this study suggests that unexpected alloresponses occur in certain HLA combinations, and further study is needed to understand the mechanisms of alloreactivity for better prediction of alloresponses in clinical settings.

Clinical associations and risk factors for bleeding from colonic angiectasia: a case-controlled study.

AIM: A case-controlled study was performed to investigate the association of colonic angiectasia with other conditions and to identify risk factors for bleeding. METHOD: Information was collected from all patients who underwent colonoscopy at our hospital between January 2008 and December 2010. Data on 90 individuals with angiectasia [58 men; median age 69 (26-92) years] were compared with those of 180 individuals without angiectasia, matched for gender and age. RESULTS: Multivariate analysis showed that occult gastrointestinal bleeding [odds ratio (OR) 2.523; 95% confidence interval (CI) 1.238-5.142], liver cirrhosis (OR 13.195; 95% CI 3.502-49.711), chronic renal failure (OR 6.796; 95% CI 1.598-28.904) and valvular heart disease (OR 6.425; 95% CI 1.028-40.165) were identified as significant predictors of the presence of colonic angiectasia. Eight patients were diagnosed with bleeding from angiectasia. Cardiovascular disease (OR 22.047; 95% CI 1.063-457.345) and multiple angiectasias (P-value 0.0019) were identified as significant risk factors for active bleeding. Medication and a large size were not associated with an increased risk of bleeding. CONCLUSION: The presence of colonic angiectasia was associated with valvular heart disease, liver cirrhosis and chronic renal failure. Valvular heart disease and multiple lesions increased the risk of bleeding.

Body cavity fluid can induce epithelial and mesothelial differentiation from CD34 positive peripheral blood stem cells in vitro.

OBJECTIVE: Primary culture of CD34 positive stem cells collected from human peripheral blood was performed with and without supplementation with concentrated ascitic fluid; morphological and immunocytochemical pictures of cultured cells were taken chronologically and compared. METHODS: CD34-positive stem cells collected from peripheral blood were cultured for 1, 24 and 48 hours. Concentrated ascitic fluid was added to the plates for the 24-and 48-hour cultures. For immunocytochemical studies, CD34, AE1/AE3, Ber-Ep4 (EA), EMA, EGFR, CD31, CA125 and D2-40 monoclonal antibodies were used. RESULTS: After culture, small round cells with naked nuclei began to enlarge and to exhibit various changes in the cytoplasm and nucleus. Supplementation with concentrated body cavity fluid enhanced these changes. CD34-positive cells with small round cell features were detected 1 hour after culture and these had no epithelial or mesothelial markers. After 24 hours, CD34-positive cells had disappeared and cells weakly positive for EGFR, EMA, CA125 and D2-40 were detected. Cells with strong and moderate positive reactions for EGFR, AE1/AE3, EA, EMA, D2-40 and CA125 were detected after 48 hours. Supplementation with concentrated body cavity fluid increased the intensity and number of positive cells for these markers compared with the control group. The positive reaction, not only for the epithelial markers such as EGFR and AE1/AE3, but also for mesothelial markers such as CA125 and D2-40, was found to be increased in small numbers of cells in direct proportion to the duration of the primary culture of the peripheral blood cells. CD31, characteristically expressed in endothelial cells, was negative in the cultured cells. CONCLUSION: Supplementation of peripheral blood CD34-positive stem cells with body cavity fluid in vitro enhanced their differentiation toward cells of an epithelial or mesothelial phenotype, concomitant with loss of immunoreactivity for CD34. It is assumed that the routine cytological observation of cells obtained from body cavity fluid might cause possible cytomorphological and immunophenotypical changes due to the action of the growth factors contained in the body cavity fluid.

Cold stress changes the concanavalin A-positive glycosylation pattern of proteins expressed in the basal parts of rice leaf sheaths.

Post-translational modifications such as glycosylation are important for changing the properties and functions of proteins. To analyze the importance of glycosylation during cold stress in rice, a proteomics approach was used. Proteins extracted from the basal part of rice leaf sheaths were separated by two-dimensional polyacrylamide gel electrophoresis, and subjected to lectin blot analysis using concanavalin A. From a total of 250 detected proteins, 22 reacted with the lectin, suggesting that they were N-glycosylated proteins. To determine how N-glycosylation of these proteins is affected by cold stress, rice seedlings were incubated at 5 degrees C for 48 h, and proteins extracted from the basal parts of leaf sheaths were analyzed by the lectin blot assay. Cold stress changed the reactivity toward the lectin for 12 of the 22 glycoproteins. The identity of the 12 proteins was determined by protein sequencing and mass spectrometry with the majority of these glycoproteins being categorized as involved in energy production. Furthermore, calreticulin, one of the 12 glycoproteins, was also phosphorylated as a result of cold stress. These results indicate that cold stress of the basal parts of rice leaf sheaths changes the glycosylation and phosphorylation profiles of calreticulin, a key protein that regulates the quality control of other proteins.

[Investigation of the local heating caused by a closed conducting loop at clinical MR imaging: Phantom study].

PURPOSE: Several reports have suggested that unusual thermal injuries in magnetic resonance (MR) imaging have occurred due to a closed conducting loop formed accidentally in a part of the patient's body. In this study, we investigated the relationship between the increases in temperature and several parameter settings for MR imaging by use of a human body-equivalent phantom. METHOD AND MATERIALS: A standard clinical 1.5T MR system (SIGNA HORIZON; GE) and a pelvic phased-array coil were used. The human body-equivalent phantom (agar, 0.9% saline, antiseptic) simulated a part of the pelvis and both femurs in a patient. A closed conducting loop could be reproduced when two ends of femurs contacted each other at a point, so that we could measure the temperature changes without and with a closed conducting loop. The temperature of the phantom was measured at the contact point of a closed conducting loop and the center of phantom by use of an optical fiber thermometer which was immune to the influences of radiofrequency (RF) and magnetic and electronic fields. We tested two imaging sequences of spin echo (SE) and fast spin echo (FSE) with 60 minutes of scanning time. In addition to the standard imaging sequences we measured temperature changes without the RF irradiation or gradient magnetic fields. The average temperature changes were recorded from five measurements which were repeated at intervals of more than one day. RESULTS: When the closed conducting loop was reproduced, the temperatures at the contact point significantly increased (p<0.001) compared with the temperatures at the center of phantom. The temperature changes at 60 minutes of scanning time were 7.0 and 8.1 degrees C by use of the SE and FSE, respectively. There were no significant temperature changes when the imaging was performed without the RF irradiation. CONCLUSION: Our result obtained by use of a human body-equivalent phantom demonstrated that local heating, which can lead to thermal injuries accidentally, could occur when a closed conducting loop was formed in part of the patient body. CLINICAL RELEVANCE/APPLICATION: Radiologists should be more careful about local heating which can occur in patients during clinical MR imaging by a closed conducting loop.

Monocyte and Basophil Counts as Predictors of Neutrophil Count Recovery in Patients with Thiamazole-Induced Agranulocytosis.

Purpose: Thiamazole (MMI) is frequently used for the treatment of Graves' disease, but it occasionally induces agranulocytosis at the beginning of the treatment. To date, the predictive factors of recovery from MMI-induced agranulocytosis remain unclear. The primary aim of this study was to evaluate the predictive factor of the recovery time from MMI-induced agranulocytosis. Method: This was a retrospective cohort study performed in a university hospital and a thyroid hospital. We included 27 Japanese patients with Graves' disease with MMI-induced agranulocytosis diagnosed during follow-up. All patients were administrated recombinant human granulocyte colony-stimulating factor daily until they had a neutrophil count>1 000/µL, which was defined as recovery. The predictive factors associated with recovery time were estimated using multivariable regression analysis. Results: At the onset of agranulocytosis, the median administration period of MMI was 33 days, the average white blood cell count was 1 896/µL, and the median neutrophil count was 22/µL. The median recovery time was 4 days. Stepwise multivariate regression analysis identified the monocyte and basophil counts to be significant predictors of MMI-induced agranulocytosis. Conclusion: Patients with agranulocytosis and decreased monocyte and basophil counts at onset may recover late and require careful treatment.

Distribution of the ATPase inhibitor proteins of mitochondria in mammalian tissues including fibroblasts from a patient with Luft's disease.

The mitochondrial ATPase inhibitor proteins--the Pullman-Monroy inhibitor (PMI) and the Ca(2+)-binding protein (CaBI)--have a wide distribution, both being present in mitochondria of bovine heart and kidney, rat liver and brain, two mitochondrial populations of rabbit skeletal muscle, and mitochondria from human fibroblasts and the human breast cancer cell line T-47-D. The ratio of CaBI to PMI was highest in heart and skeletal muscle mitochondria. The subsarcolemmal fraction of skeletal muscle had 2.6-times as much CaBI as did the intermyofibrillar. The ratio of CaBI to PMI in the mitochondria of the other normal tissues and fibroblasts was close to 1. In contrast, mitochondria from T-47D cells had 1.5-times as much PMI as CaBI whilst mitochondria from fibroblasts from a patient with Luft's disease showed a virtual lack of PMI. The specific ATPase, ATP-synthetase and succinate dehydrogenase activities of the Luft's mitochondria were, however, in the normal range. The specific ATP synthetase activity of the T-47D cells was significantly higher than normal. We conclude that tissues like heart and skeletal muscle which experience wide fluctuations in intracellular Ca2+ have a greater need for CaBI. Why lack of PMI could lead to 'loose' coupling of oxidative phosphorylation in skeletal muscle of Luft's patients, but not in fibroblasts is discussed.

Molecular cloning and characterization of a novel human STE20-like kinase, hSLK.

We have cloned a human counterpart to a guinea pig STE20-like kinase cDNA, designated human SLK (hSLK), from a human lung carcinomatous cell line A549 cDNA library. hSLK cDNA encodes a novel 1204 amino acid serine/threonine kinase for which the kinase domain located at the N-terminus shares considerable homology to that of the STE20-like kinase family. The C-terminal domain of hSLK includes both the coiled-coil structure and four Pro/Glu/Ser/Thr-rich (PEST) sequences, but not the GTPase-binding domain (GBD) that is characteristic of the p21-activated kinase (PAK) family, polyproline consensus binding sites, or the Leu-rich domain seen in the group I germinal center kinases (GCKs). Northern blot analysis indicated that hSLK was ubiquitously expressed. hSLK overexpressed in COS-7 cells phosphorylates itself as well as myelin basic protein used as a substrate. On the other hand, hSLK cannot activate any of the three well-characterized mitogen-activated protein kinase MAPK (ERK, JNK/SAPK and p38) pathways. Moreover, hSLK kinase activity is not upregulated by constitutive active forms of GTPases (RasV12, RacV12 and Cdc42V12). These structural and functional properties indicate that hSLK should be considered to be a new member of group II GCKs.

Molecular cloning of 25-hydroxyvitamin D-3 24-hydroxylase (Cyp-24) from mouse kidney: its inducibility by vitamin D-3.

A cDNA encoding a 25-hydroxyvitamin D-3 24-hydroxylase, Cyp-24, has been isolated from mouse kidney cDNA library by hybridization screening. Mouse Cyp-24, coding for 514 amino acid residues, shared 82.1 and 94.7% amino acid identity with human and rat CYP24s, respectively. Among mouse organs examined, Cyp-24 mRNA could be detected in the kidney. When mice were treated with vitamin D-3, Cyp-24 mRNA was induced in the kidney.

Mouse cytochrome P-450 linked ferredoxin: its cDNA cloning and inducibility by dibutyryladenosine 3',5'-cyclic monophosphate and forskolin.

Two full-length cDNAs (F1-1 and F41-1) complementary to mouse kidney mRNA coding for cytochrome P-450 (P450) linked ferredoxin were isolated and completely sequenced. The coding sequences between F1-1 and F41-1 were identical. However, the 3' untranslated regions of F1-1 and F41-1 were 228 and 27 bases long due to the presence of alternative polyadenylation sites, respectively. The deduced amino acid sequence of mouse cytochrome P-450 linked ferredoxin showed 92.5, 75.0, 71.2 and 71.0% identities with those of rat, human, pig and bovine cytochrome P-450 linked ferredoxin, respectively. The cytochrome P-450 linked ferredoxin mRNA was detected in adrenal, kidney and ovary among the organs examined. The treatment of Y-1 cells with dibutyryladenosine 3',5'-cyclic monophosphate or forskolin induced the transcript of cytochrome P-450 linked ferredoxin mRNA.

cDNA cloning of mouse ferredoxin reductase from kidney.

A cDNA encoding ferredoxin reductase has been isolated from a mouse kidney cDNA library using human ferredoxin reductase cDNA as a probe. Mouse ferredoxin reductase coded for 494 amino acid residues. The mouse mature enzyme which comprises 460 amino acid residues shared 87.8-89.1% amino acid identities with the bovine and human enzyme. Northern blot analysis showed that ferredoxin reductase mRNA was expressed in the adrenal, testis and ovary and to a lesser extent in the liver and kidney. However, this mRNA in the adrenal cell line, Y-1 cell, was not induced by adenosine 3',5'-cyclic monophosphate (cAMP) in contrast with ferredoxin mRNA.

Simultaneous expression of ferredoxin, ferredoxin reductase and P450 in COS7 cells.

cDNA fragments encoding mouse ferredoxin and ferredoxin reductase were simultaneously introduced into COS7 cells by using an expression vector, pUC-SR alpha plasmid. When using the mitochondrial fraction prepared from the transfected cells, cytochrome-c reductase activity was detected. This activity was highest when 7.5 micrograms of the ferredoxin expression plasmid (pSR alpha F) and 2.5 micrograms of the ferredoxin reductase expression plasmid (pSR alpha FR) were transfected into COS7 cells. In this system, NADPH could be replaced by NADH as a cofactor for the reduction of cytochrome-c although the cytochrome-c reductase was more dependent on NADPH than NADH at a low concentration. When CYP24 expression plasmid was transfected into COS7 cells along with both pSR alpha F and pSR alpha FR, the transfected cells revealed a 3-fold higher 25-hydroxyvitamin D3-24-hydroxylase activity than COS7 cells transfected with CYP24 expression plasmid.

Brain metabolism in teenagers with attention-deficit hyperactivity disorder.

OBJECTIVES: We sought to obtain and compare values of cerebral glucose metabolism in normal minors and minors with Attention Deficit Hyperactivity Disorder (ADHD). We also sought to confirm our earlier findings of reduced brain metabolism in adults with ADHD, and to examine whether these results might be diagnostically useful. DESIGN: Case-control study. SETTING: Adolescents were recruited to National Institutes of Health Clinical Center/Research Facility through advertisement at local high schools and ADHD organizations. PATIENTS: Subjects were 10 normal adolescents and 10 adolescents with ADHD diagnosed with structured interviews using DSM-III-R criteria. MAIN OUTCOME MEASURES: Positron emission tomography and fludeoxyglucose F18 were used to study cerebral glucose metabolism in minors while they performed an auditory-attention task. RESULTS: Global or absolute measures of metabolism did not statistically differ between groups, although hyperactive girls had a 17.6% lower absolute brain metabolism than normal girls. As compared with the values for the controls, normalized glucose metabolism was significantly reduced in six of 60 specific regions of the brain, including an area of the left anterior frontal lobe (P < .05). Lower metabolism in that specific region of the left anterior frontal lobe was significantly inversely correlated with measures of symptom severity (P < .001-.009, r = -.56 to -.67). CONCLUSIONS: Global or absolute measures of metabolism using positron emission tomography and fludeoxyglucose F18 did not statistically differentiate between normal adolescents with ADHD. Positron emission tomography scans can be performed and are well tolerated by normal teenagers and teenagers with ADHD. The feasibility of normal minors participating in research involving radiation was established.

Density, proportion, and dendritic coverage of retinal ganglion cells of the common marmoset (Callithrix jacchus jacchus).

We performed a quantitative analysis of M and P cell mosaics of the common-marmoset retina. Ganglion cells were labeled retrogradely from optic nerve deposits of Biocytin. The labeling was visualized using horseradish peroxidase (HRP) histochemistry and 3-3'diaminobenzidine as chromogen. M and P cells were morphologically similar to those found in Old- and New-World primates. Measurements were performed on well-stained cells from 4 retinas of different animals. We analyzed separate mosaics for inner and outer M and P cells at increasing distances from the fovea (2.5-9 mm of eccentricity) to estimate cell density, proportion, and dendritic coverage. M cell density decreased towards the retinal periphery in all quadrants. M cell density was higher in the nasal quadrant than in other retinal regions at similar eccentricities, reaching about 740 cells/mm(2) at 2.5 mm of temporal eccentricity, and representing 8-14% of all ganglion cells. P cell density increased from peripheral to more central regions, reaching about 5540 cells/mm(2) at 2.5 mm of temporal eccentricity. P cells represented a smaller proportion of all ganglion cells in the nasal quadrant than in other quadrants, and their numbers increased towards central retinal regions. The M cell coverage factor ranged from 5 to 12 and the P cell coverage factor ranged from 1 to 3 in the nasal quadrant and from 5 to 12 in the other quadrants. These results show that central and peripheral retinal regions differ in terms of cell class proportions and dendritic coverage, and their properties do not result from simply scaling down cell density. Therefore, differences in functional properties between central and peripheral vision should take these distinct regional retinal characteristics into account.

Constipation is not associated with colonic diverticula: a multicenter study in Japan.

BACKGROUND: The association of diverticula with bowel habits is unclear. We therefore analyzed the association between diverticula and bowel habits in over 1000 Japanese individuals. METHODS: Japanese subjects who underwent total colonoscopies at seven centers in Japan from June to September 2013 were analyzed. Bowel habits were evaluated using the Gastrointestinal Symptom Rating Scale, and stool form was assessed using a part of the Bristol Scale and Rome ΙΙΙ criteria. Diverticula were diagnosed by colonoscopy with a transparent soft-short hood. KEY RESULTS: The study evaluated 1066 subjects, 648 males and 418 females (ratio, 1.55 : 1), of mean age 63.9 ± 13.0 years. After adjusting for age and sex, the presence of constipation was associated with a significantly reduced likelihood of diverticula (odds ratio [OR] = 0.70, 95% confidence interval [CI] 0.52-0.93). When assessed according to the location of diverticula, the presence of constipation was associated with a significantly decreased likelihood of left-sided (OR = 0.39, 95% CI 0.16-0.93), but not right-sided (OR = 1.10, 95% CI 0.48-2.53), diverticula. Furthermore, stool form was unrelated with the presence or absence of diverticula. CONCLUSIONS & INFERENCES: The wide-spread hypothesis that constipation was associated with colonic diverticula was not supported. Rather, we found that the absence of diverticula was associated with constipation, suggesting the need to reassess the etiology of colonic diverticula.

In vivo osteogenic capability of cultured allogeneic bone in porous hydroxyapatite: immunosuppressive and osteogenic potential of FK506 in vivo.

Fischer or ACI rat marrow cells were obtained from femoral shafts and were cultured to confluence in Eagle's minimal essential medium (EMEM) supplemented with 15% fetal bovine serum. After trypsinization, the cells were subcultured on porous hydroxyapatite (HA; Interpore 500) blocks in the presence of beta-glycerophosphate and 10 nM dexamethasone (Dex). After 2 weeks of subculture, a mineralized bone matrix with osteogenic cells developed on the HA pore surfaces. ACI or Fischer cultured bone tissue/HA constructs were implanted subcutaneously into the backs of Fischer rats and the immunosuppressant FK506 was given to the rats for 4 weeks. Implants were harvested 4 weeks and 8 weeks after insertion. At 4 weeks, the ACI constructs (allografts) showed high levels of osteogenic parameters (alkaline phosphatase [ALP] activity and osteocalcin content) and bone formation was observed together with active osteoblasts without obvious accumulation of inflammatory cells. At 8 weeks, active osteoblasts and progressive bone formation were still observed, while osteogenic parameters remained high and osteocalcin messenger RNA (mRNA) was detected. Without FK506 administration, the allografts showed neither bone formation nor osteocalcin mRNA and there were only trace levels of the osteogenic parameters. In the case of Fischer constructs (isografts), extensive bone formation was detected and all the osteogenic parameters were higher with FK506 than without FK506 at both 4 weeks and 8 weeks. These results indicate that cultured bone tissue/HA constructs possess a high osteogenic potential, even as allografts, and that FK506 not only has an immunosuppressive action, but also promotes bone formation.

Ca2+-binding properties of a unique ATPase inhibitor protein isolated from mitochondria of bovine heart and rat skeletal muscle.

Previous studies showed that Ca2+ induced monomer to active dimer interconversion of a mitochondrial ATPase inhibitor protein from bovine heart or rat skeletal muscle (Yamada, E.W., Huzel, N.J. and Dickison, J.C. (1981) J. Biol. Chem. 256, 10203-10207). Initial equilibrium dialysis measurements of Ca2+ binding showed that this unique protein possesses three binding sites of high affinity with a maximum of one mol of Ca2+ bound/mol of protein monomer. Magnesium (1 mM) did not affect the first association constant but increased the second and third by about 1.2 and 1.5 fold, respectively. That the apparent association constants varied with concentration of protein monomer was in agreement with the self-associating nature of the protein. Scatchard plots at three concentrations of protein intersected at a molar ratio of about 0.5 (Ca2+/monomer). Ka1 and Ka2 values of 4.2 microM and 12.1 microM, respectively, were estimated by extra-polation of apparent constants to infinite dilution of protein. Ka3 (51.3 microM) was estimated by extrapolation of double reciprocal plots of apparent constants versus protein concentration to infinite levels of protein. A model for Ca2+ binding by this self-associating protein is described. Trifluoperazine had no effect on the activity of the inhibitor protein from either tissue.

Interleukin-6 (IL-6) inhibits thyroid function in the presence of soluble IL-6 receptor in cultured human thyroid follicles.

Interleukin-6 (IL-6), a pleiotropic cytokine, is postulated to be involved in the pathogenesis of sick euthyroid syndrome, although the direct in vitro effects of IL-6 on human thyroid function are controversial. Because IL-6 signal can be transduced when the complex of IL-6 and soluble IL-6 receptor (sIL-6R) binds to gp 130, an IL-6 signal transducer, we studied the effects of IL-6 and sIL-6R on thyroid function, using human thyroid follicles obtained from patients with Graves' disease. IL-6 alone had no inhibitory effect on TSH-induced thyroid function (125I incorporation and organic 125I release), even at supraphysiological concentrations. However, in the presence of physiological concentrations of sIL-6R (100 ng/ml), IL-6 inhibited thyroid function dose dependently and completely, accompanied with the decreased ratio of 125I-T3/125I-T4 not only in the thyroid follicles but also in the culture medium. Thyroid follicles did not secrete sIL-6R but produced IL-6 constitutively. Consistent with these findings, sIL-6R inhibited thyroid function slightly at high concentrations. Furthermore, RT-PCR analyses revealed that human thyroid follicles expressed the messenger RNAs for IL-6 and gp130 but scarcely messenger RNA for IL-6R. These in vitro findings suggest that IL-6 alone hardly affects thyroid function in thyroid follicles in which IL-6R gene is scarcely expressed. However, because sIL-6R is present abundantly in serum, IL-6 in vivo would be capable of inhibiting the synthesis and release of T4 and, to a greater extent, T3 from the thyroid gland. These in vitro findings are at least partly related to the development of sick euthyroid syndrome.

Exposure to ambient light in patients with winter seasonal affective disorder.

In a study of the quantitative relationship between ambient light and depression in winter seasonal affective disorder, 13 outpatients and 13 normal comparison subjects each wore a light monitor for 1 week. The patients and normal subjects showed similar light exposure profiles; among the patients, severity of depression was inversely related to photoperiod, and there was a trend toward a correlation between greater severity of depression and later time of onset of morning light exposure. These findings suggest that vulnerability to short photoperiods may be related to depression in winter seasonal affective disorder.