A bispecific antibody targeting HLA-DQ2.5-gluten peptides potently blocks gluten-specific T cells induced by gluten ingestion in patients with celiac disease.

The gluten-free diet for celiac disease (CeD) is restrictive and often fails to induce complete symptom and/or mucosal disease remission. Central to CeD pathogenesis is the gluten-specific CD4+ T cell that is restricted by HLA-DQ2.5 in over 85% of CeD patients, making HLA-DQ2.5 an attractive target for suppressing gluten-dependent immunity. Recently, a novel anti-HLA-DQ2.5 antibody that specifically recognizes the complexes of HLA-DQ2.5 and multiple gluten epitopes was developed (DONQ52). OBJECTIVE: To assess the ability of DONQ52 to inhibit CeD patient-derived T-cell responses to the most immunogenic gluten peptides that encompass immunodominant T cell epitopes. METHODS: We employed an in vivo gluten challenge model in patients with CeD that affords a quantitative readout of disease-relevant gluten-specific T-cell responses. HLA-DQ2.5+ CeD patients consumed food containing wheat, barley, or rye for 3 days with collection of blood before (D1) and 6 days after (D6) commencing the challenge. Peripheral blood mononuclear cells were isolated and assessed in an interferon (IFN)-γ enzyme-linked immunosorbent spot assay (ELISpot) testing responses to gluten peptides encompassing a series of immunodominant T cell epitopes. The inhibitory effect of DONQ52 (4 or 40 µg/mL) was assessed and compared to pan-HLA-DQ blockade (SPVL3 antibody). RESULTS: In HLA-DQ2.5+ CeD patients, DONQ52 reduced T cell responses to all wheat gluten peptides to an equivalent or more effective degree than pan-HLA-DQ antibody blockade. It reduced T cell responses to a cocktail of the most immunodominant wheat epitopes by a median of 87% (IQR 72-92). Notably, DONQ52 also substantially reduced T-cell responses to dominant barley hordein and rye secalin derived peptides. DONQ52 had no effect on T-cell responses to non-gluten antigens. CONCLUSION: DONQ52 can significantly block HLA-DQ2.5-restricted T cell responses to the most highly immunogenic gluten peptides in CeD. Our findings support in vitro data that DONQ52 displays selectivity and broad cross-reactivity against multiple gluten peptide:HLA-DQ2.5 complexes. This work provides proof-of-concept multi-specific antibody blockade has the potential to meaningfully inhibit pathogenic gluten-specific T-cell responses in CeD and supports ongoing therapeutic development.

[Effect of a comprehensive image processing on radiologists' performance in differential diagnosis of liver lesions using CT and MRI].

PURPOSE: To evaluate whether a comprehensive image processing method as CAD using CT and MRI can improve the radiologists' diagnosis performance in the differentiation of focal liver lesions. METHOD AND MATERIALS: A clinical image database used in this study consists of 14 cases of each lesion including hepatic cysts, hepatocellular carcinoma (HCC), metastatic liver cancer, and hemangioma. This technique by using MR images obtained with various imaging sequences and a series of dynamic MR and dynamic CT images is designed for the enhancement of liver lesions pixel by pixel. In this method, we make the pixel sizes of MR images the same size of CT image by using tri-linear interpolation technique. Then the 3D image registration technique based on mutual information is applied for the matching of images. The image intensity pattern with and without contrast enhancement is determined as the template for the differential detection of each lesion. Pixel-by-pixel cross-correlation coefficient is calculated for the enhancement of each lesion. The radiologists' performance in distinguishing between the liver lesion was evaluated by receiver operating characteristic analysis (ROC) with a continuous rating scale. RESULTS: In free-response ROC analysis, true positive fractions were 75%, 87%, 85%, and 86% for hepatic cysts, HCC, metastatic liver cancer and hemangioma, respectively. Furthermore, average number of false positive and false negatives per image was 3.4 and 0.3, respectively. When radiologists made differential diagnosis of the liver lesions with the images of this technique, diagnostic accuracy was statistically significantly improved compared to the diagnostic accuracy without the images of this technique. The average area under the ROC curve (Az value) improved from 0.881 to 0.964 (p=0.069) for the differential diagnosis of hepatic cysts. Furthermore, the Az value of HCC, metastatic liver cancer, and hemangioma improved from 0.951 to 0.979 (p=0.040), from 0.946 to 0.976 (p=0.226), and from 0.966 to 0.987(p=0.045), respectively. CONCLUSION: A comprehensive image processing method as CAD using CT and MRI can improve the radiologists' diagnostic performance in the differentiation of focal liver lesions. CLINICAL RELEVANCE/APPLICATION: This method improved the performance of differential detection of liver lesions from a large number of images and it would save radiologists' reading time, and thus could assist their diagnosis.

Expression of coxsackievirus and adenovirus receptor in hearts of rats with experimental autoimmune myocarditis.

The expression of coxsackievirus and adenovirus receptor (CAR) was dominant in the brains and hearts of mice until the newborn phase. There is no detailed information concerning the relation between the expression of CAR and development of hearts. It is also uncertain whether CAR is able to be induced in adult hearts after cardiac injury. We demonstrated that CAR was abundant in the hearts of newborn rats but was barely detectable in the hearts of adult rats. The expression of CAR in rat hearts with experimental autoimmune myocarditis, which was induced by immunization of purified cardiac myosin, was serially investigated. Active myocarditis was observed from day 15 after immunization. By immunohistochemistry, cardiomyocytes were strongly stained for CAR antibody from days 24 to 42. CAR mRNA was also detected from days 18 to 30 by using reverse transcription-polymerase chain reaction. In the next experiment, the induction of CAR on isolated cardiomyocytes was investigated. CAR was barely detectable in cultured cardiomyocytes by Western blot analysis after isolation. This molecule gradually appeared along with the creation of clusters and beating of cardiomyocytes. Furthermore, the induction of CAR in cultured cardiomyocytes increased after supplement with conditioned medium of rat splenocytes activated by concanavalin A. In conclusion, rat CAR is expressed strongly in the hearts of newborn rats and is suppressed in those of adult rats. The expression of CAR is enhanced during the active phase of experimental autoimmune myocarditis and is induced by inflammatory mediators. CAR may play a role in cell-to-cell contact and adhesion of cardiomyocytes.

Opposite effects on cholesterol metabolism and their mechanisms induced by dietary oleic acid and palmitic acid in hamsters.

The effects of dietary oleic acid on cholesterol metabolism were investigated and compared with those of palmitic acid in hamsters. Addition of 5% oleic acid to a 0.1% cholesterol-supplemented diet decreased plasma total cholesterol, very low density lipoprotein (VLDL) cholesterol, and low density lipoprotein (LDL) cholesterol, increased hepatic LDL receptor activity, and decreased plasma cholesteryl ester transfer protein (CETP) activity in comparison with 0.1% cholesterol alone. In contrast, addition of 5% palmitic acid to a 0.1% cholesterol-supplemented diet increased total cholesterol and LDL-cholesterol, increased plasma CETP activity, and suppressed hepatic LDL receptor activity to a greater extent than 0.1% cholesterol alone. Neither oleic acid nor palmitic acid altered hepatic microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity, but oleic acid increased hepatic microsomal cholesterol 7 alpha-hydroxylase activity. These results suggest that dietary oleic acid inhibits the increases in total, VLDL-, and LDL-cholesterol induced by dietary cholesterol by preventing both LDL receptor suppression and increased CETP activity, whereas dietary palmitic acid augments the cholesterol-induced increases in total and LDL-cholesterol by both further suppression of LDL receptor activity and further stimulation of CETP activity.

Predictors of disease course in patients with acute myocarditis.

BACKGROUND: Clinical manifestations of acute myocarditis, with distinct onset, vary from asymptomatic to fatal. The predictors of the course of the disease in patients with acute myocarditis at initial presentation have not yet been established. In this study, we examined the predictive values of various parameters in the disease course of patients with myocarditis. METHODS AND RESULTS: Twenty-one consecutive patients who had been diagnosed as having acute myocarditis by histological examinations were analyzed. The patients with myocarditis were divided into the survival group (n=13) and the fatal group (n=8). We examined the parameters of the clinical state, hemodynamic variables, required therapies, biochemical laboratory data, and cytokines. The control groups were composed of 23 patients with old myocardial infarction and 20 healthy volunteers. The fatal group had lower blood pressure and higher pulmonary capillary wedge pressure compared with those values in the survival group. Mechanical ventilation support was more frequently required in the fatal group. Serum levels of soluble Fas (sFas) and soluble Fas ligand (sFasL) were significantly higher in the myocarditis group than in the 2 control groups. Furthermore, levels were significantly higher in the fatal group than in the survival group for sFas (13.93+/-4.77 versus 3.77+/-0.52 ng/mL, respectively; P:<0.001) and sFasL (611.4+/-127.7 versus 269.5+/-37.3 pg/mL, respectively; P:<0.05). Other clinical states, hemodynamic variables, required therapies, and biochemical laboratory parameters were not different between the 2 groups. CONCLUSIONS: Elevation of sFas and sFasL levels at initial presentation appear to be a good serological marker to predict the prognosis of acute myocarditis.

Oxidized low-density lipoprotein is associated with apoptosis of vascular smooth muscle cells in human atherosclerotic plaques.

BACKGROUND: Cytotoxic oxidized LDL (oxLDL) has been shown to promote apoptosis in cultured vascular smooth muscle cells (VSMCs). We investigated the localization of oxLDL and its association with apoptosis and the expression of apoptosis-related proteins in early and advanced atherosclerotic lesions. METHODS AND RESULTS: Atherosclerotic plaques (n=23) from patients undergoing aortic, carotid, or femoral arterial surgery were studied. In early lesions, oxLDL was located predominantly in the superficial intima and in the media just beneath the internal elastic lamina. Medial VSMCs staining positive for oxLDL showed expression of BAX, a proapoptotic protein of the BCL-2 family. Apoptosis, as detected by DNA in situ terminal deoxynucleotidyl transferase end-labeling (TUNEL), was not present in these early lesions. In advanced plaques, areas of the intima positive for oxLDL showed lower alpha-smooth muscle actin immunoreactivity (P<0.01) and higher BAX immunoreactivity (P<0.05). Furthermore, these areas showed an increased number of apoptotic VSMCs (P<0.01). Western blot analysis revealed that oxLDL increases BAX expression in cultured human coronary VSMCs. CONCLUSIONS: We conclude that in early atherosclerotic lesions, oxLDL-positive VSMCs express BAX, which increases the susceptibility of these cells to undergo apoptosis. This could be important in our understanding of the transition of early lesions into advanced atherosclerotic plaques, which are characterized by regions of cell death. In advanced plaques, oxLDL-positive areas of the intima show higher BAX immunoreactivity and TUNEL-positive VSMCs, and this may contribute to plaque instability and rupture.

Gastric adenocarcinoma of fundic gland type (chief cell predominant type) with unique endoscopic appearance curatively treated by endoscopic submucosal resection.

Gastric adenocarcinoma of fundic gland type [chief cell predominant type; (GA-FD-CCP)] is a rare gastric cancer variant arising from non-atrophic mucosa without Helicobacter pylori infection in the upper third portion of the stomach. GA-FD-CCP originates deep in the mucosal layer; hence, endoscopic lesion detection is often difficult at an early stage because of a minimal change in the mucosal surface. Here we present a 66-year-old man with an early stage of GA-FD-CCP showing characteristic endoscopic features. Esophagogastroduodenoscopy demonstrated a flat, slightly reddish area with black pigment dispersion and irregular micro-surface structure at the gastric fornix. The tumor was resected by endoscopic submucosal dissection and was pathologically diagnosed as GA-FD-CCP. Prussian blue staining revealed that the black pigment was a hemosiderin deposition. We reported a rare case of successfully treated GA-FD-CCP with black pigmentation that aided in early lesion detection.

Immunohistochemical analysis on the rejection of xenogeneic brain grafts.

The central nervous system (CNS) of mammals has long been thought of as an immunologically privileged site. However, this concept is now changing because the rejection of histo-incompatible neural grafts has been frequently observed in the CNS. In neural transplantation used as therapy for some human neurodegenerative diseases, it is important to determine which factors are related to brain graft rejection. In this study, we examined immunological reactions in brains that had received isogeneic (rat to rat) and xenogeneic (mouse to rat) neural transplants. In the immunohistochemical analysis, antibodies against T cell receptor αß (R73), macrophage and microglia (0X42), MHC class II antigens (0X6), CD4 (W3/25), CD8 (0X8), NK cell (3.2.3), B cell (RLN-9D3), T cell receptor (TCR) Vß8.2 (R78), TCR Vß8.5 (B73) and TCR Vßl0 (G101) were used. At the early stage of both isogeneic and xenogeneic transplantation, a nonspecific inflammatory reaction characterized by macrophage infiltration was observed along the needle track which was produced by the grafting procedure. From the day 7 stage onwards, the non-specific inflammatory reaction was replaced by the specific immune reactions of T cell infiltration, neovascularization and necrosis of xenogeneic grafts. Marked T cell infiltration was detected in the lesions, whereas NK and B cells were not. Quantitative analysis of T cell subsets revealed that both CD4+ and CD8+ T cells were found in the xenogeneic transplants. Microglia became activated and strongly expressed MHC class II antigens at the time of graft rejection. Isogeneic transplants, in contrast, showed no histological characteristics of rejection, and numerous dopaminergic neurons with several neurites were observed in the grafts. Based on these findings, we concluded that T cells are the principal effectors in the rejection of xenogeneic neural grafts, and that activated microglia may have some role in presenting antigens to the infiltrating T cells during the rejection process.

Pentaerythritol tetranicotinate (niceritrol) decreases plasma lipoprotein(a) levels.

We determined the most effective dosage of pentaerythritol tetranicotinate (niceritrol) to reduce plasma lipoprotein(a) [Lp(a)] levels in 44 Japanese patients (16 men and 28 women; mean age, 59.2 +/- 10.8 years) with hyperlipidemia types IIa, IIb, and IV. Patients received oral niceritrol at a dosage of 750 mg (3 tablets)/d for 8 weeks, followed by 1,500 mg (6 tablets)/d for 8 weeks. Administration of niceritrol 750 mg/d for 8 weeks decreased total and low-density lipoprotein (LDL) cholesterol in patients with type IIa hyperlipidemia and decreased triglycerides in patients with type IV hyperlipidemia, but did not affect Lp(a). However, niceritrol 1,500 mg/d for 8 weeks decreased Lp(a) in patients with initial Lp(a) levels greater than 30 mg/dL in addition to decreasing total and LDL cholesterol and triglycerides. These results suggest that the effective dosage of niceritrol to reduce the serum Lp(a) concentration in Japanese hyperlipidemic patients with a high Lp(a) level (> or = 30 mg/dL) is greater than 1,500 mg/d.

Effect of erythropoietin treatment on blood pressure and intracellular cation concentrations in maintenance hemodialysis patients.

To assess the effect of recombinant human erythropoietin (EPO) on the factors regulating blood pressure (BP), we determined the hemoglobin level (Hgb), blood viscosity (BV), plasma renin activity (PRA), plasma concentrations of aldosterone (PAC), adrenaline (Ad), noradrenaline (NAd), and atrial natriuretic peptide (ANP), and serum and intracellular concentrations of cations before and after 3 months of EPO treatment (40 units/kg/week of EPO intravenously after each hemodialysis session) in 11 patients undergoing maintenance hemodialysis. Intracellular sodium concentration ([Na+]i) was measured using erythrocytes with flame photometry. EPO treatment was associated with significant increases in Hgb (7.1 +/- 1.4 to 8.4 +/- 1.8 g/dl, p<0.01), mean BP (103 +/- 11.4 to 116 +/- 19.9 mmHg, p<0.01), [Na+]i (4.99 +/- 0.78 to 6.22 +/- 0.96 mmol/l, p<0.01) and BV (1.39 +/- 0.14 to 1.53 +/- 0.18 c.p., p<0.05), but no significant alteration in PRA, PAC, Ad, NAd, ANP, or in the serum concentration of Na+, K+, and Ca2+. The changes in mean BP (deltaMBP) were significantly correlated with delta[Na+]i (R=0.676, p=0.022) and deltaBV (R=0.668, p=0.034), but not with deltaHgb. By multiple regression analysis, delta[Na+]q and deltaBV independently contributed to deltaMBP; deltaMBP=2.27 X delta[Na+]i+32.2 X deltaBV +3.37 (R=0.695). These data suggest that intracellular sodium accumulation as well as increased blood viscosity may be independently involved in the blood pressure elevation after EPO treatment in patients under maintenance hemodialysis. We found no evidence supporting a role of circulating hormonal factors, such as the renin-angiotensin system, adrenaline, or ANP, in the change in blood pressure.

Analysis of neuronal death in the central nervous system using a new apoptosis model.

To examine in detail neural apoptosis in the central nervous system (CNS) for the establishment of new therapies, we have developed an experimental in vitro model of neuronal death and analyzed the mechanism of apoptosis. Septal nuclei were dissected from embryonic brains (E16) of Wistar rats and cultured in chemically defined medium. Highly enriched neurons were obtained from the cultures at 4 days. Exposure to heat shock (43.0 degrees C, 60 min) between 24 and 36 h later, resulted in the death of approximately 70% of cells. Morphologically, dying neurons showed disruption of neurites, nuclear condensation, multiple nuclear fragments, condensation of cytoplasm and multiple cellular fragmentation. Agarose electrophoresis of chromosomal DNA revealed a typical ladder-pattern of fragmentation. Following heat treatment, incubation at 37 degrees C was necessary to detect DNA fragmentation. Quantitative analysis by in situ terminal deoxynucleotidyl transferase assay, revealed that the percentage of apoptotic cells markedly increased 8 h after heat treatment, and continued to gradually increase up until 30 h. Neuronal death and DNA fragmentation were prevented by inhibiting RNA and protein synthesis. Cell death and the DNA cleavage were also inhibited by cultivation in calcium-free medium. The addition of homogenous basic fibroblast growth factor to the medium markedly enhanced cell survival under these pathogenic conditions. These results suggest that neural cell death after mild heat treatment has apoptotic characteristics and may be useful for analyzing the mechanism of apoptosis. The clinical application of drugs acting against molecular components and as well as neurotrophic factors, may in the future prevent apoptosis in neural disease.

Effects of polydextrose on serum lipids, lipoproteins, and apolipoproteins in healthy subjects.

The subjects were 61 healthy volunteers who received 15 gm of polydextrose daily for two months. A significant increase in the incidence of soft feces and diarrhea and in the volume of feces was reported during polydextrose treatment. These had returned to normal one month after treatment. Serum levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol did not change during treatment. Levels of apolipoprotein (apo) A-I and A-II were significantly lowered at one month and high-density lipoprotein cholesterol (HDL-C) and apo A-I were significantly decreased at two months; these returned to normal after treatment. Levels of HDL2-C decreased and HDL3-C levels increased significantly during treatment. The results indicate that polydextrose selectively affected the metabolism of HDL and its major proteins, apo A-I and A-II.

New method for assaying free and total cholesterol in cultured cells by high-pressure liquid chromatography.

We developed a simple, sensitive and accurate method for assaying cellular free and total cholesterol by monitoring 4-cholesten-3-one, a conversion product of the cholesterol oxidase-catalyzed oxidation of the free cholesterol that has a strong chromophoric alpha, beta-unsaturated ketone at 240 nm, using a high-pressure liquid chromatographic system. This method measured picomole quantities of free and total cholesterol and precisely determined their concentrations in cells (10(4) range) in culture using 7 beta-hydroxycholesterol as an internal standard.

Effects of Chinese herbal drugs on serum lipids, lipoproteins and apolipoproteins in mild to moderate essential hypertensive patients.

We investigated the effects of the traditional Chinese herbal drugs, Dai-saiko-to (D) and Saiko-ka-ryukotsuboreito (S) on blood pressure, pulse rates, serum lipids, lipoproteins and apolipoproteins in 30 patients with mild to moderate hypertension in an open, randomised trial. After the drug treatment, BP remained unchanged, but pulse rates declined significantly after 3 months in the S treated group. Serum total cholesterol and triglyceride values did not change, but high density lipoprotein-cholesterol increased significantly (P < 0.05) in both groups. Apo-AI (P < 0.1 in S group) and apo-AII (P < 0.05 in D group, P < 0.1 in S group) tended to increase 3 months after treatment. These data indicate that both of these traditional Chinese medicines have a preferential effect on lipid metabolism with little antihypertensive action.

Clinical features of renal tubular dysplasia, a new hereditary disease in Japanese Black cattle.

A new hereditary disease characterised by renal failure, poor growth and long hooves in Japanese Black cattle (wagyu) has been recognised in a region of central Japan since 1990. The number of calves affected has increased gradually, with the incidence reaching 17 of 485 (3.51 per cent) in 1995. Almost all the calves were slightly undersized at birth, and repeatedly had diarrhoea during the neonatal period. They began to show signs of growth retardation with proportional body and elongation of the hooves from about two to five months of age, but they had an almost normal or only slightly decreased appetite. The concentrations of urea nitrogen, creatinine and inorganic phosphorus in serum were high, and the affected calves excreted diluted urine frequently. Among 25 cases, the urine of 21 contained occult blood, 24 contained protein and two contained glucose. In 29 calves observed for 30 to 130 days, the course of the disease varied; in 21 of them it remained unchanged, six became gradually worse and two became severely debilitated and died. The disease was diagnosed as renal tubular dysplasia by histopathological examination.

Pathological changes of renal tubular dysplasia in Japanese black cattle.

Pathological studies were conducted on 91 Japanese Black cattle with a hereditary disease which induced growth retardation, long hooves and renal failure. In calves one to two months old, no gross abnormalities were observed in the kidneys, but microscopical examinations revealed immature epithelia which were arranged irregularly and not attached to the basement membranes in some proximal tubules. In animals three to 36 months old, the kidneys had shrunk perceptibly and had grey-white radial streaks; microscopically they showed severe interstitial fibrosis with round-cell infiltration in the outer zone of the medulla and cortex, and reductions in the numbers of glomeruli and tubules. In the fibrotic areas there were immature epithelia with an irregular arrangement, and the basement membrane of the tubules was thickened. It was concluded that renal tubular dysplasia was the primary lesion of the disease, and that interstitial fibrosis and reductions in the numbers of nephrons were secondary lesions.

PACS linked to EPR.

We developed a new PACS linked to Electronic Patient Record system (EPR). It was a hospital-wide PACS storing all the radiological examinations. The images and reports were linked on EPR. The concept of navigation servers and segment servers was introduced for prefetchig and quick displaying. After the start of operation, increasing retrieval indicated its effectiveness on practical work in spite of remaining delivery of radiographs.

Erythrocyte sodium cannot predict hypotensive effects of calcium channel blockers.

Antihypertensive efficacy of the calcium channel blocker, nilvadipine, was investigated in 21 patients with essential hypertension in relation to the intracellular sodium concentration ([Na]i) in erythrocytes and clinical variables, such as age, body weight, pretreatment blood pressure and plasma renin activity. Dihydropyridine nilvadipine reduced mean blood pressure from 120 +/- 6 to 106 +/- 8 mmHg (p < 0.01). The hypotensive effect of nilvadipine was positively correlated with age (r = 0.67, p < 0.01) and inversely correlated with plasma renin activity (r = -0.62, p < 0.01), but was not correlated with erythrocyte [Na]i or any other indices. Erythrocyte [Na]i was decreased with nilvadipine treatment (8.27 +/- 1.69 to 7.97 +/- 1.49 mM, p < 0.01). However, no link was found between the change in [Na]i and the hypotensive effect of the drug. In conclusion, the antihypertensive efficacy of nilvadipine was predictable by age and renin status, but not by erythrocyte [Na]i. A significant role of reduction of [Na]i in the hypotensive effect of the calcium channel blockers was not detected.