Infiltrating eosinophils and eotaxin: their association with idiopathic eosinophilic esophagitis.

BACKGROUND: Idiopathic eosinophilic esophagitis (IEE) is a very rare disease characterized by thickening and eosinophil infiltration of the esophagus. The most potent chemotactic factor for eosinophils is eotaxin, and its pathophysiologic significance in IEE needs to be elucidated. OBJECTIVE: To study the association between eotaxin and IEE. METHODS: We examined eotaxin expression in the esophagus of an IEE patient in comparison to controls by immunohistochemistry using a monoclonal antibody for human eotaxin. We also measured the free eotaxin level and the total (free and bound-form) eotaxin level in blood by enzyme-linked immunosorbent assay before and after the initiation of steroid therapy. RESULTS: Most of the infiltrating eosinophils in the affected esophageal tissue showed immunohistochemical staining with anti-eotaxin antibody. In blood samples, the free eotaxin level was slightly elevated before treatment, whereas the total eotaxin level was within the normal range. Unexpectedly, the total eotaxin level increased dramatically after the initiation of steroid therapy, whereas the increase of free eotaxin was modest. CONCLUSION: Infiltrating eosinophils that express eotaxin and the changes of blood eotaxin levels during steroid therapy suggest that eotaxin may be associated with IEE.     

Increase of chemokine interferon-inducible protein-10 (IP-10) in the serum of patients with autoimmune liver diseases and increase of its mRNA expression in hepatocytes

To clarify the role of IP-10 in autoimmune liver diseases, we studied the serum levels of IP-10 in 14 patients with autoimmune hepatitis (AIH), 23 patients with primary biliary cirrhosis (PBC), and 65 patients with chronic viral hepatitis (20 type B and 45 type C). The hepatic expression of IP-10 mRNA and the correlation between the serum levels of IP-10 and clinical parameters were also evaluated. In addition to 20 healthy controls, 16 rheumatoid arthritis (RA) patients were included as an extrahepatic inflammatory disease. The serum level of IP-10 was significantly (P < 0.02) higher in patients with AIH, PBC, and chronic hepatitis B and C than in healthy controls, and it was significantly correlated (P < 0.05) with the serum levels of aspartate aminotransferase and alanine aminotransferase in patients with AIH, PBC, and chronic hepatitis B and C. The serum level of IP-10 was not elevated in RA patients. After successful treatment of AIH and chronic hepatitis C, the serum level of IP-10 decreased to the same level as in healthy volunteers. As we previously showed in cases with chronic hepatitis B or C, in situ hybridization in both AIH and PBC cases demonstrated the expression of IP-10 mRNA in hepatocytes around focal or lobular necrosis surrounded by infiltrating mononuclear cells, whereas IP-10 mRNA was not expressed in areas around the damaged bile ducts in PBC cases. The present results suggest that IP-10 is specifically produced by hepatocytes in inflammatory areas irrespective of the aetiology of hepatitis, and that IP-10 may help to recruit T cells to the hepatic lesions in autoimmune liver diseases as well as in chronic viral hepatitis.     

Four cases of wheat-dependent exercise-induced anaphylaxis with negative gluten cap-rast score]

BACKGROUND: Either omega-5 gliadin or high molecular weight glutenin is known to be a major allergen in wheat-dependent exercise-induced anaphylaxis (WDEIA). It is generally considered that gluten specific IgE score is more reliable than that of wheat specific IgE score for the diagnosis of WDEIA. Our aim was to verify the significance of gluten specific IgE in the diagnosis of WDEIA. METHODS: We evaluated the result of gluten CAP-RAST score and omega-5 gliadin specific IgE score on four WDEIA patients who visited our hospital during the years 2004 and 2005, whose diagnosis were onfirmed by prick tests, immunoblot tests and provocation tests. RESULTS: Contrary to our expectations, all four patients showed negative gluten CAP-RAST scores, however all patient's omega-5 gliadin specific IgE scores were positive. CONCLUSION: Our results suggest that gluten specific CAP-RAST score is unreliable in the diagnosis of WDEIA. On the other hand omega-5 gliadin specific IgE score is possibly a better candidate as a diagnostic tool for WDEIA.     

A Comparative Analysis of the Murine Thymic Microenvironment in Normal,Autoimmune, and Immunodeficiency States

It is widely accepted that the thymic microenvironment regulates normal thymopoiesis through a highly coordinated and complex series of cellular and cytokine interactions. A direct corollary of this is that abnormalities within the microenvironment could be of etiologic significance in T-cell-based diseases. Our laboratory has developed a large panel of monoclonal antibodies (mAbs) that react specifically with epithelial or nonepithelial markers in the thymus. We have taken advantage of these reagents to characterize the thymic microenvironment of several genetic strains of mice, including BALB/cJ, C57BL/6J, NZB/BlnJ, SM/J, NOD/Ltz, NOD/Ltz-scid/sz, C57BL/6J-Hcph m^e/Hcph m^e, and ALY/NscJcl-aly/aly mice, and littermate control animals. We report herein that control mice, including strains of several backgrounds, have a very consistent phenotypic profile with this panel of monoclonal antibodies, including reactivity with thymic epithelial cells in the cortex, the medulla and the corticomedullary junction, and the extracellular matrix. In contrast, the disease-prone strains studied have unique, abnormal staining of thymic cortex and medulla at both the structural and cellular levels. These phenotypic data suggest that abnormalities in interactions between developing thymocytes and stromal cells characterize disease-prone mice.     

Age-dependent change in activities of lysosomal enzymes in rat brain

The age-dependent change in activities of seven lysosomal enzymes (cathepsin D, β-glucuronidase, acid phosphatase, acid/alkaline DNases and acid/alkaline RNases) was studied in four brain regions (cerebrum, hippocampus, pons and cerebellum) of Wistar rats. The activity of cathepsin D was significantly increased with aging in the four regions. The age-dependent change in activities of acid and alkaline DNases showed the characteristic regional difference, and the ratio of acid to alkaline DNases was increased with aging in all regions. Acid RNase showed the lowest activity in 18-month-old rats, and alkaline RNase activity was decreased with aging. The activity of β-glucuronidase was higher in 2-month-old rats in all of the regions studied. Acid phophatase showed no significant age-dependent change except in pons. The study demonstrated that all of the lysosomal enzyme activities do not change in parallel with aging, and that the age-dependent change showed the characteristic regional difference.     

Structural basis for distinct roles of Lys63- and Lys48-linked polyubiquitin chains

Ubiquitination, a modification in which single or multiple ubiquitin molecules are attached to a protein, serves as a signalling function that controls a wide variety of cellular processes. To date, two major forms of polyubiquitin chain have been functionally characterized, in which the isopeptide bond linkages involve Lys48 or Lys63. Lys48-linked polyubiquitin tagging is mostly used to target proteins for degradation by the proteasome, whereas Lys63-linked polyubiquitination has been linked to numerous cellular events that do not rely on degradative signalling via the proteasome. Apparently linkage-specific conformations of polyubiquitin chains are important for these cellular functions, but the structural bases distinguishing Lys48- and Lys63-linked chains remain elusive. Here, we report NMR and small-angle X-ray scattering (SAXS) studies on the intersubunit interfaces and conformations of Lys63- and Lys48-linked di- and tetraubiquitin chains. Our results indicate that, in marked contrast to Lys48-linked chains, Lys63-linked chains are elongated molecules with no stable non-covalent intersubunit interfaces and thus adopt a radically different conformation from that of Lys48-linked chains.     

Stimulatory effect of vasoactive intestinal polypeptide on catecholamine secretion from isolated guinea pig adrenal chromaffin cells

The stimulatory effect of vasoactive intestinal polypeptide (VIP) on catecholamine (CA) secretion from isolated guinea pig adrenal chromaffin cell was studied. VIP (1-10 microM) induced dose-dependent CA secretion, which was slow and continued for at least 30 min. This VIP-induced CA secretion was dependent on the presence of Ca2+ in the medium, but no significant increase in Ca2+ uptake by the cells was observed during their stimulation with VIP. Studies on the intracellular free Ca2+ level ([Ca2+]i) using fura-2 showed that acetylcholine and muscarine induced a marked increase in the [Ca2+]i, but that VIP induced only a slight increase. Thus VIP may induce CA secretion by increasing the sensitivity of the secretion of CA to Ca2+.     

Role of apoptosis controlled by cytochrome c released from mitochondria for luteal function in human granulosa cells

PROBLEM: The aim of this study was to investigate the relationship between apoptosis by the mitochondrial pathway and luteal function in human granulosa cells. METHOD OF STUDY: Granulosa cells were obtained by ultrasound-guided follicular aspiration from patients undergoing in vitro fertilization and embryo transfer. After the addition of RU486, cells were stained with a mitochondria-specific fluorescent dye, MitoTracker Red CM x Ros. Using flow cytometry and National Institute of Health image, the mitochondrial fluorescent area was measured. After staining with Hoechst 33258 dye, the number of apoptotic bodies per 1000 cells were counted at random on photomicrographs. Homogenates were used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis using antibodies against cytochrome c or caspase-3. RESULTS: The incidence of apoptotic bodies increased and the mitochondrial membrane potential decreased time dependently. The opposite effect was observed dose dependently with RU486 treatment. Western blot analysis showed increased cytochrome c expression, after treatment with 1-2 microg/mL of RU486 which then decreased with 5-10 microg/mL of RU486. Caspase-3 expression increased dose dependently with RU486. CONCLUSIONS: These results suggest that the activation of caspase-3 caused by cytochrome c released from mitochondria plays an important role in apoptosis-related luteal function in human granulosa cells.     

Tissue-engineered vascular autograft: inferior vena cava replacement in a dog model

Tissue-engineered vascular autografts (TEVAs) were made by seeding 4-6 x 10(6) of mixed cells obtained from femoral veins of mongrel dogs onto tube-shaped biodegradable polymer scaffolds composed of a polyglycolid acid (PGA) nonwoven fabric sheet and a copolymer of L-lactide and caprolactone (n = 4). After 7 days, the inferior vena cavas (IVCs) of the same dogs were replaced with TEVAs. After 3, 4, 5, and 6 months, angiographies were performed, and the dogs were sacrificed. The implanted TEVAs were examined both grossly and immunohistologically. The implanted TEVAs showed no evidence of stenosis or dilatation. No thrombus was found inside the TEVAs, even without any anticoagulation therapy. Remnants of the polymer scaffolds were not observed in all specimens, and the overall gross appearance similar to that of native IVCs. Immunohistological staining revealed the presence of factor VIII positive nucleated cells at the luminal surface of the TEVAs. In addition, lesions were observed where alpha-smooth muscle actin and desmin positive cells existed. Implanted TEVAs contained a sufficient amount of extracellular matrix, and showed neither occlusion nor aneurysmal formation. In addition, endothelial cells were found to line the luminal surface of each TEVA. These results strongly suggest that "ideal" venous grafts with antithrombogenicity can be produced.