Study of CYP2D6 gene in children with autoimmune hepatitis and P450 IID6 autoantibodies.

Cytochrome P450 IID6 is an autoantigen recognized by the sera of children affected with a subtype of autoimmune hepatitis. It was hypothesized that a mutation in the CYP2D6 gene could explain the autoimmune response in these patients. To examine this question, genomic DNA from peripheral lymphocytes (n = 9) and liver (n = 1) of 10 patients with anti-LKM-1 antibody was analysed by Southern blot for genetic association studies between a particular CYP2D6 haplotype and autoimmune hepatitis. In addition, a region of CYP2D6, from the same genomic DNA, was amplified by polymerase chain reaction (PCR) and digested by BstNI, in a search for the most prevalent 29B mutation, described in subjects who do not express the P450 IID6. Total RNA and proteins, prepared from the liver of an anti-LKM-1+ patient, were analysed by Northern and Western (immunoblot) blots respectively. Our results do not reveal any major structural change in the DNA of this patient at the CYP2D6 locus that could explain their autoimmune response. Corroborating this observation, no changes were noted either in P450 IID6 mRNA size or in the corresponding protein. However, these data do not exclude the possibility of subtle changes in the protein due to point mutations in critical regions that might trigger an autoimmune response.     

Morphology and Thermal Properties of MCPA6/ABS by in situ Polymerization of ε‐Caprolactam

Summary: In this work, blends of monomer casting polyamide 6 (MCPA6) and acrylonitrile‐butadiene‐styrene (ABS) were successfully prepared by in situ polymerization via the application of ε‐caprolactam as a reactive solvent. The morphology and thermal properties of MCPA6/ABS were investigated by means of wide angle X‐ray diffraction (WAXD), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM), respectively. The domain sizes of the ABS phase in MCPA6/ABS blends were much finer than those in corresponding polyamide 6 (PA6)/ABS blends prepared by simple melt blending. With an increased amount of ABS in MCPA6, the melt enthalpy (ΔH_f), the rate of crystallization (T_c) and the degree of crystallinity (X_c(DSC)) of MCPA6 in MCPA6/ABS blends were all decreased. The degree of supercooling (ΔT_d) showed a contrary trend. However, the melting temperatures of these blends were almost unchanged. All the results could be attributed to in situ polymerization and the hydrolysis reaction of ABS that occurred during the polymerization process. Furthermore, WAXD results showed that only α‐form crystals existed in the MCPA6/ABS blends, despite the ABS content and heat treatment.

SEM micrograph of the fractured surface of an MCPA6/ABS blend with an ABS content of 20 wt.‐% (×10 000).     

The intra-cortical trajectory of the coeruleo-cortical projection in the rat: A tangentially organized cortical afferent

Cortical and sub-cortical lesions in the rat were used to analyze the intracortical trajectory of the noradrenergic axons, which were visualized by aldehyde-induced catecholamine histofluorescence and by immunohistochemistry using an antibody directed against rat dopamine-β-hydroxylase. Following subcortical lesions there is a slowly progressive reduction in the density of cortical noradrenergic axons, indicating that they undergo asynchronous anterograde degeneration. By 2 weeks after transection of the dorsal noradrenergic bundle, no dopamine β-hydroxylase-immunoreactive fibers are detectable in the ipsilateral cortex. Neither transection of the cingulum bundle, nor parasagittal incisions through the dorsal cortex lateral to the cingulum, diminished the noradrenergic innervation of medial or dorso-lateral cortex. A cortical lesion medial to the cingulum bundle markedly reduced the density of noradrenergic fibers in cingulate cortex caudal to the lesion, but did not affect the innervation of dorso-lateral cortex. In contrast, dorso-lateral frontal incisions and decortication (frontal lobotomy) produced a marked ipsilateral decrease in the noradrenergic fiber density throughout the remaining dorso-lateral cortex, while sparing the innervation of cingulate and infra-rhinal cortex.These results demonstrate that the dorso-lateral cortex is innervated by noradrenergic fibers in the medial forebrain bundle that reach the frontal pole, turn dorsally over the anterior portion of the forceps minor and continue caudally within the deep layers of frontal and dorso-lateral cortex, supplying the noradrenergic innervation throughout their trajectory. The medial cortex is innervated by a separate group of noradrenergic fibers that ascend through the septum, curve over the genu of the corpus callosum, and run caudally in the supracallosal stria.The present results show that the cingulum bundle is not a major intra-cortical noradrenergic pathway and does not provide branches that contribute significantly to the innervation of dorsal or lateral cortex. Thus the medial and lateral cortex can be selectively and differentially denervated of noradrenergic fibers and a coarse topographic order exists in the noradrenergic innervation of cortex. Since noradrenergic fibers travel long distances within the cortical grey matter, a small lesion of frontal cortex can have far-reaching effects on the innervation of distant, more caudal regions of cortex. The coeruleocortical projection has properties that differ from those of the best characterized cortical afferents and may be a useful model for the study of other ascending monoamine systems. The tangential, intracortical trajectory of the noradrenergic fibers would confer upon the coeruleo-cortical system the capacity to modulate neuronal activity simultaneously through a vast expanse of neocortex. A formulation of cortical organization is presented which integrates the tangential organization of the coeruleo-cortical projection with the concept of columnar organization of cortex.     

Morphine-6beta-glucuronide modulates the expression of inducible nitric oxide synthase

The immunomodulatory effects of morphine are well established; however, suprisingly little is known about the immunomodulatory properties of the major metabolites of morphine. The present study tests the hypothesis that expression of inducible nitric oxide synthase (iNOS) is modulated by the administration of the morphine metabolite, morphine-6-glucuronide. The initial study using rats shows that morphine-6-glucuronide administration (0, 1.0, 3.163, 10 mg/kg s.c.) results in a pronounced reduction in lipopolysaccharide (LPS)-induced expression of iNOS (inducible nitricoxide synthease) in spleen, lung, and liver tissue as measured by western blotting. Morphine-6-glucuronide also produces a reduction in the level of plasma nitrite/nitrate, the more stable end-product of nitric oxide degradation. In a subsequent study, administration of the opioid receptor antagonist, naltrexone (0.1 mg/kg) prior to the injection of morphine-6-glucuronide (10 mg/kg) blocks the morphine-6-glucuronide induced reduction of iNOS expression and plasma nitrite/nitrite levels indicating that the effect is mediated via the opioid-receptor. This study provides the first evidence that morphine-6-glucuronide alters the expression of iNOS.     

A mammary gland adenomyoepithelioma in a C57BL/6 mouse

Mammary gland adenomyoepitheliomas are benign complex mammary gland tumors composed of neoplastic cells of epithelial and myoepithelial origins, described in many species (humans, dogs, cats, rats) and rarely in mice. We report here an adenomyoepithelioma in a C57BL/6 female mouse. Histologically, tubes and cords formed by neoplastic epithelial cells were separated by bundles of neoplastic myoepithelial cells in a clear and partially mucinous matrix. The tumor displayed characteristics of a benign neoplastic proliferation with a compressive growth pattern, and moderate cellular pleomorphism and mitotic index. At immunohistochemistry, the epithelial cells were strongly cytokeratin positive; the myoepithelial cells were weakly cytokeratin positive and strongly smooth muscle actin positive. This is to our knowledge, the first report of a mammary gland adenomyoepithelioma in a C57BL/6 mouse.     

Acute-phase protein synthesis in human hepatoma cells: differential regulation of serum amyloid A (SAA) and haptoglobin by interleukin-1 and interleukin-6.

Interleukin-6 (IL-6, BSF-2 or IFN-beta 2) is thought to be the major regulator of the acute-phase protein response that follows tissue injury and inflammation, with interleukin-1 (IL-1), tumour necrosis factor and more recently, LIF or HSF III, slightly stimulatory on only certain acute phase proteins. The synthesis of the major acute-phase protein SAA, originally described as being synthesized in response to IL-1, has been claimed recently to be mainly under IL-6 regulation. Our results show that in the human hepatoma cell line HuH-7, IL-1 is the major stimulating cytokine increasing SAA synthesis by a factor in excess of 100-fold. We also show that under most conditions interleukin-6 and tumour necrosis factor stimulate additively in combination with IL-1. Isoelectric focusing has demonstrated that SAA1 and SAA2 alpha are expressed but not SAA2 beta. The HuH-7 cell line is IL-6 responsive since haptoglobin is stimulated mainly by IL-6.     

Acidic ATP activates lymphocyte outwardly rectifying chloride channels via a novel pathway

Using whole-cell patch-clamp techniques we found that ATP activated an outwardly rectifying current in Daudi human B lymphoma cells under acidic conditions. The substitution of Cl^– for gluconate^– shifted the reversal potential, while Cl^– channel blockers, 4,4-diisothiocyanostibene-2,2-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC), blocked the current, indicating that ATP induces this current by activating the outwardly rectifying chloride channel (ORCC). The effect of ATP on ORCC was mimicked by ADP, but not by other P2 receptor agonists such as ATPS (a poorly hydrolyzable analog of ATP), 2,3-O-benzoyl-4-benzoyl-ATP (BzATP), and UTP. The ATP-induced ORCC current was completely blocked by 100 M suramin (a P2 receptor antagonist), and was partially blocked by 100 M pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid tetrasodium (PPADS), which is another P2 receptor antagonist. Neither inactivation of G proteins nor elimination of extracellular Ca²⁺ affected the ATP-induced current, indicating that G protein-coupled P2Y receptors and Ca²⁺-permeable P2X receptors are not involved. Based on the pharmacological profile and the fact that acidic conditions are required for ATP to activate the ORCC, we suggest that acidic ATP activates the lymphocyte ORCC via a novel pathway, which is not associated with any previously described purinergic receptors.     

Idiotypic characterization of antibody-induced antibody responses

Anti-idiotypic antisera were produced in syngeneic (C57BL/6) mice against a monoclonal anti-Dextran B512 (Dex) antibody (38-13). In radioimmunoassays, anti-idiotypic antibodies were shown to react with the homologous idiotype, while failing to recognize another monoclonal anti-Dex antibody, independently derived from C57BL/6 mice (D-16). Plaque inhibition tests confirmed the specificity of the anti-idiotypic antibodies and revealed that the 38-13 idiotype is expressed by about half of all anti-Dex antibodies produced in C57BL/6, but not in CBA mice. Injection of normal (but not athymic) C57BL/6 mice with low doses of 38-13 monoclonal antibodies, contained culture supernatants or ascitic fluids, resulted in a 10-20 fold increase in the numbers of anti-Dex PFC detected in the spleen 5 days later, the majority of which carried the 38-13 idiotype.     

NF-κB哑铃形诱骗剂ODN对骨髓瘤细胞生长及IL-6表达的抑制作用

目的设计合成靶向NF-κB的哑铃形诱骗剂ODN,分析靶向NF-κB的哑铃形诱骗剂对NF-κB转录活性、多发性骨髓瘤细胞8266的生长及其分泌的IL-6的抑制效应。方法采用电泳迁移率变动分析(EMSA)体外检测诱骗剂ODN对NF-κB转录活性的抑制效应。将8266细胞随机分为传代培养的8266细胞组;诱骗剂ODN处理组及脂质体处理组。通过阳离子脂质体以2mg/L、4mg/L、8mg/L不同剂量诱骗剂ODN转染8266细胞。转染后8、12、18h,ELISA法检测8266细胞培养上清中IL-6的表达。用MTT比色检查诱骗剂ODN对IL-6刺激的8266细胞生长的影响。结果硫代磷酸的诱骗剂ODN在体外能有效地抑制NF-κB与其顺式元件的结合;2mg/L、4mg/L、8mg/L等不同浓度的脂质体-ODN复合物对8266细胞表达IL-6的抑制程度不同。脂质体-ODN复合物对8266细胞的生长及IL-6的活性均有抑制作用。结论靶向NF-κB的诱骗剂ODN在体外可抑制NF-κB的转录活性,从而抑制8266细胞的生长,降低瘤细胞中IL-6的表达。