Increased circulating interleukin-12 (IL-12) p40 in pulmonary sarcoidosis

In sarcoidosis, a T helper 1 (Th1) response is an essential event and the up-regulation of interleukin-12 (IL-12) has been detected in affected disease sites. In order to investigate the clinical usefulness of circulating IL-12, we measured the serum concentrations of IL-12 by ELISA and performed immunohistochemistry using specific MoAbs for IL-12 in the lungs and scalene lymph nodes of patients with sarcoidosis. The serum concentration of IL-12 p40 was detectable in all 45 patients with pulmonary sarcoidosis and 18 normal controls, whereas that of IL-12 p70 was undetectable. The serum concentrations of IL-12 p40 in pulmonary sarcoidosis were significantly higher than those of the normal controls, especially in cases with abnormal intrathoracic findings detected by chest roentogenogram. The serum concentrations of interferon-gamma (IFN-gamma) also increased compared with those of normal controls and there was a significant positive correlation between the serum concentrations of IL-12 p40 and IFN-gamma. Furthermore, serum angiotensin-converting enzyme (ACE) and lysozyme, which are known to be useful markers for disease activity in sarcoidosis, correlated well with the serum concentrations of IL-12 p40. The positive 67Ga scan group (for lung field) had significantly elevated serum IL-12 p40 levels compared with those of the negative group. No bioactivity of IL-12 p70 was detected in three sarcoid cases sera by using the IL-12 responsive cell line. Finally, the immunohistochemical approach revealed that IL-12 p40 was expressed in the epithelioid cells and macrophages of sarcoid lungs and lymph nodes. We concluded that the production of IL-12 p40 was far greater in the sera and we have demonstrated this to be a useful clinical marker for disease activity and the Th1 response in pulmonary sarcoidosis.     

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.     

Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. .     

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.     

Korrelation zwischen der Blutkörperchensenkung und Plasmaproteinen

Zusammenfassung Die BKS und 12 Plasmaproteine von 21 Kranken mit unterschiedlichen Krankheiten wurden statistisch miteinander verglichen. Dabei ergaben sich positive Korrelationen zwischen der BKS und dem sauren ₁-Glykoprotein und der BKS und dem ₁-Antitrypsin, eine negative Korrelation zwischen der BKS und dem Transferrin. Außerdem korrelierten das saure ₁-Glykoprotein und das ₁-Antitrypsin sowie das saure ₁-Glykoprotein und das Transferrin miteinander. Bei der Deutung dieser Ergebnisse müssen einerseits Gemeinsamkeitskorrelationen in Betracht gezogen werden, zum anderen die Möglichkeit, daß zwischen bestimmten Proteinmustern und der BKS Zusammenhänge bestehen.     

肺腺癌细胞微环境及转移与黏附分子表达的关系

目的研究肺腺癌细胞生长环境及转移性与黏附分子CD44v6和CD29的表达关系。方法将起源相同、转移性不同的两个肺腺癌细胞系AGZY和Anip分别用简便肿瘤多细胞球体(MTS)培养法培养，并设常规单层贴壁细胞培养对照。通过倒置显微镜、扫描及透射电镜观察MTS形成情况，并用免疫组化法分别对MTS及贴壁细胞上CD44v6和CD29表达进行检测。结果MTS培养成功，贴壁细胞与MTS在细胞结构及细胞连接结构上相似，两种MTS在形态及结构上差异无显著性。免疫组化结果显示，CD29在高转移性的Anip细胞及其MTS上呈阳性表达；在低转移性的AGZY细胞及其MTS上阴性表达。CD44v6在Anip和AGZY细胞及MTS上均呈阳性表达，差异无显著性。贴壁细胞与MTS上两种黏附分子表达均无差异。结论成功建立了一种简易制备MTS的方法。细胞生长方式(单层贴壁与MTS)可能不影响CD44v6和CD29的表达。CD29表达可能与肺腺痛转移性相关；CD44v6表达可能与肺腺癌转移无关。     

TGF-β1 Null Mutation Leads to CD154 Upregulated Expression in Affected Tissues

The TGF-1(–/–) mouse is a murine model for systemic autoimmune disease. The aim of this study is to elucidate the immunological mechanism that leads to multifocal tissue inflammation and autoantibody production in TGF-1(–/–) mice. Heart, lung, liver, and salivary gland from TGF-1(–/–) were assessed for CD154 expression by RT-PCR and immunohistochemistry. Compared to wild-type littermates, CD154 expression was elevated in all tissues studied. Furthermore, IL-12 mRNA was expressed in the salivary gland and heart of TGF-1(–/–) mice and not in wild-type littermates. This suggests that the CD154 pathway is activated in these tissues. This shows that TGF-1 regulates CD154 expression leading to spontaneous IL-12 production and autoimmunity.