Identification of a new autoantibody in patients with chronic hepatitis

To comprehensively study autoantibodies in patients with chronic hepatitis (CH), especially those with hepatitis C virus (HCV) infection, proteins extracted from HepG2 cells were separated by two-dimensional electrophoresis. Spots reacting with sera from 15 patients with CH-C were detected by Western blotting. Proteins extracted from the spots were subjected to mass spectroscopy for identification by mass fingerprinting. Antigenicity of the proteins identified was confirmed by Western blotting and enzyme-linked immunoabsorbent assay. The localization of the autoantigens so detected was investigated by immunohistochemistry. Among 20 protein spots detected, four were identified as actin, heat shock protein (HSP) 70, HSP60, and a novel protein (hepalaminin). Hepalaminin consists of two domains of laminin β-2 and a specific domain. Autoantibodies against the specific domain were detected in 60.8% of patients with CH-C, 37.7% of those with CH-B, 42.3% of those with autoimmune CH, 28.6% of nonalcoholic steatohepatitis, 10.0% of asymptomatic HCV carriers, but in no healthy volunteers. Antihepalaminin positivity in CH-C and CH-B was related to histologic grading. Immunohistochemical staining demonstrated that hepalaminin is present in the cytoplasm of hepatocytes and cholangiocytes but not of fibroblasts or the vascular epithelium. Hepalaminin is a novel protein expressed in hepatocytes and cholangiocytes. Autoimmunity to this protein may exacerbate inflammation in chronic viral hepatitis.     

An immunohistochemical study of extracellular matrix proteins laminin,fibronectin and type IV collagen in paediatric glioblastoma multiforme

Summary Aims. In the recent decades many studies have been addressed in the literature to assess specific factors related to glioblastoma multiforme (GBM) invasion. However, few studies have evaluated tumour cells interaction with specific extracellular matrix (ECM) components, and, moreover, there is a lack of information regarding the occurrence of these phenomena in paediatric GBM.Methods and results. ECM proteins were evaluated in six cases of paediatric GBM assessing the immunohistochemical expression of laminin, fibronectin, and type IV collagen. We used a semiquantitative scale, ranging from not detected (zero) to marked (3). Laminin expression was minimal in three cases, moderate in one case, marked and generalised in one patient and marked and focal in the last case. Fibronectin expression was minimal in three patients; moderate immunoreactivity was documented in one case. Conversely, one case was classified as marked with generalised distribution and the remaining case as marked with focal immunostaining. Type IV collagen expression was minimal in three cases, moderate in one, marked with focal reaction in one and marked with generalised reaction in the remaining case.Conclusions. This study provides additional insights into tumour invasion features of paediatric GBM, as ECM plays a pivotal role in numerous cellular functions during normal and pathological processes. Although based on a limited number of patients, this investigation may serve as a challenge for the management of paediatric GBM, stimulating trials with larger patient numbers aimed at documenting specific factors influencing GBM prognosis.     

Laminin- and fibronectin-like molecules produced by periodontal ligament fibroblasts under serum-free culture are potent chemoattractants for gingival epithelial cells

Previously, we revealed that hepatocyte growth factor (HGF) or an HGF-like factor secreted by periodontal ligament fibroblasts (PLF) and gingival fibroblasts cultured in the presence of serum was a major chemoattractant for gingival epithelial cells, and suggested that it might play a role in epithelial invasion. However, our recent study showed that serum-free culture of PLF and gingival fibroblasts produced potent chemoattractants other than HGF for gingival epithelial cells. To identify these chemoattractants, PLF-conditioned medium (PLF-CM) from serum-free cultures was obtained, concentrated, and separated by gel filtration column chromatography, and the chemotactic activity for gingival epithelial cells of each eluted fraction was monitored by a modified Boyden chamber assay. The chemoattractant activity was eluted at a molecular mass of around 600 kDa, which would include laminin and fibronectin, but not HGF, determined by ELISA. The chemotactic activity was reduced by treatment with antilaminin and/or antifibronectin polyclonal antibodies. Western blots using both antibodies revealed that the PLF-CM contained laminin- and fibronectin-like molecules. Along with HGF, these large glycoprotein molecules produced by PLF may be involved in the pathogenesis and progression of periodontitis by inducing the apical migration of epithelial cells.     

Tumor cell invasion and survival in head and neck cancer

Squamous cell carcinoma (SCC) is the primary tumor type in head and neck cancer. Typically, these tumor cells show persistent invasion that frequently leads to local recurrence and distant lymphatic metastasis. The process of invasion involves concurrent infiltration and destruction of adjacent tissues. As with normal mucosal epithelium, SCC cells express receptors that mediate cell-extracellular matrix (ECM) adhesion (integrins) and cell-cell adhesion (cadherins). Both receptor families represent important signaling devices that are capable of promoting survival and proliferation. Recent results indicate that integrins and cadherins cooperate to regulate invasive behavior. During SCC invasion, cells actively migrate through the surrounding ECM with the simultaneous remodeling of their intercellular adhesions. During invasion, integrin receptor engagement with specific ECM ligands along with concurrent remodeling of cadherin adhesions induces changes in the cytoskeleton though modulation of the activities of Rho family members. Tumor development and progression of SCC proceeds with the generation of variant cells with potential alterations in expression of adhesion receptors, and their associated signaling pathways lead to a highly invasive and metastatic phenotype. Understanding the molecular events that define this subset of invasive cells will facilitate the development of new treatment strategies.     

Laminin在大白鼠胚脑的分布

本文用免疫组化ABC法观察了大白鼠胚脑中Laminin的分布。结果发现12天胚鼠脑泡全层神经上皮细胞均呈Laminin阳性反应,以内、外界膜处较深染。在15天以后的胚鼠,仅室周区神经上皮细胞深染,其它部分可见细胞外基质中淡染的Laminin阳性物质;还可见血管从软膜面伸入神经细胞之间分支成网状,其基膜呈Laminin阳性反应。从室腔面向软膜面见有Laminin阳性反应的放射胶质纤维,在中脑尤以中线处为密集,并向腹外侧放射。19天以后的胚鼠脑则未见放射胶质纤维。正常羊血清孵育的对照片上未见上述结构。实验表明Laminin在胚脑中主要存在于神经上皮细胞以及放射胶质纤维中。本文还探讨了Laminin与发育中神经细胞迁移的关系。     

层粘连蛋白、Ⅳ型胶原和纤维连接蛋白在大鼠实验矽肺形成过程中分布的变化

用免疫荧光和免疫酶标组织化学技术对大鼠实验性矽肺形成过程中的Ⅳ型胶原、层粘连蛋白(LM)和纤维连接蛋白(FN)三者的分布变化进行了动态观察。发现FN从病变初始的炎症期就出现在肺泡上皮周围炎症灶内疏散分布,随后明显增多及至包裹在所有胶原纤维的表面。LM是一种专一的基底膜成份,与FN的变化完全相反,随肺泡结构的破坏和纤维化病变的形成逐渐减少以致消失。Ⅳ型胶原也称基底膜胶原,但不是专一的基底膜成份,在肉芽肿期肺泡结构被破坏,网状纤维取而代之时,它并未减少却呈纤维走向,当病变发展成胶原纤维结节时,它才趋于消失。作者对观察到的上述现象及其在矽肺纤维化过程中的意义进行了讨论。     

Reactivity of chagasic sera with crude and highly purified glycosphingolipid fractions from Trypanosoma cruzi epimastigotes

The reactivities of sera from patients with Chagas disease or from T. cruzi-immunized rabbits with two different lipid preparations of T. cruzi were assessed using epimastigote antigens. Serum reactivities were determined using a quantitative enzyme-linked immunosorbent assay (ELISA). Antigen 1 represents the lower phase obtained from crude lipid extract after Folch partition (LCL). Antigen 2 is a highly purified glycosphingolipid fraction (GSL). The LCL antigen discriminated quite well the reactivities of Chagasic patients' sera and sera from healthy individuals, as well as between the serum from a T. cruzi-immunized rabbit (TIRS) and normal rabbit serum (NRS). A strong reactivity with GSL was obtained with TIRS. Reactivity with GSL was also obtained with human Chagasic sera. Compared to a group of normal individuals, the reactions of antibodies directed against lipid antigens were considerably increased in sera of patients with Chagas disease. Chagasic sera did not differentiate between glycolipids with terminal β-glucosyl or β-galactosyl nonreducing units. They discriminated, however, glucosylceramides with differences in the ceramide structure. To determine the specificity of Chagasic sera, antibodies isolated on LCL-immunosorbent (LCL-Ch Abs) as well as on laminin-immunosorbent (Lam-Ch Abs) were tested against laminin and LCL antigens. We found that Lam-Ch Abs reacted with murine laminin, whereas the reaction was negative with LCL. In contrast, the LCL-Ch Abs reacted either with LCL antigens or with laminin. The reactivity with laminin was strong in comparison with LCL. Results suggest that although the glycolipids are recognized by Chagasic patients' sera, the reactive antibodies are not specific since they reacted quite well with murine laminin. Polyspecific antibodies from Chagasic sera have already been described using other unrelated antigens. © 1994 Wiley-Liss, Inc.     

Laminin binding to Prevotella intermedia

The interaction of laminin (Lm), a basement membrane protein abundant in the periodontium, with 66 strains of Prevotella intermedia isolated from diseased pockets, was tested in a 125I-labeled protein binding assay. The mean binding value was 28% of the total protein added. The binding significantly increased to 35% when the environmental pH decreased from 7 to 6. The Lm interaction was characterized in a highly binding (about 65%) strain, OMGS105. The binding was rapid and required about 1 min and 1-2 h for 50% and 100% equilibrium respectively. The 125I-Lm binding was maximum in the pH interval 3.0 to 6.5 and could not be displaced by unlabeled Lm or inhibited by other proteins and carbohydrates. The interaction was stable in the presence of NaCl or urea (concentrations up to 4 M) but was dissociated by > or = 1 M KSCN. The Lm-binding component was thermolabile and sensitive to proteolytic enzymes. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and Western blot analysis revealed a approximately 62 kDa Lm-binding protein, both in the whole cell extract and the outer membrane preparation. Weaker binding was also observed to other proteins. These data establish the ability of P. intermedia to interact with Lm via certain cell surface proteins, a property that might contribute to the colonization of this bacterium in the periodontal pocket.     

An evaluation of neural invasion in esophageal cancer

P < 0.05). Examining the types of recurrence, namely hematogenous, lymphogenous, and local/stump, as well as pleural or peritoneal dissemination, a relationship was observed between lymphogenous recurrence and N or ly, and between local/stump recurrence and NI. The prognosis of the NI(+) patients was significantly different from that of the NI(−) patients. According to a multivariate analysis, NI and N were significant prognostic factors. These findings demonstrate that NI is an important prognostic factor closely related to local recurrence in patients with esophageal cancer. Thus, when treating advanced esophageal cancer with T2 or greater depth of invasion, NI and lymph node excision should be considered. (Received for publication on May 29, 1997; accepted on Jan. 6, 1998)     

Passages: 1996

The spatial and temporal expression patterns of several extracellular matrix molecules—laminin and fibronectin and cell surface molecules, neural cell adhesion molecule (NCAM), L1, tenascin, chondroitin sulfate proteoglycan, and peanut agglutinin (PNA) binding sites—were investigated during early olfactory nerve development. NCAM and L1 have similar patterns: They are expressed in the olfactory nerve and on the olfactory receptor neurons (ORNs) commencing with the earliest olfactory axon outgrowth (E12-E15). Their expression patterns suggest that both NCAM and L1 are associated with extension and fasciculation of olfactory axons. A comparison of L1 and olfactory marker protein suggests that L1 is expressed predominantly on immature ORNs. Laminin has an unique punctate staining pattern in the developing olfactory pathway as early as E12. These laminin puncta might play a role in olfactory neurite outgrowth and guidance. At E14, when pioneer olfactory axons enter the brain, the laminin-positive meninges on the surface of the olfactory bulb primordium break down but remain intact in the rest of the telencephalon. This suggests a functional interaction between the olfactory axons and the glial-pial barrier. Fibronectin staining is diffuse throughout the cranial mesenchyme but is absent from the olfactory nerve pathway. No specific patterns of tenascin or chondroitin sulfate, were observed during early olfactory development. PNA binding sites were associated with olfactory axon fasciculation. The expression of several extracellular matrix molecules and cell surface molecules is spatially and temporally regulated in the developing olfactory system. These molecules, thus, may play functional roles in olfactory axon outgrowth, fasciculation, and/or guidance. ©1996 Wiley-Liss, Inc.