A new autoantibody was detected by immunoprecipitation in the serum of 21 patients with chronic active hepatitis. The antibody reacted against a soluble cytosolic antigen in liver. The antibody was organ specific but not species specific and was therefore called anti-liver cytosol antibody Type 1 (anti-LC1). In seven of 21 cases, no other autoantibody was found; the remaining 14 cases had anti-liver/kidney microsome antibody Type 1 (anti-LKM1). With indirect immunofluorescence, a distinctive staining pattern was observed with the seven sera with anti-LC1 and without anti-LKM1. The antibody stained the cytoplasm of hepatocytes from four different animal species and spared the cellular layer around the central veins of mouse and rat liver that we have called juxtavenous hepatocytes. The immunofluorescence pattern disappeared after absorption of sera by a liver cytosol fraction. The 14 sera with both antibodies displayed anti-LC1 immunofluorescent pattern after absorption of anti-LKM1 by the liver microsomal fraction. The anti-LC1 was found in the serum only in patients with chronic active hepatitis of unknown cause. Anti-LC1 antibody was not found in sera from 100 patients with chronic active hepatitis associated with anti-actin antibody classic chronic active hepatitis Type 1, 100 patients with primary biliary cirrhosis, 157 patients with drug-induced hepatitis and a large number of patients with liver and nonliver diseases. This new antibody was considered a second marker of chronic active hepatitis associated with anti-LKM1 (anti-LKM1 chronic active hepatitis) or autoimmune chronic active hepatitis Type 2.

Constitutive activation of the MAP kinase kinase MEK1 induces oncogenic transformation in intestinal epithelial cells. Loss of cellcell adhesion followed by the dissociation of epithelial structures is a prerequisite for increased cell motility and tumor invasion. This phenotypic switch is designated epithelial-to-mesenchymal transition (EMT). EMT also plays an important role in determining the dissemination of tumors. However, the role of MEK1 in intestinal EMT, tumor invasion and metastasis has not been elucidated. To determine the functions of activated MEK1 in intestinal tumorigenesis, we established intestinal epithelial cell lines that overexpress wild-type MEK1 (wtMEK) or activated MEK1 (caMEK). Our results indicate that expression of caMEK is sufficient to induce EMT as confirmed with the induction of N-cadherin, vimentin, Snail1 and Snail2, whereas a reduction in E-cadherin, occludin, ZO-1 and cortical F-actin was noted. The Snail1 and Snail2 promoter analyses revealed that Egr-1 and Fra-1, an AP-1 protein, are responsible for MEK1-induced Snail1 and Snail2 expression, respectively. Cells expressing activated MEK1 clearly acquired an invasive capacity when compared to wtMEK-expressing cells. Zymography studies confirmed elevated levels of MMP2 and MMP9 activities in media of caMEK-expressing cells. Importantly, cells expressing activated MEK1 induced tumors with short latency in correlation with their ability to induce experimental metastasis in vivo and to express factors known to promote colorectal cancer cell metastasis. In conclusion, our results demonstrate, for the first time, that constitutive activation of MEK1 in intestinal epithelial cells is sufficient to induce an EMT associated with tumor invasion and metastasis. ' UICCKey words: MEK; ERK; intestinal epithelium; epithelialmesenchymal transition; snail; metastasis Ras proteins act as molecular switches that cycle between active GTP-bound and inactive GDP-bound forms and function as essential components of signal transduction pathways regulating cell growth. Activating mutations of the Ras family members are among the most common genetic events in human tumorigenesis. 1 For example, K-ras is mutated in nearly 50% of colorectal tumors at a relatively early stage of the carcinogenic process 2 and despite extensive research, the primary reason for this high frequency remains unclear.The most studied downstream effector pathway of K-ras is the mitogenic serine/threonine kinase cascade called Raf/MEK/mitogen-activated protein kinase (MAPK). Indeed, upon activation by growth factor-stimulated receptors, activated Ras complexes with and promotes Raf kinases, which in turn activate MAPK kinases (MEK1 and MEK2), resulting in activation of extracellular signal-regulated kinases (ERK1 and ERK2). 3 Activated ERKs then translocate into the nucleus where they phosphorylate and activate nuclear transcription factors, such as Elk-1, ATF-2 and ETS1/2 resulting in immediate-early gene induction. 4 Studies on cultured intestinal epithelial cells and many othe...

BackgroundAmong the most harmful of all genetic abnormalities that appear in colorectal cancer (CRC) development are mutations of KRAS and its downstream effector BRAF as they result in abnormal extracellular signal-related kinase (ERK) signaling. In a previous report, we had shown that expression of a constitutive active mutant of MEK1 (caMEK) in normal rat intestinal epithelial cells (IECs) induced morphological transformation associated with epithelial to mesenchymal transition, growth in soft agar, invasion and metastases in nude mice. Results from microarrays comparing control to caMEK-expressing IECs identified the gene encoding for serpinE2, a serine protease inhibitor, as a potential target of activated MEK1.Results1- RT-PCR and western blot analyses confirmed the strong up-regulation of serpinE2 expression and secretion by IECs expressing oncogenic MEK, Ras or BRAF. 2- Interestingly, serpinE2 mRNA and protein were also markedly enhanced in human CRC cells exhibiting mutation in KRAS and BRAF. 3- RNAi directed against serpinE2 in caMEK-transformed rat IECs or in human CRC cell lines HCT116 and LoVo markedly decreased foci formation, anchorage-independent growth in soft agarose, cell migration and tumor formation in nude mice. 4- Treatment of CRC cell lines with U0126 markedly reduced serpinE2 mRNA levels, indicating that expression of serpinE2 is likely dependent of ERK activity. 5- Finally, Q-PCR analyses demonstrated that mRNA levels of serpinE2 were markedly increased in human adenomas in comparison to healthy adjacent tissues and in colorectal tumors, regardless of tumor stage and grade.ConclusionsOur data indicate that serpinE2 is up-regulated by oncogenic activation of Ras, BRAF and MEK1 and contributes to pro-neoplastic actions of ERK signaling in intestinal epithelial cells. Hence, serpinE2 may be a potential therapeutic target for colorectal cancer treatment.

Immunoglobulin A (IgA), which is heavily glycosylated, interacts with a variety of receptors, e.g. the asialoglycoprotein receptor (ASGP-R), which binds terminal galactose residues, and the Fcalpha receptor (FcalphaRI). It has thus been proposed that elevated serum levels of IgA in primary Sjögren's syndrome (pSS) are caused by its defective clearance. To test this hypothesis, we developed a method (based on sialyl transferases eluted from a hepatoma cell line) to increase the amount of sialic acid (SA) on IgA, and used a battery of IgA1- and IgA2-specific glycosidases to reduce this amount. Binding of IgA1 and IgA2 to ASGP-R and FcalphaRI was found to be sugar dependent because oversialylated IgA bound less than native or desialylated IgA. However, individual sugars did not play a direct role in this binding. Given that IgA are oversialylated in pSS, defective clearance of IgA may indeed be ascribed to an excess of SA in IgA1 and IgA2.

No abstract

We have reported that human autoantibodies reacting with the polymorphonuclear neutrophil (PMN)-anchored FcγRIIIb (CD16) protect these cells from spontaneous apoptosis. In this study, we used anti-CD16 F(ab′)2 to delineate the mechanism(s) whereby the PMN life span is extended. As documented using four methods, CD16 cross-linking impeded spontaneous apoptosis, whereas anti-CD18 F(ab′)2 exerted no effect. Incubation of PMNs with anti-CD16 prevented the up-regulation of β2 integrins, particularly CD11b, which is the α-chain of complement receptor type 3, but also CD18, which is its β-chain, as well as CD11a and CD11c. Anti-CD16-conditioned supernatant of PMNs diminished the percentage of annexin V-binding fresh PMNs after another 18 h in culture, whereas the negative control anti-CD18 had no effect. The expression of mRNA for G-CSF and GM-CSF was induced by anti-CD16, followed by the release of G-CSF and GM-CSF in a dose-dependent manner. Anti-G-CSF and anti-GM-CSF mAbs abrogated the antiapoptotic effect of the related growth factors. The delay in apoptosis was accompanied by a down-regulated expression of Bax, and a partial reduction of caspase-3 activity. These data suggest an autocrine involvement of anti-CD16-induced survival factors in the rescue of PMNs from spontaneous apoptosis. Thus, apoptosis of aged PMNs can be modulated by signaling through FcγRIIIb, which may occur in patients with PMN-binding anti-FcγRIIIb autoantibodies.

Lymphocytes are very sensitive to radiation. Our aim was to test the possibility of detecting apoptosis in lymphocytes as a potential short-term biomarker of ionizing radiation exposure. Our in vitro data confirmed the dose-time-effect relationships involved in radiation-induced apoptosis. The detection of in vivo induction of apoptosis in circulating lymphocytes after exposure of animals to radiation appears to depend critically on the technique used to measure apoptosis. Among the different techniques we investigated, mitochondrial modification was the most appropriate; they allowed establishment of dose-time-effect relationships when animals were observed for 72 h. A model of in vitro phagocytosis of apoptotic lymphocytes by macrophages was developed to mimic clearance of apoptotic cells occurring in vivo. Together, our data show that mitochondrial labeling may make it possible to detect ex vivo radiation-induced apoptosis of lymphocytes before macrophage ingestion occurs. We propose the measurement of apoptosis in lymphocytes as a potential short-term biomarker of ionizing radiation exposure.

SUMMARYSome cd T cells express a receptor for the Fc portion of immunoglobulin G (FccRIII ± CD16). The relevance of this Fc receptor to cd T-cell function is at present unclear. Our previous studies have shown that cd T cells express activation markers in patients with rheumatoid arthritis (RA). In this study we have examined the relative proportions of CD16+ cd T cells in the blood and synovial¯uid of these patients compared with control blood. CD16+ cd T cells from RA patients were signi®cantly reduced in synovial¯uid compared with the circulation. That this was due to blocking of antibody binding to CD16 was unlikely as treatment of blood cd T cells with RA synovial¯uid (known to contain immune complexes) failed to alter expression of CD16. Treatment of blood cd T cells with phytohaemagglutinin in vitro, resulted in a time-dependent decrease in expression of CD16, with a concomitant increase in expression of human leucocyte antigen-DR, at the single cell level. We conclude that expression of CD16 by cd T cells is lost in the synovial compartment as the result of activation.