concanavalin-a and Liver-Cirrhosis

Concanavalin A (Con A) affinity electrophoresis of serum alpha-fetoprotein (AFP) can distinguish hepatocellular carcinoma (HCC) from other malignancies when the serum AFP concentration is elevated. However, Con A has not been able to distinguish HCC from benign chronic liver disease such as cirrhosis or chronic hepatitis.. The Con A affinity electrophoresis of serum AFP was analyzed in patients with a serum AFP concentration greater than 50 ng/mL by antibody affinity electrophoresis and Western blotting in an attempt to distinguish hepatocellular carcinoma from benign chronic liver disease. Before the assay, the serum AFP concentrations were adjusted between 100 ng/ml and 300 ng/ml by concentrating or diluting the samples.. Of 180 patients with HCC, 44 (24%) had a single band and 91 (51%), 35 (19%), and 10 (6%) had 2, 3, and 4 bands, respectively. All 35 patients with chronic hepatitis had a single band. All but 1 of 72 patients with cirrhosis had a single band. Multiple AFP bands on Con A affinity electrophoresis appear to be diagnostic of HCC. This method has a sensitivity of 76%, a specificity of 99%, a positive predictive value of 99%, and a negative predictive value of 71% for detecting HCC. The number of AFP bands correlated with serum AFP concentration and tumor size in patients with HCC.. This assay is useful for distinguishing HCC from benign chronic liver diseases.

Topics: Adult; Aged; alpha-Fetoproteins; Blotting, Western; Carcinoma, Hepatocellular; Chi-Square Distribution; Chronic Disease; Concanavalin A; Diagnosis, Differential; Electrophoresis; Female; Hepatitis; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Predictive Value of Tests; Sensitivity and Specificity

Objective To investigate how mutation of nuclear autoantigenic sperm protein (NASP) gene affects mouse liver fibrosis induced by concanavalinA (ConA) and its mechanism. Methods The wild-type B6 (B6-WT) mice were used as a control group, and the NASP mutant B6 (B6-NASP

Topics: Animals; Autoantigens; Cell Cycle Proteins; Collagen Type I; Concanavalin A; Liver Cirrhosis; Male; Mice; RNA, Messenger; Semen; Spermatozoa; Tumor Necrosis Factor-alpha

Serotonin exerts important functions in several liver pathophysiological processes. In this study, we investigated the role of serotonin in concanavalin A (Con A)-induced liver fibrosis (LF) in mice and the underlying mechanisms. To establish the mouse model of LF, mice of wild-type (WT) and tryptophan hydroxylase 1 (Tph1) knockout (serotonin depletion) received Con A for 8 successive weeks. Degree of fibrosis was assessed by Sirius red staining, as well as the measurements of alpha smooth muscle actin (α- SMA), hydroxyproline (Hyp) and type I collagen in liver tissues. To elucidate the potential mechanisms, we assessed the effect of serotonin depletion on inflammatory, oxidative stress as well as TGF-β1/Smads signaling pathway. We found that serotonin depletion significantly inhibited collagen deposition as evaluated by less collagenous fiber in Sirus Red staining and reduced contents of Hyp and type I collagen. In addition, the absence of serotonin significantly inhibited the release of several inflammatory cytokines, including interleukin-6 (IL-6), interferon-gamma (IFN-γ), tumor necrosis-alpha (TNF-α), and transforming growth factor β1 (TGF-β1). Oxidative stress was also largely mitigated in LF mice with serotonin deficiency as manifested by the decreases of oxidative stress markers (malonaldehyde (MDA) and myeloperoxidase (MPO)), as well as the increases of antioxidant stress indicators (glutathione (GSH), and GSH-px, catalase (CAT), superoxide dismutase (SOD)) in liver tissues. Moreover, the lack of serotonin may provide an antifibrotic role by inhibiting the intrahepatic expressions of TGF-β1, phosphorylated-smad2 (p-smad2), and phosphorylated-smad3 (p-smad3). These results indicated that, serotonin depletion attenuates Con A-induced LF through the regulation of inflammatory response, oxidative stress injury, and TGF-β1/Smads signaling pathway.

Topics: Animals; Concanavalin A; Humans; Inflammation; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogens; Oxidative Stress; Serotonin; Signal Transduction; Smad Proteins; Transforming Growth Factor beta1; Tryptophan Hydroxylase

Aim Liver fibrosis represents a massive global health burden with limited therapeutic options. Thus, the need for curative options is evident. Thus, this study aimed to assess the potential antifibrotic effect of honokiol in Concanavalin A (Con A) induced immunological model of liver fibrosis as well the possible underlying molecular mechanisms.. Male Sprague-Dawley rats were treated with either Con A (20 mg/kg, IV) and/or honokiol (10 mg/kg, orally) for 4 weeks. Hepatotoxicity indices were as well as histopathological evaluation was done. Hepatic fibrosis was assessed by measuring alpha smooth muscle actin (α-SMA) expression and collagen fibers deposition by Masson's trichrome stain and hydroxyproline content. To elucidate the underlying molecular mechanisms, the effect of honokiol on oxidative stress, inflammatory markers as well as transforming growth factor beta (TGF-β)/SMAD and mitogen-activated protein kinase (MAPK) pathways was assessed.. Honokiol effectively reversed the hepatotoxicity indices elevations and abnormal histopathological changes induced by Con A. Besides, honokiol attenuated Con A-induced liver fibrosis by down-regulation of hydroxyproline levels, α-SMA expression together with a marked decrease in collagen fibers deposition. Mechanistically Con A induced oxidative stress, provocation of inflammatory responses and activation of TGF-β/SMAD/MAPK pathways. Contrariwise, honokiol co-treatment significantly restored antioxidant defence mechanisms, down-regulated inflammatory cascades and inhibited TGF-β/SMAD/MAPK signaling pathways.. The results provide an evidence for the promising antifibrotic effect of honokiol that could be partially due to suppressing oxidative stress and inflammatory processes as well as inhibition of TGF-β/SMAD/MAPK signaling pathways.

Topics: Actins; Animals; Biphenyl Compounds; Concanavalin A; Hydroxyproline; Inflammation; Lignans; Liver Cirrhosis; Male; Mitogen-Activated Protein Kinases; Oxidative Stress; Rats; Rats, Sprague-Dawley; Signal Transduction; Smad Proteins; Survival Analysis; Transforming Growth Factor beta

Kupffer cells are a major producer of reactive oxygen species and have been implicated in the development of liver fibrosis during chronic hepatitis in non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH). We recently reported on the development of a polythiolated and mannosylated human serum albumin (SH-Man-HSA) that functions as a Kupffer cell-targeting nanoantioxidant. In this material, the albumin is mannosylated, which permits it to be taken up by mannose receptor C type 1 expressed on Kupffer cells, and is also polythiolated to have antioxidant activity. To clarify the anti-fibrotic property of this nanoantioxidant, we repeatedly administered SH-Man-HSA to a liver fibrosis mouse model that was induced by the repeated treatment of the concanavalin-A, which mimics the liver fibrosis observed in NASH and ASH. SH-Man-HSA dramatically improved the survival rate and suppressed liver fibrosis in the experimental model. In addition, SH-Man-HSA suppressed hepatic oxidative stress levels, thereby decreasing the numbers of apoptotic cells. In contrast, N-acetylcysteine, which contains the same thiol content as the SH-Man-HSA, failed to show a substantial therapeutic effect in these mice. The expression levels of inflammatory genes including epidermal growth factor module-containing mucin-like receptor (Emr-1/F4/80), Toll-like receptor-4 (TLR-4), high mobility group box-1 (HMGB-1), CC chemokine ligand-5 (CCL-5), tumor necrosis factor-α (TNF-α), CCL-2, interleukin-6 (IL-6), and IL-1β, as well as fibrotic (α-smooth muscle actin (α-SMA), transforming growth factor-β (TGF-β), and Snail) and extracellular matrix genes (collagen, type Iα2 (Col1α2), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase 1 (TIMP-1)), showed some decreasing trends by the SH-Man-HSA administration. These findings suggest that the repeated administration of the Kupffer cell-targeting nanoantioxidant, SH-Man-HSA, ameliorates liver fibrosis in mice by suppressing the level of oxidative stress and a portion of the inflammation, and has a potential therapeutic effect against NASH and ASH.

Topics: Albumins; Animals; Antioxidants; Concanavalin A; Disease Models, Animal; Fatty Liver, Alcoholic; Female; Gene Expression; Glycoproteins; Kupffer Cells; Liver Cirrhosis; Mice, Inbred BALB C; Non-alcoholic Fatty Liver Disease; Oxidative Stress

The present study investigated the improving effect of cucurbitacin B on liver fibrosis induced by concanavalin A in mice and explored its possible mechanism. AST, ALT and TB were detected by kits. ELISA was performed to detect the levels of IL 5, IL 6, IL 13 and TNF-α in serum. Haematoxylin-eosin (HE) staining and Masson's trichrome staining were used to evaluate pathological changes. Western blotting was performed to observe expression levels of sirtuin (SIRT) 1, insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) and TGF β1. The activity of SIRT 1 also was detected. Results showed that cucurbitacin B could effectively improve the abnormal liver function, inhibit liver fibrosis and suppress releases of inflammatory factors in mice induced by concanavalin A. Furthermore, cucurbitacin B could down-regulate the expressions of TGF β1 and IGFBPrP1, increase the expression and activity of SIRT 1. Interestingly, when SIRT1 activity was inhibited by EX 527, a selective inhibitor of SIRT 1, the preventive effect of cucurbitacin B was significantly attenuated. Taken together, the above results showed that cucurbitacin B could significantly suppress releases of inflammatory cytokines and improve liver fibrosis induced by concanavalin A in mice, and those may be achieved through SIRT1/IGFBPrP1/TGF β1 axis.

Topics: Animals; Body Weight; Concanavalin A; Insulin-Like Growth Factor Binding Proteins; Interleukins; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred BALB C; Sirtuin 1; Transforming Growth Factor beta1; Triterpenes; Tumor Necrosis Factor-alpha

Liver fibrosis is defined as excessive extracellular matrix deposition in the hepatic parenchyma as a consequence of complex interactions among matrix-producing hepatic stellate cells (HSCs) and liver-resident and infiltrating cells. In addition to the liver, the process of fibrosis may represent end-stage disease of several diseases including kidneys, lungs, spleens, heart, muscles and at certain extent, the central nervous system and the peripheral nerves. To date, antifibrotic treatment of fibrosis represents an unconquered area for drug development. The aim of the present study was to test the efficacy of a new drug combination for the treatment of hepatic fibrosis in order to provide a proof-of-concept for the use of therapeutic agents in clinical practice. For this purpose, we have studied the effects of the PDGF inhibitor imatinib and the angiogenesis inhibitor sorafenib, administered alone or in combination, in reducing the progression of the fibrogenetic process in a pre-clinical model of liver damage induced in mice by repeated administration of Concanavalin A (ConA), resembling long-tern autoimmune hepatitis. Our results suggest that treatments with imatinib and sorafenib can modulate potently and, in a superimposable fashion, the fibrinogenic process when administered alone. However, and in agreement with the computational data presently generated, they only exert partial overlapping antifibrotic effects in modulating the main pathways involved in the process of liver fibrosis, without significant additive or synergist effects, when administered in combination.

Topics: Angiogenesis Inhibitors; Animals; Computer Simulation; Concanavalin A; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Female; Hepatic Stellate Cells; Humans; Imatinib Mesylate; Liver; Liver Cirrhosis; Mice; Mice, Inbred BALB C; Protein Kinase Inhibitors; Receptors, Platelet-Derived Growth Factor; Sorafenib

Hepatitis is an important health problem worldwide. Novel molecular targets are in demand for detection and management of hepatitis. Hepatoma-derived growth factor (HDGF) has been delineated to participate in hepatic fibrosis and liver carcinogenesis. However, the relationship between hepatitis and HDGF remains unclear. This study aimed to elucidate the role of HDGF during hepatitis using concanavalin A (ConA)-induced hepatitis model. In cultured hepatocytes, ConA treatment-elicited HDGF upregulation at transcriptional level and promoted HDGF secretion while reducing intracellular HDGF protein level and cellular viability. Similarly, mice receiving ConA administration exhibited reduced hepatic HDGF expression and elevated circulating HDGF level, which was positively correlated with serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. By using HDGF knockout (KO) mice, it was found the ConA-evoked cell death was prominently alleviated in KO compared with control. Besides, it was delineated HDGF ablation conferred protection by suppressing the ConA-induced neutrophils recruitment in livers. Above all, the ConA-mediated activation of tumor necrosis factor-α (TNF-α)/interleukin-1β (IL-1β)/interleukin-6 (IL-6)/cyclooxygenase-2 (COX-2) inflammatory signaling was significantly abrogated in KO mice. Treatment with recombinant HDGF (rHDGF) dose-dependently stimulated the expression of TNF-α/IL-1β/IL-6/COX-2 in hepatocytes, further supporting the pro-inflammatory function of HDGF. Finally, application of HDGF antibody not only attenuated the ConA-mediated inflammatory cascade in hepatocytes, but also ameliorated the ConA-induced hepatic necrosis and AST elevation in mice. In summary, HDGF participates in ConA-induced hepatitis via neutrophils recruitment and may constitute a therapeutic target for acute hepatitis.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Cells, Cultured; Concanavalin A; Hepatitis, Animal; Hepatocytes; Intercellular Signaling Peptides and Proteins; Interleukin-1beta; Interleukin-6; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophil Infiltration; Rats; Signal Transduction; Tumor Necrosis Factor-alpha; Up-Regulation

Hepatic iron overload is one of the causative factors for chronic liver injury and fibrosis. The present study aimed to investigate the potential antifibrotic effect of the iron chelator; deferasirox (DFX) in experimentally-induced liver fibrosis in rats. Male Sprague-Dawley rats were administered concanavalin A (Con A) and/or DFX for 6 consecutive weeks. Con A injection induced significant hepatotoxicity as was evident by the elevated transaminases activity, and decreased albumin level. Also, it disturbed the iron homeostasis through increasing C/EBP homologous protein (CHOP), decreasing phosphorylated cAMP responsive element binding protein(P-CREB) and hepcidin levels leading to significant serum and hepatic iron overload. In addition, it induced an imbalance in the oxidative status of the liver via upregulating NADPH oxidase 4 (NOX4), together with a marked decrease in anti-oxidant enzymes' activities. As a consequence, upregulation of nuclear factor-kappa b (NF-κB) and the downstream inflammatory mediators was observed. Those events all together precipitated in initiation of liver fibrosis as confirmed by the elevation of alpha-smooth muscle actin (α-SMA) and liver collagen content. Co-treatment with DFX protected against experimentally-induced liver fibrosis in rats via its iron chelating, anti-oxidant, and anti-inflammatory properties. These findings imply that DFX can attenuate the progression of liver fibrosis.

Topics: Animals; Concanavalin A; Deferasirox; Iron Chelating Agents; Iron Overload; Liver Cirrhosis; Male; Rats; Rats, Sprague-Dawley

Liver fibrosis (LF) is a life-threatening complication of most chronic liver diseases resulting from a variety of injurious agents and hepatotoxic insults. To date, there are no specific therapies for LF, and all the currently available drugs have been developed for other indications. Thus, there is a pressing need to develop new drugs for treatment of LF. Therefore, the current study aimed to elucidate the potential antifibrotic effect of Pirfenidone (PFD) against concanavalin A (ConA)-induced immunological model of liver fibrosis in mice.. Hepatic fibrosis was induced in mice by injecting ConA (10 mg/kg/wk./i.v) for 4 weeks. Then, the mice were treated with or without PFD (125 mg/kg/ip/day) for 2 weeks. Hepatic fibrosis was determined by Masson Trichrome staining; Haematoxylin & eosin (H&E) staining, immunohistochemistry staining of type II and IV collagens, and colorimetric assessment of hydroxyprolline (HP) content in the liver tissues. In addition, the expression of α-SMA mRNA was determined by real time RT-PCR. The serum levels of TGF-β, TNF-α, TIMP-1 and MMP-2 were measured by ELISA.. Treatment with PFD significantly reduced ConA-induced expression of type II and IV collagens, α-SMA mRNA expression, and HP content and decreased inflammatory cells infiltration in hepatic tissues. Furthermore, serum levels of TGF-β, TNF-α, and TIMP-1 were significantly reduced with concomitant increase in MMP-2 expression.. Treatment with PFD ameliorates concanavalin A-induced hepatic inflammation and fibrosis in mice. Thus, PFD may represent a promising therapeutic option for hepatic fibrosis and its related complications.

Topics: Animals; Collagen Type II; Collagen Type IV; Concanavalin A; Disease Models, Animal; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred BALB C; Pyridones; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

MicroRNAs (miRNAs) have been confirmed to participate in liver fibrosis progression and activation of hepatic stellate cells (HSCs). In this study, the role of miR-193a/b-3p in concanavalin A (ConA)-induced liver fibrosis in mice was evaluated. According to the results, the expression of miR-193a/b-3p was down-regulated in liver tissues after exposure to ConA. Lentivirus-mediated overexpression of miR-193a/b-3p reduced ConA-induced liver injury as demonstrated by decreasing ALT and AST levels. Moreover, ConA-induced liver fibrosis was restrained by the up-regulation of miR-193a/b-3 through inhibiting collagen deposition, decreasing desmin and proliferating cell nuclear antigen (PCNA) expression and lessening the content of hydroxyproline, transforming growth factor-β1 (TGF-β1) and activin A in liver tissues. Furthermore, miR-193a/b-3p mimics suppressed the proliferation of human HSCs LX-2 via inducing the apoptosis of LX-2 cells and lowering the levels of cell cycle-related proteins Cyclin D1, Cyclin E1, p-Rb and CAPRIN1. Finally, TGF-β1 and activin A-mediated activation of LX-2 cells was reversed by miR-193a/b-3p mimics via repressing COL1A1 and α-SMA expression, and restraining the activation of TGF-β/Smad2/3 signalling pathway. CAPRIN1 and TGF-β2 were demonstrated to be the direct target genes of miR-193a/b-3p. We conclude that miR-193a/b-3p overexpression attenuates liver fibrosis through suppressing the proliferation and activation of HSCs. Our data suggest that miR-193a-3p and miR-193b-3p may be new therapeutic targets for liver fibrosis.

Topics: Alanine Transaminase; Animals; Apoptosis; Aspartate Aminotransferases; Cell Line; Cell Proliferation; Concanavalin A; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Male; Mice; Mice, Inbred BALB C; MicroRNAs; Signal Transduction; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Up-Regulation

Fibrosis induces a progressive loss of liver function, thus leading to organ failure. Activins are secreted proteins that belong to the transforming growth factor (TGF)‑β superfamily, which initiate signaling by binding to their two type II receptors: Activin A receptor type 2A (ACVR2A) and activin A receptor type 2B. Previous studies that have explored the mechanisms underlying immune‑induced hepatic fibrosis have mainly focused on TGF‑β signaling, not activin signaling. To investigate the role of the activin pathway in this disease, adenovirus particles containing short hairpin (sh)RNA targeting ACVR2A mRNA (Ad‑ACVR2A shRNA) were administered to mice, which were chronically treated with concanavalin A (Con A). The pathological changes in the liver were evaluated with hematoxylin/eosin staining, Masson trichrome staining and immunohistochemical assay. The results detected an increase in serum activin A and liver ACVR2A in Con A‑treated animals. Conversely, liver function was partially restored and fibrotic injury was attenuated when activin signaling was blocked. In addition, the activation of hepatic stellate cells (HSCs) in response to Con A was suppressed by Ad‑ACVR2A shRNA, as evidenced by decreased α‑smooth muscle actin, and type I and IV collagen expression. Furthermore, primary mouse HSCs (mHSCs) were activated when exposed to interleukin (IL)‑17A or IL‑17F, which are two major cytokines produced by cluster of differentiation 4+ T helper 17 cells. The levels of activin A, type I and IV collagen were determined with ELISA kits and the expression of fibrotic molecules was determined with western blot analysis. Conversely, blocking activin/ACVR2A impaired the potency of HSCs to produce collagens in response to IL‑17s. In addition, C terminus phosphorylation of Smad2 on Ser465 and Ser467, induced by either Con A in the liver or by IL‑17s in mHSCs, was partly inhibited when activin A/ACVR2A signaling was suppressed. Collectively, the present study demonstrated an involvement of activated activin A/ACVR2A/Smad2 signaling in immune‑induced hepatic fibrosis.

Topics: Activin Receptors, Type II; Activins; Adenoviridae; Animals; Cells, Cultured; Concanavalin A; Gene Knockdown Techniques; Hepatic Stellate Cells; Interleukin-17; Liver; Liver Cirrhosis; Mice, Inbred BALB C; RNA, Small Interfering; Signal Transduction

To explore the exact interaction between Notch and transforming growth factor (TGF)-β signaling in liver fibrosis.. We established a rat model of liver fibrosis induced by concanavalin A. Peripheral blood mononuclear cells (PBMCs) were isolated from the modeled rats, and cultured with γ-secretase inhibitor DAPT and TGF-β inhibitor for 24 h. The mRNA levels of Notch and TGF-β signaling were detected by quantitative real-time polymerase chain reaction. Expression of Notch and TGF-β proteins was analyzed by western blotting.. Compared to control rats, Notch and TGF-β signaling was activated in PBMCs of model rats. Administration of DAPT and TGF-β inhibitor suppressed Notch and TGF-β signal transducer in PBMCs of model rats. DAPT reduced mRNA and protein expression of TGF-β signaling, such as TGF-β1 and Smad3. TGF-β inhibitor also downregulated Notch1, Hes1 and Hes5, and mRNA and protein expression of the Notch signaling pathway.. Notch and TGF-β signaling play a role in liver fibrosis. TGF-β signaling upregulates Notch signaling, which promotes TGF-β signaling.

Topics: Animals; Concanavalin A; Diamines; Liver Cirrhosis; Male; Primary Cell Culture; Rats, Wistar; Receptors, Notch; Smad Proteins; Thiazoles; Transforming Growth Factor beta

To investigate the preventive and therapeutic effects of rapamycin (RAPA) on autoimmune hepatitis and liver fibrosis induced by concanavalin A (ConA) and possible mechanisms.. Female C57BL/6 mice aged 8 weeks were randomly divided into normal control group, ConA model group, and ConA+RAPA treatment group. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured; hematoxylin-eosin and Masson staining and Knodell HAI and Ishak scoring systems were used to evaluate the degrees of liver inflammation and fibrosis. Gradient centrifugation was used to separate mononuclear cells, flow cytometry was used to measure CD4(+)/CD8(+) ratio, and intracellular cytokine staining was performed to measure the levels of interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-10 (IL-10), and transforming growth factor β (TGF-β) in immune cells. The t-test was used for data comparison between groups.. The RAPA treatment group showed a significant reduction in serum ALT level compared with the ConA model group (P < 0.05). Liver inflammatory injury was reduced significantly, and there was no obvious fibrous tissue proliferation. The level of TGF-β in mononuclear cells was reduced significantly, and the treatment group had a significantly lower level of TGF-β than the model group (8.91%±1.25% vs 16.65%±2.05%, P < 0.05). The proportions of CD4(+) and CD8(+)T cells in the liver were reduced, and the treatment group had significantly lower proportions of CD4(+) and CD8(+)T cells than the model group (proportion of CD4(+)T cells: 4.09%±1.20% vs 8.91%±0.69%, P < 0.05; proportion of CD8(+)T cells: 3.28%±0.66% vs 9.68%±1.46%, P < 0.05). The proportion of Th1 cells was reduced, and the treatment group had a significantly lower proportion of Th1 cells than the model group (1.02%±0.06% vs 2.83%±0.21%, P < 0.05); the proportions of Th3 and Tr1 regulatory T cells were increased, and the treatment group had significantly higher proportions of Th3 and Tr1 regulatory T cells than the model group (proportion of Th3 regulatory T cells: 59.53%±9.82% vs 47.13%±4.79%, P < 0.05; proportion of Tr1 regulatory T cells: 10.63%±2.27% vs 7.09%±1.66%, P < 0.05), but the proportion of Th2 cells showed no significant difference between the two groups (P > 0.05).. RAPA can promote the differentiation of Th3/Tr1 cells, reduce the expression of TGF-β in mononuclear cells, slow down the progression of chronic hepatitis induced by ConA into liver fibrosis, and thus prevent liver fibrosis.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Concanavalin A; Female; Hepatitis, Autoimmune; Interferon-gamma; Interleukin-10; Interleukin-4; Liver Cirrhosis; Mice; Mice, Inbred C57BL; Random Allocation; Sirolimus; T-Lymphocytes, Regulatory; Th1 Cells; Th2 Cells; Transforming Growth Factor beta

Iron-overload is a well-known factor of hepatotoxicity and liver fibrosis, which found to be a common finding among hepatitis C virus patients and related to interferon resistance. We aimed to elucidate the potential antifibrotic effect of deferoxamine; the main iron chelator, and its additional usefulness to interferon-based therapy in concanavalin A-induced immunological model of liver fibrosis. Rats were treated with deferoxamine and/or pegylated interferon-α for 6 weeks. Hepatotoxicity indices, oxidative stress, inflammatory and liver fibrosis markers were assessed. Concanavalin A induced a significant increase in hepatotoxicity indices and lipid peroxidation accompanied with a significant depletion of total antioxidant capacity, glutathione level and superoxide dismutase activity. Besides, it increased CD4(+) T-cells content and the downstream inflammatory cascades, including NF-κB, TNF-α, iNOS, COX-2, IL-6 and IFN-γ. Furthermore, α-SMA, TGF-β1 and hydroxyproline were increased markedly, which confirmed by histopathology. Treatment with either deferoxamine or pegylated interferon-α alone reduced liver fibrosis markers significantly and improved liver histology. However, some of the hepatotoxicity indices and oxidative stress markers did not improve upon pegylated interferon-α treatment alone, besides the remarkable increase in IL-6. Combination therapy of deferoxamine with pegylated interferon-α further improved all previous markers, ameliorated IL-6 elevation, as well as increased hepcidin expression. In conclusion, our study provides evidences for the potent antifibrotic effects of deferoxamine and the underlying mechanisms that involved attenuating oxidative stress and subsequent inflammatory cascade, as well as the production of profibrogenic factors. Addition of deferoxamine to interferon regimen for HCV patients may offer a promising adjuvant modality to enhance therapeutic response.

Topics: Animals; Antiviral Agents; Concanavalin A; Deferoxamine; Interferon alpha-2; Interferon-alpha; Iron; Liver; Liver Cirrhosis; Mitogens; Oxidative Stress; Polyethylene Glycols; Rats; Rats, Wistar; Recombinant Proteins; Siderophores

Anti-inflammation strategy is one of the proposed therapeutic approaches to hepatic fibrosis. T helper (Th) 17 cells, which play a detrimental role in experimental murine models of inflammatory diseases, have been demonstrated to participate in the pathogenesis of liver damage. The inhibitory effect of halofuginone (HF), an active component of extracts derived from the plant alkaloid febrifugine, on collagen synthesis has been shown in animal models of the fibrotic disease. The aim of this study was to clarify the in vivo effect of HF on Th17 cells in concanavalin A-induced fibrosis rats. Haematoxylin-eosin (HE) staining and Masson staining were performed to observe collagen deposition. The presence of INF-gamma, TNF-alpha, IL-6, IL-17, IL-1beta, IL-33 and IL-10 in serum and the presence of ROR-γt, IL-17, TGF-β1 and α-SMA in liver tissue were detected. Flow cytometry was performed to analyse the percentage of Th17 cells. We observed significantly lower levels of INF-gamma, TNF-alpha, IL-6, IL-17, IL-1beta, TGF-β1 and α-SMA in HF-treated group of rats, and the percentage of Th17 cells in splenic lymphocyte was decreased well. Histological examination demonstrated that HF significantly reduced the severity of liver fibrosis in HF-treated rats. We concluded that HF (10 mg/kg) exerts an antifibrotic impact on Th17 cells and its relative cytokines in rats with ConA-induced fibrosis.

Topics: Actins; Alanine Transaminase; Albumins; Animals; Aspartate Aminotransferases; Cell Differentiation; Concanavalin A; Disease Models, Animal; Interferon-gamma; Interleukin-10; Interleukin-17; Interleukin-1beta; Interleukin-33; Interleukin-6; Interleukins; Liver; Liver Cirrhosis; Male; Nuclear Receptor Subfamily 1, Group F, Member 3; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Rats; Rats, Wistar; Th17 Cells; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Immune response plays an important role in the development of hepatic fibrosis. In the present study, we investigated the effects of quercetin on hepatitis and hepatic fibrosis induced by immunological mechanism. In the acute hepatitis model, quercetin (2.5 mg/kg) was injected iv into mice 30 min after concanavalin A (Con A) challenge. Mice were sacrificed 4 or 24 h after Con A injection, and aminotransferase tests and histopathological sections were performed. Treatment with quercetin significantly decreased the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Consistent with this observation, treatment with quercetin markedly attenuated the pathologic changes in the liver. A hepatic fibrosis model was also generated in mice by Con A challenge once a week for 6 consecutive weeks. Mice in the experimental group were treated with daily iv injections of quercetin (0.5 mg/kg). Histopathological analyses revealed that treatment with quercetin markedly decreased collagen deposition, pseudolobuli development, and hepatic stellate cells activation. We also examined the effects of quercetin on the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and transforming growth factor beta (TGF-β) pathways by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). NF-κB and TGF-β production was decreased after treatment with quercetin, indicating that the antifibrotic effect of quercetin is associated with its ability to modulate NF-κB and TGF-β production. These results suggest that quercetin may be an effective therapeutic strategy in the treatment of patients with liver damage and fibrosis.

Topics: Alanine Transaminase; Animals; Antioxidants; Aspartate Aminotransferases; Collagen; Concanavalin A; Disease Models, Animal; Female; Hepatic Stellate Cells; Hepatitis; Liposomes; Liver Cirrhosis; Mice, Inbred BALB C; Mitogens; NF-kappa B; Quercetin; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta

Halofuginone (HF) is an active component of extracts derived from the plant alkaloid febrifugine and has shown therapeutic promise in animal models of fibrotic disease. Our main objectives were to clarify the suppressive effect of HF on concanavalin A (ConA)-induced liver fibrosis. ConA injection into the tail vein caused a great increase in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, while orally administration of HF significantly decreased the levels of the transaminases. In addition, the levels of hyaluronic acid (HA), procollagen III (PCIII) and TGF-β1 in the serum and collagen I, α-SMA, tissue inhibitors of metalloproteinase 2 (TIMP2) and Smad3 in the liver tissue were significantly lowered with the treatment of HF. Histological examination also demonstrated that HF significantly reduced the severity of liver fibrosis. Since ConA-induced liver fibrosis is caused by the repeated activation of T cells, immunomodulatory substances might be responsible for the suppressive effect of HF. We found that the production of nuclear factor (NF)-kB in the serum was increased in ConA-treated group, while decreased significantly with the treatment of HF. The changes of inflammatory cytokines tumor necrosis factor (TNF-α), IL-6 and IL-1β in the serum followed the same rhythm. All together, our findings indicate that orally administration HF (10ppm) would attenuate the liver fibrosis by suppressing the synthesis of collagen I and inflammation-mediated liver injury.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Concanavalin A; Drugs, Chinese Herbal; Interleukin-1beta; Interleukin-6; Liver Cirrhosis; Male; Piperidines; Quinazolinones; Rats, Wistar; Transforming Growth Factor beta1

The aims of this study were to elucidate the immunomodulatory effects of glycyrrhizin (GL) on CD4(+)T cell responses during liver fibrogenesis. To obtain in vivo evidence about the effects of GL on CD4(+)T cells in livers and spleens of concanavalin A (ConA)-induced mouse model, mice were administrated with ConA together with or without GL for 8 weeks. Mice treated with GL dramatically prevented liver inflammation and fibrosis. Besides, GL inhibited the infiltration of T helper (Th) cell type 1, Th2, Th17 and regulatory T cells (Treg) in livers and spleens of mouse fibrosis models, and regulated the Th1/Th2 and Treg/Th17 balances respectively to a relative dominance of Th1 and Treg lineages in livers. Moreover, GL dramatically enhanced the antifibrotic cytokine interferon (IFN)-γ and interleukin (IL)-10. GL at a concentration of 10 or 100 μg/mL was respectively incubated with ConA-stimulated splenic CD4(+)T cells in vitro, and JNK inhibitor (SP600125), ERK inhibitor (U0126), p38 inhibitor (SB203580) or PI3K/AKT inhibitor (LY29400225) was added during the incubation. Notably, GL not only inhibited ConA-induced proliferation of splenic CD4(+)T cells but also enhanced the mRNAs of IFN-γ and IL-10 in these cells. Be similar to the effects of GL, SP600125, U0126 and LY29400225, however not SB203580, also inhibited ConA-induced CD4(+)T cell proliferation, indicating the involvement of JNK, ERK and PI3K/AKT in this process. Moreover, GL significantly inhibited ConA-induced phosphorylation of JNK, ERK and PI3K/AKT in vitro. Collectively, GL might alleviate liver injury and fibrosis progression via regulation of CD4(+)T cell response in JNK, ERK and PI3K/AKT-dependent pathways.

Topics: Animals; CD4-Positive T-Lymphocytes; Cell Proliferation; Concanavalin A; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation; Glycyrrhizic Acid; Interferon-gamma; Interleukin-10; Liver Cirrhosis; MAP Kinase Kinase 4; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Phosphatidylinositol 3-Kinases

Alpha-calcitonin gene-related peptide (alphaCGRP) is a 37-amino acid pleiotropic peptide that we previously showed to exert a hepatoprotective effect during concanavalin A (Con A)-induced acute hepatitis. In the present study, we used alphaCGRP(-/-) mice to further investigate the antifibrogenic and hepatoprotective effects of endogenous alphaCGRP in Con A-induced chronic hepatitis.. Chronic hepatitis was induced in alphaCGRP(-/-) and wild-type mice by repeated administration of Con A. Serum transaminases were measured to assess hepatic injury. The severity of fibrosis and the activation of hepatic stellate cells (HSCs) were analysed by Masson trichrome staining and immunohistochemical staining of alpha-smooth muscle actin (alpha-SMA) respectively. Altered expression of fibrosis- and inflammation-related genes was evaluated using a quantitative real-time polymerase chain reaction. Activation and proliferation of HSCs were analysed using both primary cultured HSCs from the mice and the LI90 HSC cell line.. alphaCGRP(-/-) mice showed more severe liver fibrosis than wild-type mice in a Con A-induced chronic hepatitis model. In histological and gene expression analyses, alphaCGRP(-/-) mice showed greater inflammatory and fibrotic changes, greater HSC activation and a higher incidence of apoptosis among nonparenchymal cells than wild-type mice.. Endogenous alphaCGRP mitigates liver fibrosis in chronic hepatitis induced by repeated administration of Con A. alphaCGRP could be a useful therapeutic target for the treatment of chronic hepatitis.

Topics: Animals; Calcitonin Gene-Related Peptide; Cell Line; Chemical and Drug Induced Liver Injury, Chronic; Concanavalin A; DNA Primers; Hepatic Stellate Cells; Humans; Immunohistochemistry; Liver Cirrhosis; Mice; Mice, Knockout; Reverse Transcriptase Polymerase Chain Reaction

Previous studies have shown that transforming growth factor-beta 1 (TGF-beta1) is the most potent means of stimulating liver fibrogenesis by myofibroblast-like cells derived from hepatic stellate cells. Thus, TGF-beta1 could be a target for treating hepatic fibrosis. This study aimed to investigate the inhibitory effects of specific TGF-beta1 small interference RNA (siRNA) on immune hepatic fibrosis induced by Concanavalin A (Con A) in mice.. Three short hairpin RNAs targeting different positions of TGF-beta1 were designed and cloned to the plasmid pGenesil-1 to obtain three recombinant expression vectors (pGenesil-TGF-beta1-m1, pGenesil-TGF-beta1-m2 and pGenesil-TGF-beta1-m3). Thirty male Kunming mice were randomly divided into 6 groups: normal, model, control, and three treatment groups. The immune hepatic fibrosis models were constructed by injecting Con A via the tail vein at 8 mg/kg per week for 6 weeks. At weeks 2, 4 and 6, pGenesil-TGF-beta1-m1, pGenesil-TGF-beta1-m2 or pGenesil-TGF-beta1-m3 was injected by a hydrodynamics-based transfection method via the tail vein at 0.8 ml/10 g within 24 hours after injection of Con A in each of the three treatment groups. The mice in the control group were injected with control plasmid pGenesil-HK at the same dose. All mice were sacrificed at week 7. The levels of hydroxyproline in liver tissue were determined by biochemistry. Liver histopathology was assessed by Van Gieson staining. The expression levels and localization of TGF-beta1, Smad3, and Smad7 in liver tissue were detected by immunohistochemistry. The expression of TGF-beta1, Smad3, Smad7 and alpha-smooth muscle actin (alpha-SMA) mRNAs in the liver were assessed by semi-quantitative RT-PCR.. The levels of hydroxyproline in the liver tissue of the treatment groups were lower than those of the model group (P<0.01). Histopathologic assay showed that liver fibrogenesis was clearly improved in the treatment groups compared with the model group. The expression levels of TGF-beta1 and Smad3 of liver tissue were also markedly lower in the treatment groups than in the model group (P<0.01), while the levels of Smad7 were higher in the treatment groups than in the model group (P<0.01). RT-PCR further showed that the expression of TGF-beta1, Smad3 and alpha-SMA mRNA was significantly inhibited in the treatment groups compared with the model group, while the levels of Smad7 were increased. There was no difference in the above parameters among the three treatment groups or between the control and model groups (P>0.05), but the inhibitory effect of pGenesil-TGF-beta1-m1 was the highest among the treatment groups.. Specific siRNA targeting of TGF-beta1 markedly inhibited the fibrogenesis of immune hepatic fibrosis induced by Con A in mice. The anti-fibrosis mechanisms of siRNAs may be associated with the down-regulation of TGF-beta1, Smad3 and alpha-SMA expression and up-regulation of Smad7 expression in liver tissue, which resulted in suppressing the activation of hepatic stellate cells.

Topics: Actins; Animals; Concanavalin A; Down-Regulation; Hydroxyproline; Immunohistochemistry; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred Strains; Plasmids; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; RNA, Small Interfering; Smad3 Protein; Transforming Growth Factor beta1

A pivotal role of phosphoinositide 3-kinase-gamma (PI3Kgamma) in inflammatory cell activation and recruitment makes it an attractive target for immunomodulatory therapy. In present study we investigated the therapeutic efficiency of AS605240, a selective PI3Kgamma inhibitor, on hepatitis and liver fibrosis in murine models induced by concanavalin A (ConA). Orally administration of AS605240 significantly improved survival, decreased the serum levels of alanine aminotransaminase (ALT), prevented inflammatory infiltration to liver in ConA-induced hepatitis. TNF-alpha and IFN-gamma at protein levels in serum and mRNA levels in liver were markedly reduced. Downregulated phospho-Akt level of inflammatory cells infiltrating the liver by AS605240 treatment was detected by immunohistochemistry analysis in liver and further confirmed by Western blotting analysis in splenocytes. In ConA-induced chronic liver fibrosis model, accumulation of smooth-muscle actin (SMA)-expressing cells was partially inhibited by AS605240 treatment. These observations suggest that AS605240 might be of therapeutic value for the treatment of ConA-induced hepatic injury.

Topics: Acute Disease; Animals; Chemical and Drug Induced Liver Injury; Class Ib Phosphatidylinositol 3-Kinase; Concanavalin A; Cytokines; Enzyme Inhibitors; Isoenzymes; Liver; Liver Cirrhosis; Mice; Mitogens; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Quinoxalines; Thiazolidinediones

The in vitro response of peripheral blood mononuclear cells (PBMCs) to the suppressive effects of calcineurin inhibitors is known to correlate with the clinical efficacy of drugs used in renal transplantations. The present study was conducted to examine the differences of PBMC responses to calcineurin inhibitors between chronic renal failure (CRF) patients awaiting renal transplantation and cirrhosis patients awaiting liver transplantation. The study included 99 CRF patients awaiting renal transplantation and 27 cirrhosis patients awaiting liver transplantation. Twenty milliliters of venous blood was taken 1-7 days before transplantation. The in vitro drug concentrations giving 50% inhibition of PBMC blastogenesis stimulated with concanavalin A (IC(50)s) were calculated. The suppressive effects of tacrolimus against PBMC blastogenesis were more than 10-100 times stronger than those of cyclosporine. The median IC(50) value for cyclosporine against the CRF PBMCs was not significantly different from the median IC(50) value against the cirrhosis PBMCs. In contrast, tacrolimus sensitivity in cirrhosis PBMCs is approximately seven times higher than that in CRF PBMCs. The median IC(50) value for tacrolimus against cirrhosis PBMCs was significantly lower and therefore the effect was stronger in comparison to the CRF PBMCs (p < 0.001). These data suggest that the PBMCs of cirrhosis patients, in comparison to those of CRF patients, are highly sensitive to the suppressive effect of tacrolimus. However, PBMC sensitivity to cyclosporine was not significantly different between the CRF and cirrhosis patients. These observations raise the possibility that treatment with tacrolimus, rather than cyclosporine, may therefore be a better choice to reduce the risks of allograft rejection in liver transplantation.

Topics: Aged; Calcineurin; Calcineurin Inhibitors; Concanavalin A; Cyclosporine; Enzyme Inhibitors; Female; Humans; Immunosuppressive Agents; Kidney Failure, Chronic; Kidney Transplantation; Liver Cirrhosis; Liver Transplantation; Lymphocyte Activation; Male; Middle Aged; Tacrolimus

To investigate the molecular mechanism of the inhibitory effect of curcumine on the migration and invasion of hepatic stellate cells (HSC).. Rat hepatic stellate cells were cultured and activated with ConA. Matrix metalloproteinase-2 (MMP-2) expression and activity was determined by Western blot and gelatin zymography. Migration and invasion of HSC was assessed by wound healing assay and modified Boyden chamber assay.. Curcumine reduced the level and activity of MMP-2 expression in activated HSC in a dose-dependent manner. When treated with 25, 50 or 100 micromol/L curcumine, the expression of MMP-2 was reduced by 21.8%+/-5.1%, 65.5%+/-9.2% or 87.9%+/-11.5% (P < 0.05), and the activity of MMP-2 was also significantly reduced by curcumine. Migration and invasion of activated HSC was also inhibited by curcumine in a dose-dependent way. When treated with 25, 50 or 100 micromol/L curcumine, the migration of activated HSC was reduced by 27.5%+/-5.8%, 54.4%+/-7.6% or 67.1%+/-9.3% (P < 0.05), and the invasion of activated HSC was also significantly reduced by curcumine.. Curcumine inhibits migration and invasion of activated HSC by reducing MMP-2 expression and activity.

Topics: Animals; Blotting, Western; Cell Movement; Cells, Cultured; Concanavalin A; Curcumin; Dose-Response Relationship, Drug; Hepatic Stellate Cells; Liver Cirrhosis; Matrix Metalloproteinase 2; Rats; Tissue Inhibitor of Metalloproteinase-2

Tenascin-C (TNC), an extracellular matrix glycoprotein, is upregulated in chronic liver disease. Here, we investigated the contribution of TNC to liver fibrogenesis by comparing immune-mediated hepatitis in wild-type (WT) and TNC-null (TNKO) mice. Eight-week-old BALB/c mice received weekly intravenous injections of concanavalin A to induce hepatitis, and were sacrificed one week after the 3rd, 6th, 9th, and 12th injections. In WT livers, immunohistochemical staining revealed a gradual increase in TNC deposition. TNC mRNA levels also increased sequentially and peaked after the 9th injection. Collagen deposition, stained with picrosirius red, was significantly less intense in TNKO mice than in WT mice, and procollagen I and III transcripts were significantly upregulated in WT mice compared with TNKO mice. Inflammatory infiltrates were most prominent after the 3rd-6th injections in both groups and were less intense in TNKO mice than in WT mice. Interferon-gamma, tumour necrosis factor-alpha, and interleukin-4 mRNA levels were significantly higher in WT mice than in TNKO mice, while activated hepatic stellate cells (HSCs) and myofibroblasts, a cellular source of TNC and procollagens, were more common in WT livers. Transforming growth factor (TGF)-beta1 mRNA expression was significantly upregulated in WT mice, but not in TNKO mice. In conclusion, TNC can promote liver fibrogenesis through enhancement of inflammatory response with cytokine upregulation, HSC recruitment, and TGF-beta expression during progression of hepatitis to fibrosis.

Topics: Animals; Concanavalin A; Female; Hepatitis, Chronic; Immunohistochemistry; Interferon-gamma; Interleukin-4; Liver Cirrhosis; Lymphocytes; Mice; Mice, Inbred BALB C; Mice, Knockout; Procollagen; Reverse Transcriptase Polymerase Chain Reaction; RNA; Tenascin; Transforming Growth Factor beta1

The administration of concanavalin A (ConA) induces severe hepatic fibrosis in mice. Tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and interleukin 4 (IL-4) were the key cytokines involved in the process. The aim of this research was to explore the effects and the mechanisms of IL-18 and anti-IL-18 on hepatic fibrosis in a ConA induced hepatic fibrosis model in BABL-C mice.. One hundred eighty BABL-C mice were randomly divided into five groups (Group a, b, c, d, e). The mice were administered saline, immunoglobulin G, ConA, IL-18 + ConA, Anti-IL-18 + ConA, respectively. At 1, 7, 14, 21 wk, the levels of serum alanine aminotransferase, TNF-alpha, IFN-gamma, IL-4, matrix metalloproteinase (MMP)-2-RNA, and tissue inhibitor of metalloproteinase-1-mRNA were measured.. The levels of serum TNF-alpha and IFN-gamma detected in the IL-18 + ConA group was higher than in the anti-IL-18 + ConA group (P < 0.05). Similarly, the levels of MMP-2-RNA and tissue inhibitor of metalloproteinase-1-mRNA expressed in IL-18 + ConA group was higher than in the anti-IL-18 + ConA group (P < 0.05). A majority of these cytokines was secreted by CD4(+)T cells.. The immunological response to hepatic fibrosis by repeated injection of ConA in the mouse model was aggravated by IL-18 and blocked by anti-IL-18.

Topics: Animals; Antibodies; Apoptosis; CD4-Positive T-Lymphocytes; Cell Proliferation; Concanavalin A; Disease Models, Animal; Interferon-gamma; Interleukin-18; Interleukin-4; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred BALB C; Mitogens; Random Allocation; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1; Tumor Necrosis Factor-alpha

Topics: Animals; Concanavalin A; Immunoglobulin G; Interleukin-18; Liver Cirrhosis; Mice; Mice, Inbred BALB C

The acute phase response to injury or infection results in alterations in the expression of the plasma proteins produced by the liver. Many of these biomolecules are glycosylated with oligosaccharide chains covalently attached to the polypeptide backbone and the extent and composition of this glycosylation can be altered in a disease-dependent manner. Of particular interest is the observation that the acute phase glycoprotein, alpha-1-acid glycoprotein (AGP) has altered glycosylation in several physiological and pathological conditions. It is posited that changes induced in liver diseases may reflect disease severity and may therefore act as a non-invasive marker of fibrosis. This study has investigated the glycosylation of AGP in the plasma of people with varying degrees of cirrhosis and fibrosis. Hyperfucosylation was observed in all disease samples in comparison to normal plasma and was significantly increased in cirrhosis. Both sialic acid and N-acetylgalactosamine (GalNAc) were negatively associated with fibrosis. Two samples were found to express GalNAc, which as a constituent of the glycosylation of serum proteins is rare. In conclusion, fucose, sialic acid and other aspects of the glycosylation of AGP are influenced by the degree of fibrosis and as such may prove a valuable prognostic indicator of the development of cirrhosis.

Topics: Acetylgalactosamine; Adult; Biomarkers; Chromatography, Ion Exchange; Concanavalin A; Enzyme-Linked Immunosorbent Assay; Fucose; Glycosylation; Humans; Liver Cirrhosis; Middle Aged; N-Acetylneuraminic Acid; Orosomucoid

Apoptosis of T cells contributes to the immune homeostasis in inflamed organs. A prominent T-cell infiltration is usually seen in human chronic active hepatitis, being associated with liver fibrosis. In order to demonstrate T-cell apoptosis in the hepatic fibrotic tissue, we induced T-cell infiltration in the fibrotic liver of the rat by injecting concanavalin A (Con A), a T-cell mitogen. Lymphocytes increased in number with a peak at 1 day, preferentially distributing in the fibrotic tissue rather than the parenchyma. They consisted of CD4-positive and CD8-positive cells, and gave the feature of lymphoblasts. Double staining for CD3 and TUNEL demonstrated that T cells underwent apoptosis. Apoptotic cells were more frequent in the fibrotic livers than the normal livers, and were spatially associated with alpha-smooth muscle actin-positive myofibroblast-like cells that possibly derived from hepatic stellate cells (HSCs) and portal fibroblasts through activation. In vitro experiments demonstrated that lymphocyte apoptosis was more frequently induced in the co-culture of Con A-activated splenic T cells/activated HSCs compared to that induced in activated T cells/quiescent HSCs or resting T cells/activated HSCs. The present results indicate that T cells which have extravasated and infiltrated the hepatic fibrotic tissue undergo apoptosis probably through an interaction with myofibroblast-like cells, suggesting the regulatory role of the latter cells in T-cell accumulation in the fibrotic liver.

Topics: Actins; Animals; Apoptosis; CD3 Complex; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Communication; Cell Differentiation; Chemotaxis, Leukocyte; Concanavalin A; Disease Models, Animal; Fibroblasts; Hepatitis; In Situ Nick-End Labeling; Liver Cirrhosis; Male; Microscopy, Electron; Mitogens; Rats; Rats, Wistar; T-Lymphocytes

Heterogeneous reactivity of human serum transferrin (Tf) with lectins was analysed using patient sera to determine whether it can be used to distinguish patients with hepatocellular carcinoma (HCC) from those with liver cirrhosis (LC). Microheterogeneity of Tf was analysed by crossed immunoaffinity electrophoresis (CIAE) with concanavalin A (Con A) and Lens culinaris agglutinin (LCA). Sample sera from 58 patients with HCC, 43 patients with LC and 10 normal controls were used in this study and the results were evaluated statistically. The increments of Con A-non-reactive (C1) and -weakly reactive (C2) species of Tf were observed in HCC compared with those of LC and Norm. Significant increase in the combined percentage of Con A- C1 + C2 species was also revealed in HCC (35.5 +/- 8.5%, mean+/-s.d.) compared with those of LC (29.1 +/- 6.8%; P < 0.001) and normal controls (17.1 +/- 2.3%; P < 0.001). The elevation of LCA-reactive (L2) species of Tf was recognized in HCC (8.2 +/- 3.8%) in comparison with those of LC (4.8 +/- 3.1%; P < 0.001) and normal controls (1.3 +/- 1.7%; P < 0.001). The increment of C1 + C2 species and/or L2 species of Tf was observed in 78% (sensitivity) of patients with HCC. The specificity, the positive predictive value and the overall accuracy were 81, 88 and 72%, respectively. Positive ratio of C1 + C2 and/or L2 species was 77 and 70% in alpha-fetoprotein low and -high producing HCC patients, respectively. These results indicate that the microheterogeneity analysis of human serum Tf is useful for distinguishing patients with HCC from those with LC and normal controls.

Topics: Aged; alpha-Fetoproteins; C-Reactive Protein; Carcinoma, Hepatocellular; Concanavalin A; Electrophoresis; Female; Humans; Lectins; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Plant Lectins; Reference Values; Transferrin

We analyzed the Con A affinity of serum AFP in patients with a serum AFP concentration greater than 50ng/ml by antibody affinity electrophoresis and Western blotting to distinguish hepatocellular carcinoma (HCC) from benign chronic liver diseases (CLD). Of 164 patients with HCC, 48 (29.3%) had a single band, while 116 (70.7%) had multiple bands. All but three of 65 patients with cirrhosis had a single band. All but one of 32 patients with chronic hepatitis had a single band. We concluded that multiple AFP bands are diagnosis of HCC. This method is a useful assay for distinguishing HCC from CLD.

Topics: Adult; Aged; alpha-Fetoproteins; Blotting, Western; Carcinoma, Hepatocellular; Chronic Disease; Concanavalin A; Diagnosis, Differential; Electrophoresis, Agar Gel; Female; Hepatitis; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Sensitivity and Specificity

1. Using crossed immunoaffinity electrophoresis with free concanavalin A in the first dimension, we studied the microheterogeneity of alpha 1-antichymotrypsin due to various glycoforms in sera from patients with various liver diseases and after liver transplantation. 2. Studies by isoelectric focusing and immunoblotting and by crossed immunoelectrophoresis without concanavalin A in the first dimension allowed us to show that there is no dramatic variation in electrophoretic heterogeneity of alpha 1-antichymotrypsin in the serum of patients with liver diseases or after liver transplantation when compared with that of normal subjects. Therefore the heterogeneity observed in crossed immunoaffinity electrophoresis is due to various interactions with concanavalin A. 3. The results were expressed as the ratio of concanavalin A non-reactive glycoforms plus concanavalin A weakly reactive glycoforms to concanavalin A reactive glycoforms, called R alpha 1-ACT. R alpha 1-ACT was significantly higher in patients with cirrhosis (n = 53) when compared with normal subjects (n = 30). The median R alpha 1-ACT was 1 (range 0.72-1.25) in normal subjects. It was 1.6 (range 1.18-3.02), 1.45 (range 0.65-4.12) and 2.24 (range 1.03-19) in cirrhosis of Child's grade A, B and C, respectively. There was a dramatic decrease in glycoforms with mostly biantennary glycans in some patients with Child's grade C cirrhosis. Serum levels of alpha 1-antichymotrypsin were lower than normal only in some patients with Child's grade C cirrhosis. 4. Among the patients with acute viral hepatitis studied (n = 17), five were studied longitudinally.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Aged; Aged, 80 and over; alpha 1-Antichymotrypsin; Concanavalin A; Female; Glycosylation; Hepatitis, Viral, Human; Humans; Immunoelectrophoresis, Two-Dimensional; Isoelectric Focusing; Liver Cirrhosis; Liver Diseases; Liver Transplantation; Male; Middle Aged

Isozymic alteration of serum cholinesterase (ChE) was investigated in patients with chronic liver diseases using affinity electrophoresis with concanavalin A (Con A) or wheat germ agglutinin (WGA). On Con A-containing agarose gel electrophoresis, three bands with enzyme activity (named bands I to III, from the anodic side to the cathodic) were observed in sera of normal controls. Disappearance of band II was observed in 50% (15/30) of cirrhotic patients, but only one of 20 patients with chronic hepatitis lacked band II of the serum ChE isozymes. Meanwhile, WGA-containing agarose gel electrophoresis revealed that normal controls had four ChE isozymes (named bands I to IV from the anodic side to the cathodic). These four isozymes were also observed in patients with chronic hepatitis. However approximately 67% (20/30) of cirrhotic patients lacked band II of ChE isozymes. When these two affinity electrophoreses were used in combination, 22 (73%) of 30 cirrhotic patients had isozymic alteration of their serum ChE on either Con A-containing or WGA-containing agarose gel electrophoresis, or both. Thus, affinity electrophoreses with Con A and WGA seemed to be useful methods in differentiating liver cirrhosis from chronic hepatitis.

Topics: Cholinesterases; Clinical Enzyme Tests; Concanavalin A; Diagnosis, Differential; Electrophoresis, Agar Gel; Hepatitis, Chronic; Humans; Isoenzymes; Liver Cirrhosis; Wheat Germ Agglutinins

Topics: Carcinoma, Hepatocellular; Chronic Disease; Concanavalin A; Diagnosis, Differential; Electrophoresis; Electrophoresis, Cellulose Acetate; gamma-Glutamyltransferase; Hepatitis; Humans; Isoenzymes; Liver Cirrhosis; Liver Neoplasms; Octoxynol; Polyethylene Glycols; Sensitivity and Specificity

1. alpha 1-acid glycoprotein (AAG) concentration and molecular heterogeneity, and oxprenolol protein binding were studied in serum of 15 healthy volunteers, 14 patients with lung carcinoma and 17 patients with liver cirrhosis. 2. The AAG serum concentration was increased to 180.7% in patients with lung cancer and decreased to 73.4% in cirrhotic patients as compared with controls (P less than 0.05). 3. The concanavalin A (conA) dependent heterogeneity of serum AAG was very similar in controls and patients with lung cancer: a ratio of 9/9/2 was obtained for the conA nonreactive, the conA weakly reactive and the conA strongly reactive subfraction respectively; in cirrhotic patients, the ratio shifted to 11/7/1. 4. The heterogeneity in electric charge, demonstrated by isoelectric focusing, was similar in the three groups of subjects: 70-80% of the focussed bands were found in the main three bands. 5. The binding of oxprenolol to serum proteins was increased in lung tumour patients and decreased in liver cirrhotic patients as compared with controls (P less than 0.05). There was no change in binding affinity and oxprenolol binding was significantly correlated to total AAG serum concentration and to the concentration of each of the conA dependent subtypes, in controls as well as in both patients groups.

Topics: Adult; Blood Proteins; Concanavalin A; Electrophoresis; Female; Humans; Immunoelectrophoresis; Isoelectric Focusing; Liver Cirrhosis; Lung Neoplasms; Male; Middle Aged; Orosomucoid; Oxprenolol; Protein Binding

We previously have shown [Takahashi & Kobayashi (1982) Hepatology 2, 249-254] that the administration of concanavalin A to mice with schistosomiasis caused liver collagen content to be reduced by 50%. Here we report the effects of concanavalin A and aggregated mouse myeloma IgG on liver lysyl oxidase activity and present further evidence concerning the possible mechanism by which the liver collagen content was decreased in infected-treated mice. The lysyl oxidase activity at 8 weeks after infection in both treated mice and untreated infected controls was about 28-fold greater than in the age-matched uninfected controls. The specific radioactivity of intracellular free [14C]proline, the rate of collagen synthesis, the ratio of collagenase-sensitive, protein-bound, hydroxyproline to proline of collagen and the intracellular degradation of newly synthesized collagen were similar in treated animals and in untreated infected controls. In contrast, the extracellular degradation of newly secreted collagen and the specific radioactivity of protein-bound [14C]hydroxyproline in the agent-treated groups were about 2-fold greater than those in the untreated infected controls. These results suggest that the observed 50% decrease in content of liver collagen of mice treated with the agents apparently was due to the increased extracellular degradation of newly secreted collagen.

Topics: Animals; Collagen; Concanavalin A; Female; Immunoglobulin G; Immunoglobulins; Liver; Liver Cirrhosis; Macromolecular Substances; Mice; Myeloma Proteins; Protein-Lysine 6-Oxidase

Topics: alpha-Fetoproteins; Concanavalin A; Hepatitis; Humans; Immunoelectrophoresis; Lectins; Liver Cirrhosis; Liver Neoplasms; Phytohemagglutinins

Mitogen-induced suppressor T lymphocyte function was evaluated in patients with chronic active hepatitis (CAH). The in vitro effect of the biological response modifier, thymosin fraction 5, on the suppressive activity of peripheral blood mononuclear cells (PBM) was also assessed. Suppressor cell activity was significantly decreased in patients with CAH when compared to controls (P less than 0.001). In the absence of the inducing mitogen, thymosin-treated PBM from both patients and controls promoted enhancement of tritiated thymidine uptake by cocultured allogeneic lymphocytes. When thymosin-treated mononuclear cells were mitogen-activated; patients, but not the controls, showed a marked increase in suppressor activity (P less than 0.001). These results indicate that the polypeptides contained in thymosin fraction 5 can promote a helper effect in patients and controls. Furthermore, PBM from patients with CAH contain a subset of lymphocytes that can express a suppressive function following thymosin treatment. We conclude that thymosin fraction 5 can promote an in vitro restoration of suppressor T cell function in patients with CAH.

Topics: Adolescent; Adult; Concanavalin A; Dose-Response Relationship, Drug; Female; Hepatitis, Chronic; Humans; Liver Cirrhosis; Liver Function Tests; Male; Middle Aged; Mitogens; Monocytes; Prednisone; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory; Thymosin; Thymus Hormones

Peripheral blood B cell response to staphylococcus aureus (SpA) Cowan I was evaluated in 8 healthy subjects, 8 patients with liver cirrhosis (LC), and 2 patients with chronic active hepatitis (CAH) with hypergammaglobulinemia (greater than 2 g/dl). In control studies it was shown that stimulation by SpA Cowan I was much less T cell-dependent than that induced by pokeweed mitogen (PWM) or con-canavalin A (ConA) when the amounts of immunoglobulins (Ig) secreted into culture supernatants were measured by radioimmunoassay (RIA) and the blastogenic response was measured by incorporation of tritiated thymidine. B cells from only 2 patients revealed increased Ig synthesis by SpA Cowan I stimulation. In the study of blastogenic response, increased DNA synthesis of B cells by SpA Cowan I stimulation was observed in 2 patients and decreased DNA synthesis in 3 patients. The remaining patients demonstrated normal range response. There was no correlation between B cell response to SpA Cowan I and clinical data such as gammaglobulin level in the patients studied. These studies indicate that B cell function remains intact in many patients with chronic liver disease with hypergammaglobulinemia.

Topics: Adult; Aged; B-Lymphocytes; Chronic Disease; Concanavalin A; Female; Hepatitis, Chronic; Humans; Immunoglobulin G; Immunoglobulin M; Liver Cirrhosis; Lymphocyte Activation; Male; Middle Aged; Pokeweed Mitogens; Staphylococcus aureus

Topics: Adult; Aged; alpha-Fetoproteins; Chronic Disease; Concanavalin A; Female; Ferritins; Hepatitis B; Humans; Liver Cirrhosis; Liver Cirrhosis, Alcoholic; Liver Diseases; Liver Neoplasms; Male; Middle Aged

Hypergammaglobulinaemia (HGG) is frequently found in patients with hepatic cirrhosis (HC). Using an assay system of in vitro PWM-stimulated immunoglobulin (Ig) production, the amounts of IgG, IgA, and IgM produced by peripheral blood lymphocytes (PBL) from 15 HBs Ag-negative patients with HC and from 16 age-matched healthy subjects were quantitated by radioimmunoassay. We found that PBL from patients with HC produced significantly greater amounts of IgG (P less than 0.05) but not IgA or IgM than did those from control subjects. This increased IgG production by PBL from patients with HC was attributed to enhanced T helper activity and not to enhanced B cell function. We also searched for defects in naturally occurring suppressor T cell activity which is sensitive to irradiation. Irradiation-induced enhancement for IgG production was significantly lower in patients with HC compared with age-matched control subjects (P less than 0.01). Similarly, we examined the effect of Con A-induced suppressor T cells on the in vitro PWM-stimulated IgG production by allogeneic PBL and observed the decrease of Con A-induced suppressor T cell activity in patients with HC (P = 0.01). We conclude, therefore, that the increased serum levels of Ig, particularly IgG in patients with HC may result from in part on the basis of depressed ability of naturally occurring suppressor T cells or Con A-induced suppressor T cells to suppress Ig production.

Topics: Adult; B-Lymphocytes; Concanavalin A; Female; Humans; Immunoglobulins; Liver Cirrhosis; Lymphocyte Activation; Male; Middle Aged; Pokeweed Mitogens; T-Lymphocytes; T-Lymphocytes, Regulatory

Topics: Concanavalin A; Hepatitis; Humans; Liver Cirrhosis; Liver Diseases; Lymphocyte Activation

The carcinoembryonic antigen (CEA) of normal and pathological tissue extracts was separated into two variants, Concanavalin A reactive (CEAr) and non-reactive CEA (CEAn) by affinity chromatography on Con A Sepharose columns. CEAr was the quantitatively predominant variant. CEAn varied in concentration between 0.2 and 6 percent of the total CEA activity. The affinity of CEAn for anti-CEA antibodies was significantly lower than that of CEAr. Pooled extracts of primary adenocarcinomas of the colon contained CEAn in the lowest concentration and with the least affinity for antibodies. It is suggested that a deficiency and/or steric blocking of alpha-D-mannopyranosyl residues in CEAn reduce the affinities for both antibodies and Con A.

Topics: Adenocarcinoma; Carcinoembryonic Antigen; Chromatography, Affinity; Colonic Neoplasms; Concanavalin A; Fetus; Genetic Variation; Humans; Liver; Liver Cirrhosis; Liver Neoplasms; Lung; Neoplasm Metastasis; Organ Specificity; Radioimmunoassay; Spleen