This report describes surface properties of several cell membranes tested by lectininduced agglutination reactions which were quantitated using the microquantitative particle counter agglutination assay of Davis et al. (1976). The quantitative assays of concanavaalin A (con A) induced agglutination were performed for rabbit erythrocyte, rat erythrocyte, human erythrocyte, and sarcoma 180 mouse ascites cells. The percent agglutination versus the con A concentration revealed a sigmoid curve in all cases, but the steepness of the sigmoid curve is variable depending on the cell types. It varies even with the same cell but in different species. Optimum cell concentration was (0.92-0.95) x 10(7) cells/ml final concentration in the hanging drop, for rabbit erythrocytes, (0.77-1.64) x 10(7) cells/ml for rat erythrocytes, (1.59-2.7) x 10(7) cells/ml for human erythrocytes and (0.23-0.39) x 10(7) cells/ml for sarcoma 180 mouse ascites cells. When minimal and maximal agglutination percentages were defined as the concentration of con A/ml/1 x 10(6) cells corresponding to 10% and 95% agglutination, minimal and maximal agglutination occured at 0.56 ug, 19.98 ug for human erythrocytes at 0.56 ug, 224 ug for rat erythrocytes at 0.08 ug, 1.43ug for rabbit erythrocytes at 0.12 ug, 14.8 ug for sarcoma 180-mouse ascites cells respectively. The order of inhibitory activity of alpha-methyl-D-mannopyranoside (alphaMM) for each corresponding cells from the highest inhibition was human erythrocytes, rat erythrocytes, sarcoma 180 mouse ascites cells and rabbit erythrocytes. The concentrations of alphaMM required for 50% inhibition per ml of the final concentration in the hanging drop per 1 x 10(7) cells were 0.565 umoles for rabbit erythrocytes, 0.072 umoles for rat erythrocytes, 0.018 umoles for human erythrocytes and 3.677 umoles for sarcoma 180 mouse ascites cells, respectively. From our experimental results we conclude that the cytoagglutination activity was increased with con A, the inhibitory activity with alphaMM in the presence of con A was decreased, however the sarcoma 180 mouse ascites cells revealed a contradictory result, and might be due to the topological distribution of agglutination site changes to a distribution more favorable for agglutination.
Agglutination ; Agglutination Tests ; Animal ; Cell Membrane/immunology* ; Concanavalin A/pharmacology* ; Erythrocytes/immunology ; Human ; Methylmannosides/pharmacology ; Mice ; Rabbits ; Rats ; Sarcoma 180/immunology ; Surface Properties

AIMTo observe the effects of acute hypoxia and adenosine on splenic T lymphocyte proliferation.

METHODSWistar rats were divided into control and hypoxic group, and the latter were exposed to hypoxia (5000 m simulated high altitude, 23 h/d). Three days later, spleen cells were collected and stimulated by 5.0 microg/ml and 2.5 microg/ml concanavalin A (ConA) to determine the splenocyte proliferation. The proliferation was also observed after addition of different amount of adenosine to culture medium.

RESULTSAcute hypoxia and adenosine had marked inhibitory effect on mitogenic response to Con A in splenic T cells, and the inhibitory effect induced by adenosine displayed concentration-dependent.

CONCLUSIONAcute hypoxia may impair the T cell function and adenosine could be involved in this process.

Adenosine ; pharmacology ; Animals ; Cell Proliferation ; drug effects ; Concanavalin A ; pharmacology ; Culture Media ; chemistry ; Hypoxia ; Male ; Rats ; Rats, Wistar ; Spleen ; cytology ; T-Lymphocytes ; cytology ; drug effects

Animals ; Concanavalin A ; adverse effects ; Immunoglobulin G ; blood ; Interleukin-18 ; blood ; immunology ; pharmacology ; Liver Cirrhosis ; blood ; chemically induced ; prevention & control ; Mice ; Mice, Inbred BALB C

OBJECTIVETo investigate the effect of membrane type-1 matrix metalloproteinase (MTI-MMP) on the invasive potential of breast cancer cell and analyze its mechanisms.

METHODSAfter treatment of breast cancer MDA-MB-453 cell line with concanavalin A ( ConA, 20 microg/ml) for 24 h, MT1-MMP protein was detected in cancer cells by Western analysis and immunocytochemistry. MDA-MB-453 cells were cultured with exogenous latent proMMP-2 and MMP-2 activity was analyzed by gelatin zymography. The invasive potential of the tumor cells was measured with a membrane invasion culture system. Cancer cells of the cell line were divided into four groups: the control group treated by neither reagent, group ConA was only treated by ConA, group MMP-2 was treated only by MMP-2, and group ConA + MMP-2 was treated by both ConA and MMP-2. RESULTS The expression of MTI-MMP protein could be detected in groups ConA and ConA + MMP-2, but nothing was detected in control and group MMP-2. There was only 72 000 precursor form of MMP-2 in group MMP-2 and there were both 72 000 precursor form and 64 000 active enzyme form of MMP-2 in group ConA + MMP-2, but there was no forms of MMP-2 in the other two groups detected by gelatin zymography. The largest amount of cells penetrated through Matrigel was observed in group ConA + MMP-2 than in the other three groups.

CONCLUSIONMTI-MMP can remarkably promote the invasive potential of breast cancer cells mainly through its ability of activating latent proMMP-2 to degrade

Blotting, Northern ; Blotting, Western ; Breast Neoplasms ; enzymology ; genetics ; pathology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Concanavalin A ; pharmacology ; Female ; Humans ; Immunohistochemistry ; Matrix Metalloproteinase 14 ; genetics ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Neoplasm Invasiveness ; RNA, Messenger ; genetics ; metabolism

AIMTo provide further evidence for the synthesis of catecholamines (CAs) in lymphocytes and to investigate the effect of the endogenous CAs synthesized by lymphocytes on function of the lymphocytes themselves and the receptor mechanisms involved in the effect.

METHODSRT-PCR was performed to detect the expression of TH mRNA in the lymphocytes from the mesenteric lymph nodes of rats. Different concentrations of pargyline, an inhibitor of monoamine oxydase, and antagonists of alpha1-, alpha2-, beta1-, and beta2-adrenergic receptor (AR) were added to the lymphocyte cultures, and then proliferative response of the lymphocytes to mitogen concanavalin A (Con A) were measured via methyl-thiazole-tetrazolium (MTT) assay.

RESULTSThe lymphocytes could express TH mRNA, and the expression of TH mRNA was significantly higher in the Con A-activated lymphocytes than in the resting ones. The treatment of pargyline of 10(-6) and 10(-5) mol/L (not 10(-7) mol/L) notably attenuated Con A-induced lymphocyte proliferation. Beta2-AR antagonist ICI118551 (10(-7) and 10(-6) mol/L) completely blocked, but alpha1-AR antagonist corynanthine and alpha2-AR antagonist yohimbine (10(-7) and 10(-6) mol/L) partly blocked the suppressive effect of pargyline on the Con A-induced lymphocyte proliferation. Nevertheless, atenolol, an antagonist of beta1-AR, had no blocking effect on pargyline inhibition of lymphocyte proliferation.

CONCLUSIONLymphocytes have the ability to synthesize CAs and the ability is enhanced in the activated lymphocytes. The endogenous CAs synthesized by lymphocytes can inhibit T cell proliferation and the inhibition of T cells by the CAs is mediated predominantly by beta2-AR on the lymphocytes.

Animals ; Catecholamines ; biosynthesis ; physiology ; Cell Proliferation ; drug effects ; Concanavalin A ; pharmacology ; Female ; Lymphocyte Activation ; Lymphocytes ; metabolism ; Male ; Neuroimmunomodulation ; physiology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Adrenergic, beta ; physiology ; T-Lymphocytes ; cytology ; immunology ; Tyrosine 3-Monooxygenase ; genetics ; metabolism

The aim of this study was to investigate the effect of the novel antihepatitis drug bicyclol on the gene expression profiles in concanavalin A (Con A) intoxicated mice by using cDNA microarray analysis. Bicyclol (250 mg x kg(-1)) was given orally to mice three doses before Con A intravenous injection (26.5 mg x kg(-1)). Serum levels of aminotransferases were examined by biochemical methods. Liver mRNA was extracted and reversely transcribed to cDNA with the incorporation of labeled Cy3-dUTP and Cy5-dUTP, separately. The probes were hybridized to the cDNA microarray. The acquired image was scanned and analyzed by Cenepix Pro 3.0 software. Microarray analysis showed that 287 genes exhibited differential expression in bicyclol group, in which 121 genes were up-regulated and 166 genes were down-regulated comparing with that of untreated Con A intoxicated mice. The differential gene expression after bicyclol treatment was involved in the biotransformation, protein synthesis, degradation and circadian rhythm, proliferation and signal transduction. Bicyclol might regulate a series of genes expressions in Con A intoxicated mice.
Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Biphenyl Compounds ; pharmacology ; Chemical and Drug Induced Liver Injury ; blood ; etiology ; genetics ; Concanavalin A ; Gene Expression Profiling ; Male ; Mice ; Mice, Inbred ICR ; Oligonucleotide Array Sequence Analysis ; Random Allocation

The purpose of the present study is to explore the protective effects of sodium butyrate (SB) pretreatment on concanavalin A (Con A)-induced acute liver injury in mice. The model animals were first administered intraperitoneally with SB. Half an hour later, acute liver injury mouse model was established by caudal vein injection with Con A (15 mg/kg). Then, levels of serous alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using standard clinical method by an automated chemistry analyzer, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were measured by ELISA, and pathological changes in hepatic tissue were observed by using HE staining and light microscopy. The expression and release of high-mobility group box 1 (HMGB1) were assessed by using reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry and ELISA. The results showed that the pretreatment of SB significantly protected Con A-treated mice from liver injury as evidenced by the decrease of serum ALT, AST (P < 0.01) and reduction of hepatic tissues necrosis. SB also decreased levels of serous TNF-α and IFN-γ (P < 0.01). Furthermore, the expression and release of HMGB1 were markedly inhibited by SB pretreatment (P < 0.05 or P < 0.01). These results suggest that the attenuating effect of SB on Con A-induced acute liver injury may be due to its role of reducing the TNF-α and IFN-γ production, and inhibiting HMGB1 expression and release.
Alanine Transaminase ; metabolism ; Animals ; Aspartate Aminotransferases ; metabolism ; Butyric Acid ; pharmacology ; Chemical and Drug Induced Liver Injury ; drug therapy ; Concanavalin A ; adverse effects ; Disease Models, Animal ; HMGB1 Protein ; metabolism ; Interferon-gamma ; metabolism ; Liver ; pathology ; Mice ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo explore the effect of Ronggan Mixture (RM) on immunoregulation and hepatocyte apoptosis-related factors in concanavalin A (Con A) induced acute immunological liver injury mice.

METHODSTotally 60 hepatitis B virus (HBV) transgenic mice were randomly divided into 6 groups, i.e., the blank control group, the model group, the RM group, the Herba Artemisiae Scopariae (HAS) group, the Yinchenhao Decoction (YD) group, and the Bifendate group, 10 mice in each group. The acute immunological liver injury model was established by tail vein injection of ConA. Fourteen days before modeling, normal saline was administered to mice in the blank control group and the model group. RM, YD, HAS decoction, and Bifendate solution was respectively given to mice in the RM group, the YD group, the HAS group, and the Bifendate group. The medication was performed once daily. One h after the last gastrogavage, phosphate buffer solution (PBS) was injected to mice in the blank control group from the tail vein. Modeling was conducted by injecting Con A at 3 microg/g body weight from the tail vein. Mice were sacrificed 8 h after modeling. Blood or tissue samples were collected to detect lab indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), tumor necrosis factor alpha (TNF-alpha), interferon gamma (INF-gamma), IL-4, IL-10, Fas, FasL, Bax, and bcl-2.

RESULTSThere was significant difference in all lab indicators between the normal group and the blank control group (P < 0.05, P < 0.01). Compared with the model group, ALT and AST levels were significantly lower in the RM group and the Bifendate group (P < 0.01); TBil significantly decreased in the RM group (P < 0.01). The expression level of TNF-alpha decreased in the RM group (P <0.05). The expression level of IFN-gamma decreased in the RM group and the YD group (P < 0.05). The expression level of IL-4 could be elevated in all medicated groups (P < 0.05). RM could elevate the expression level of IL-10 (P < 0.05). The expression level of Fas in the liver tissue decreased in the RM group and the YD group (P < 0.05). The expression level of FasL decreased and the expression of bcl-2 gene increased in the RM group (both P < 0.05). The expression level of Bax was down-regulated in the RM group and the YD group (P < 0.05). The ratio of bcl-2/Bax was up-regulated in the RM group (P < 0.05). Meanwhile, RM showed better effect in decreasing expressions of ALT and AST than HAS (P < 0.05). The effect of increasing IL-10 expression levels was better in the RM group than in the YD group (P < 0.01). The effect of decreasing expressions of Fas and FasL was better in the RM group than in the HAS group, the YD group, and the Bifendate group (P < 0.05). The effect of enhancing the expression of IL-10 in the liver tissue was better in the RM group than in the HAS group (P < 0. 05).

CONCLUSIONRM had protective effect on Con A induced acute immunological liver injury mice, which might be achieved by changing the immunological balance of Thl/Th2 factors (decreasing expressions of TNF-alpha and IFN-gamma, elevating expressions of IL-10 and IL-4) and regulating hepatocyte apoptosis-related factors (down-regulating gene expressions of Fas, FasL, and Bax; up-regulating bcl-2 gene expression, and up-regulating the bcl-2/Bax ratio).

Animals ; Apoptosis ; drug effects ; Chemical and Drug Induced Liver Injury ; immunology ; pathology ; Concanavalin A ; adverse effects ; Cytokines ; immunology ; Drugs, Chinese Herbal ; pharmacology ; Female ; Gene Expression ; Hepatocytes ; cytology ; drug effects ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic

OBJECTIVETo investigate the gene expression of MAPEG in the cortex of concanavalin A (Con A)-induced mouse immune inflammatory model and the effect of cyclosporine A (Cs A).

METHODSMale Balb/c mouse immune inflammation model was developed by intravenous injection of Con A (20 mg/kg). Cs A (150 mg/kg) was intravenously infected prior to Con A administration. The MAPEG expressions were determined by RT-PCR.

RESULTmGST1, mGST3, LTC(4)S, FLAP and mPGES-1 were detected by RT-PCR but not mGST2. Eight hours after Con A treatment, mGST1 level was up-regulated to 1.2 approximately 1.5 folds of control with or without Cs A treatment. mGST3ìLTC(4)S, FLAP and mPGES-1 mRNA levels were not influenced by Con A administration.

CONCLUSIONImmune mechanism may be not involved in mGST1 up-regulation in this model and Con A does not alter arachidonic acid metabolism in cortex.

5-Lipoxygenase-Activating Proteins ; Animals ; Brain ; metabolism ; Carrier Proteins ; genetics ; metabolism ; Concanavalin A ; toxicity ; Cyclosporine ; pharmacology ; Eicosanoids ; metabolism ; Glutathione ; metabolism ; Glutathione Transferase ; genetics ; metabolism ; Intramolecular Oxidoreductases ; genetics ; metabolism ; Male ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Prostaglandin-E Synthases

OBJECTIVETo evaluate the role of adiponectin in regulating tumor necrosis factor alpha (TNF alpha) production and preventing fulminant autoimmunological damage of hepatocytes following concanavalin A (Con A) injection into mice.

METHODSThree days after recombinant plasmids pAA-neo-mAd were injected into the mice via the tail veins, Con A was injected into the mice. Mice transfected with empty pAA-neo vector served as controls. The serum levels of alanine aminotransferase (ALT), TNF alpha and adiponectin were detected, and histological examination of livers was carried out at different time points after the Con A injection. All results were subjected to statistical analyses.

RESULTSHistological examinations showed that the damage in livers of mice with high serum adiponectin levels was milder than that of the controls. The serum levels of ALT and TNF alpha were both lower than those of the controls (P less than 0.01, respectively). Statistical analyses showed the serum levels of ALT was negatively related to the levels of adiponectin in the sera (r=-0.5034).

CONCLUSIONAdiponectin is effective in protecting hepatocytes from Con A-induced immunological injury. The mechanism of this protective effect may be caused by inhibiting the synthesis and/or release of TNF alpha.

Adiponectin ; blood ; pharmacology ; Alanine Transaminase ; blood ; Animals ; Concanavalin A ; adverse effects ; Female ; Immune System Diseases ; chemically induced ; pathology ; prevention & control ; Liver ; drug effects ; pathology ; Liver Diseases ; pathology ; prevention & control ; Mice ; Mice, Inbred BALB C ; Tumor Necrosis Factor-alpha ; blood