Human menstrual blood-derived stem cells alleviate autoimmune hepatitis via JNK/MAPK signaling pathway in vivo and in vitro / 医学前沿

Autoimmune hepatitis (AIH) is a severe globally distributed liver disease that could occur at any age. Human menstrual blood-derived stem cells (MenSCs) have shown therapeutic effect in acute lung injury and liver failure. However, their role in the curative effect of AIH remains unclear. Here, a classic AIH mouse model was constructed through intravenous injection with concanavalin A (Con A). MenSCs were intravenously injected while Con A injection in the treatment groups. The results showed that the mortality by Con A injection was significantly decreased by MenSCs treatment and liver function tests and histological analysis were also ameliorated. The results of phosphoproteomic analysis and RNA-seq revealed that MenSCs improved AIH, mainly by apoptosis and c-Jun N-terminal kinase/mitogen-activated protein signaling pathways. Apoptosis analysis demonstrated that the protein expression of cleaved caspase 3 was increased by Con A injection and reduced by MenSCs transplantation, consistent with the TUNEL staining results. An AML12 co-culture system and JNK inhibitor (SP600125) were used to verify the JNK/MAPK and apoptosis signaling pathways. These findings suggested that MenSCs could be a promising strategy for AIH.

Establishment of Acute Immunological Liver Injury Wistar Rat Model Induced by Concanavalin A / 广州中医药大学学报

Objective To explore the dosage and injection method of concanavalin A(Con A) for inducing Wistar rats into the acute hepatic injury model. Methods (1)According to the dosage of Con A, 42 Wistar rats were randomly divided into groups A, B, C, D, E, N, 7 rats in each group. Group N was given tail intravenous injection of normal saline as normal control group. Groups A, B, C, D, E were given intravenous injection of 4, 8, 16, 30, 40 mg/kg of Con A respectively. At the 8th hour after modeling, the levels of alanine transaminase(ALT), aspartate aminotransferase(AST), albumin(ALB), interleukin(IL)-2 , IL-10, interferon (IFN)-γ, and tumor necrosis factor(TNF)-αwere detected. And HE staining was used to observe the pathological feature of hepatic tissue. (2)According to the injection method of Con A, 21 Wistar rats were randomly divided into normal control group, intraperitoneal injection group and tail intravenous injection group, 7 rats in each group. The dosage of Con A for the rats in intraperitoneal injection group and tail intravenous injection group was 16 mg/kg. At the 8th hour after modeling, the levels of serum ALT, AST, and ALB were determined. Results The number of abnormal deaths in various dose Con A groups at the end of each experiment was 0 in groups A, B, C, and 2 in group D, and 7 in group E. A small amount of spotty necrosis, inflammatory cell infiltration, and hepatic lobule with almost integrity of structure were found in groups A, B, while obvious bridging-like necrosis was seen in groups C, D. Serum ALT, AST, and ALB levels in intraperitoneal injection group had no statistically significant difference as compared with the normal control group. Conclusion Tail intravenous injection of 16 mg/kg of Con A can be used to induce an acute immunological liver injury rat model successfully.

The effection of heat stress on cell immunology in rats / 重庆医学

Objective To investigate the effect of heat stress on the expression of Toll-like receptor 4 (TLR4) and peripheral blood T regulatory cells (Treg) in splenic cells of rats.Methods Senventy-two SD rats were divided into 20 ℃ control group and 37 ℃ group.Each group was divided into non stimulation,bacterial lipopolysaccharide (LPS) stimulation and concanavin A (Con-A) stimulation subgroup.Each subgroup had 1,12,48 h and 168 h observation points,and flow cytometry was used to determine the level of TLR4 and Treg.Results The TLR4+ immunocompetent cells in the spleen was decreased from 1 h to 168 h in every subgroup of the 37 ℃ group compared to the 20 ℃ control group (P＜0.05).The level of CD4+CD25+ Treg in the peripheral blood in rats from 1 to 48 h was significantly decreased (P＜0.05) and slightly increased at 168 h in LPS stimulation subgroup in the 37 ℃ group compared to the 20 ℃ control group.The level of CD4+ CD25+ Foxp3+ Treg in the peripheral blood in rats at 12 h were significantly increased in non-stimulation and concanavalin A (Con-A) stimulation subgroups in the 37 ℃group,and were significantly decreased at 168 h in every subgroup of the 37 ℃ group compared with 20 ℃ control group (P＜0.05).The level of CD8+CD25+ Treg in the peripheral blood in rats was significantly increased at 1 h and 168 h in the 37 ℃ hot and humid group in the every subgroup whencompared with the 20 ℃ control group (P＜0.05).The level of CD8+ CD25+Foxp3+ Treg in the peripheral blood in rats was significantly decreased at 1 h and 48 h in the every subgroup,and was significantly increased at 12 h and 168 h in the non-stimulation and LPS stimulation subgroups in the 37 ℃ group compared to the 20 ℃ control group (P＜0.05).Conclusion High temperature and damp heat can destroy the innate immunity and alter the functional state of adaptive immunity in rats.

Protective effects of artesunate against Con A-induced autoimmune liver injury in mice / 中国中药杂志

Autoimmune liver disease is a refractory disease clinically, and there is no particularly effective drug at present. Therefore, it is of important clinical value to develop new effective intervention drugs for the prevention and treatment of autoimmune liver disease. In order to investigate the potential protective effect of artesunate (Art) on concanavalin A (Con A)-induced autoimmune liver injury, different doses of Art (27, 54, 108 mg·kg⁻¹) were orally administered to mice for consecutive 7 days, respectively. Then the Con A was injected into mice via tail vein to induce liver injury models. 8 h after modeling, the mice were sacrificed. The serum and liver tissue were collected for detecting the level of alanine aminotransferase (ALT), and aspartate transaminase (AST), liver pathological histopathology, inflammatory cytokines and nuclear factor (NF-κB) key protein expression level. The results showed that 108 mg·kg⁻¹ Art remarkably reduced Con A-induced liver indexes and serum transaminase levels (ALT and AST) as compared with model group(<0.01). Meanwhile, the liver histopathological changes were obviously alleviated with a significant decrease of pro-inflammatory cytokine including tumor necrosis factor (TNF-α), interferon (IFN-γ), interleukin (IL-6), IL-17 and a higher increase of anti-inflammatory cytokine IL-10 (<0.01). Western blot results showed that 108 mg·kg⁻¹ Art markedly inhibited the expressions of p-p65 and p-IκBα proteins (<0.01). The specific inhibitor of NF-κB, pyrrolidinedithiocarbamate (PDTC) could also significantly inhibit the expressions of p-p65 and p-IκBα with and alleviate liver injuries. Therefore, our results indicated that Art may have a protective action against Con A-induced autoimmune liver injury mainly by suppressing NF-κB signal pathway in mice. The study provides scientific reference for artesunate usage in preventing autoimmune liver injury.

PVA-Glutaraldehyde as support for lectin immobilization and affinity chromatography / PVA-Glutaraldeído como suporte para imobilização de lectina e cromatografia de afinidade

Immobilized lectins are a powerful biotechnological tool for separation and isolation of glycoconjugates. In the present study, polyvinyl alcohol (PVA) and glutaraldehyde (GA) were used as a support for Concanavalin A (Con A) covalent immobilization and for entrapment of Parkia pendula seed gum (PpeG). Con A immobilization yielded approximately 30% and 0.6 M glucose solution was the minimum concentration able to elute fetuin from column. PVA-GA-PpeG column was efficiently recognized by pure P. pendula lectin (PpeL) . These findings indicate that PVA-GA interpenetrated network showed to be an efficient support for lectin covalent immobilization and as affinity chromatography matrix after trapping of PpeG.

Lectinas imobilizadas são uma poderosa ferramenta biotecnológica para a separação e isolamento de glicoconjugados. No presente trabalho álcool polivinílico (PVA) e glutaraldeído (GA) foram utilizados como um suporte para a imobilização covalente da Concanavalina A (Con A) e para aprisionamento da goma de semente de Parkia pendula (PpeG). A eficiência da imobilização da Con A foi aproximadamente 30 % e a concentração mínima de glucose capaz de eluir a fetuína da coluna foi 0,6 M. Coluna de PVA - GA - PpeG foi eficientemente reconhecida pela lectina de P. pendula (PpeL) pura. Estes resultados indicam que a rede interpenetrada de PVA-GA mostrou-se um suporte eficiente para a imobilização covalente de lectina e como matriz de cromatografia de afinidade após aprisionamento de PpeG.

Análise da marcação de células da linhagem C6 de glioma com as lectinas vegetais CPL, WGA, e Con A / cell marking analysis of C6 glioma line with plant lectins CPL, WGA and Con A

Introdução e objetivos: O glioblastoma multiforme é um glioma de alto grau que apresenta um prognóstico ruim. O diagnóstico definitivo é estabelecido pela avaliação histológica, porém este pode apresentar conflitos na classificação, com isso surge à necessidade de ferramentas que auxiliem o patologista em sua análise. Atualmente, maior ênfase tem sido dada a alterações na glicosilação, pois estão associadas a neoplasias, e a descoberta da capacidade de lectinas em reconhecer tais alterações fez destas, ferramentas aplicáveis para o diagnóstico biomédico. Dessa forma, o objetivo deste trabalho é analisar a marcação das lectinas CpL, WGA e Con A em células da linhagem C6...

Introduction and objectives: Glioblastoma multiforme is a high-grade glioma that has a poor prognosis. The definitive diagnosis is established by histological assessment. However, this can present conflicts in grading gliomas, which justifies new tools to assist the pathologist in his analysis. Currently, it is known that there are changes in glycosylation pattern of molecules associated with cancer, and the discovery of the ability of lectins to recognize these changes made these tools applicable for biomedical diagnosis. Thus, the aim of this study is to analyze the labelling of C6...

Bacteriostatic activity of con a lectin from Canavalia ensiformis.

The aim of this work was to explore the therapeutic applications of Con A lectin from Canavalia ensiformis and to explore its antibacterial activity. Activity of lectin was quantified by their ability to agglutinate erythrocytes using Hemagglutination assay. Characterization and purity of Con A lectin was evaluated by using SDS-PAGE analysis. The reversal of hemagglutination activity of lectin was evaluated by using the sugars namely; mannose, galactose, lactose, fructose, glucose. The antibacterial activity of lectins was tested against Streptococcus mutans, Staphylococcus aureus, Bacillus subtilis, Escherichia coli using pour plate method. Amoxycillin was used as standard. At 250mg/ml concentration Con A lectin showed good bacteriostatic activity.

The Role of Nitric Oxide on Cataractogenesis in Uveitis Model Induced by Concanavalin A and Lipopolysaccharide

Nitric oxide[NO]plays a diverse role in many biological systems such as vascular, nervous and respiratory system. Recently, many studies have been performed to define the relationship between NO and physiological and pathophysiological processes. This study was conducted to evaluate the role of NO on cataractogenesis in uveitis model induced by Con A and LPS. Forty-five white male rabbits were divided into five experimental groups[ten rabbits for each group I, II, III, IV;five rabbits for group V, control]. The rabbits were received intravitreal injection of Con A[I], Con A &L-NAME [II], LPS[III], LPS &L-NAME[IV], BSS[Vfor control], respectively. After injection, the author evaluated the inflammatory grade, cataractous lens changes, NO[nitrite+nitrate]concentration in response to each material in aqueous humor and vitreous cavity and MDA concentration of lens with time interval. Other 7 rabbits 'eyes were enucleated and 14 fresh lenses were extracted to investigate the correlation with in vivo study via lens culture in 12-well plate. These results suggested that NO was closely related to Con A and LPS induced cataractogenesis in uveitis model and Con A was a more reliable candidate due to its prolonged effect. NOS inhibitors could suppress both inflammation and cataract formation. Potent oxidant, peroxynitrite played an important role on NO related cataractogenesis. So, on the clinical basis, NOS inhibitors may be useful in prevention of cataract in inflammatory condition of the eyes but further studies are needed for the confirmation of stability and selectivity of the NOS inhibitors.

The Role of Nitric Oxide on Cataractogenesis in Uveitis Model Induced by Concanavalin A and Lipopolysaccharide

Nitric oxide[NO]plays a diverse role in many biological systems such as vascular, nervous and respiratory system. Recently, many studies have been performed to define the relationship between NO and physiological and pathophysiological processes. This study was conducted to evaluate the role of NO on cataractogenesis in uveitis model induced by Con A and LPS. Forty-five white male rabbits were divided into five experimental groups[ten rabbits for each group I, II, III, IV;five rabbits for group V, control]. The rabbits were received intravitreal injection of Con A[I], Con A &L-NAME [II], LPS[III], LPS &L-NAME[IV], BSS[Vfor control], respectively. After injection, the author evaluated the inflammatory grade, cataractous lens changes, NO[nitrite+nitrate]concentration in response to each material in aqueous humor and vitreous cavity and MDA concentration of lens with time interval. Other 7 rabbits 'eyes were enucleated and 14 fresh lenses were extracted to investigate the correlation with in vivo study via lens culture in 12-well plate. These results suggested that NO was closely related to Con A and LPS induced cataractogenesis in uveitis model and Con A was a more reliable candidate due to its prolonged effect. NOS inhibitors could suppress both inflammation and cataract formation. Potent oxidant, peroxynitrite played an important role on NO related cataractogenesis. So, on the clinical basis, NOS inhibitors may be useful in prevention of cataract in inflammatory condition of the eyes but further studies are needed for the confirmation of stability and selectivity of the NOS inhibitors.

Effect of Lectin on Fine Structure and Extracellular Matrix Protein of Cancer Cell Lines / 대한해부학회지

Lectins are glycoproteins that bind specifically to carbohydrates. Considerable interests in the lectins were encouraged by several reports that certain members of the family bind to the extracellular matrix proteins (ECM), such as fibronectin and laminin. However, the relations between lectin and ECM protein remain unclear. To elucidate the relations of lectin-matrix-cell, we treated three cancer cell lines, HeLa, L929, and EATC with ConA and PHA-P at low dose (4 microgram/ml) and high dose (20 microgram/ml) for 1, 3, 5 days. 1. Whether or not lectins significantly regulate the cell proliferation was evaluated by MTT assays. 2. Whether the amount of fibronectin and laminin which of cancer cells can be influenced by lectins was confirmed by immunocytochemical staining. 3. Whether, in turn, the lectins which can change the morphology were observed under inverted and electron microscopes. ConA and PHA-P inhibited cell proliferation rate of all cell lines in a dose- and time- dependent manner. The amount of fibronectin and laminin considerably reduced in the three cell lines after the lectins treatment in a dose- and time-dependent manner. The cancer cell lines showed various morphological changes such as cell aggregation, irregular-shaped cellular processes, rounded cells, cytoplasmic vacuolation, swollen RERs, dilation of mitochondria, margination of chromatin and cell death. In conclusion, our results showed ConA and PHA-P caused damages of the three cancer cell lines, but the effect of PHA-P was much stronger than ConA. Taken together, the present data strongly indicate that ConA and PHA-P influence the cell proliferation rate, reduce the amount of fibronectin and laminin and induce cell injuries of HeLa, L929, and EATC cell lines. Our results also suggest that the cancer cell proliferation and the morphological changes might be modulated by the specific interaction between lectins and ECM proteins associated with the cell surface.

Protective Effect of Isodon lophanthoides var.gerardianus (Benth.) Hara on Hepatic Injury Induced by Concanavalin A in NIH mice / 中药新药与临床药理

Objective To study the protective effects of aqueous extract and alcohol-soluble extract of Isodon lophanthoides var.gerardianus (Benth.)Hara (ILVG) on immunity hepatic injury induced by concanavalin A (Con A) in mice.Methods One hundred and eight NIH mice were allocated into normal control group,model group,ILVG groups of aqueous extract and alcohol-soluble extract (low-,middle-and high-dosage),and bifendate group randomly.In the experimental groups,mice received either ILVG aqueous extract or alcohol-soluble extract (18.20 g?kg-1,9.10 g?kg-1,4.55 g?kg-1 respectively) or Bifendate (45 mg?kg-1) by gastric perfusion daily for consecutive 5 days.In the 5th day,Con A (20 mg?kg-1) was injected into mice via the tail vein 4h after administration.And then the blood was obtained by picking out the eyeball and the serum was separated after 8-hour fasting.The serum levels of ALT and AST were analyzed,the body weight as well as the weight of liver,spleen and thymus were measured,and pathological features of hepatic tissue were observed.Results ILVG can decrease the ALT and AST,restrain the enlargement of liver and the shrinkage of thymus and reduce the necrosis of hepatic tissue.Conclusion Aqueous extract and alcohol-soluble extract of ILVG possess the effects of protecting liver from immunity injury induced by Con A in NIH mice.

The Role of Membrane TNF-a in Murine Con A-LAK Cell Mediated MHC Non-restricted Tumoricidal Effect / 中国肿瘤生物治疗杂志

This essay investigated the in vitro tumoricidal activity of Con A primed murine LAK cells (Con A-LAK), discussed the role of mTNF-? in the killing effect. The results showed that Con A-LAK cells could kill NK sensitive YAC-1, TNF sensitive L929 and H-2 low/non expression HCa-F target cells; The cytotoxicitity of Con A-LAK cells was performed by direct contact, the non-secretary pathway mediated by interaction of surface ligands of effector cells and receptors of target cells play an important role; Con A-LAK cells expressed mTNF-?, which participated in the killing of TNF sensitive tumor cells and might be one of the important effector molecules in MHC non-restricted, antigen non-specific cvtotoxicitv.

Mucin histochemistry by paradoxical concanavalin A staining in early gastric carcinomas

Phenotypic expression of tumor cells was investigated in 33 early gastric carcinomas by mucin histochemistry using paradoxical concanavalin A staining. This staining method had been developed to differentiate 3 classes of mucins located at various sites of the alimentary tract. Twenty-five (76%) tumors contained mixtures of neutral or acid class II mucin and class III mucin, suggesting the origin of multipotential stem cells. The surface mucous cell expression was more dominant than the pyloric gland or intestinal phenotypes in the well-and poorly differentiated adenocarcinomas. The intestinal properties of the tumor cells were noted not only in the well-differentiated but also in the poorly differentiated or signet ring cell carcinomas, not closely being related to the presence of background intestinal metaplasia. Signet ring cell carcinomas revealed a distinct pattern of mucin histochemistry compared with the other types.

Requirements for in vitro growth of human thymocytes

Since it is difficult to study human thymocyte maturation in vitro, we have developed an in vitro thymocyte culture system which has allowed us to select the optimal growth conditions for thymocyte subpopulations. Three thymocyte subpopulations (CD3-CD1-, CD1+CD3-, and CD3+CD1-) were isolated by a single step percoll density gradient centrifugation and indirect panning procedure using anti-CD1 and anti-CD3 monoclonal antibodies, and their purity was checked by flow cytometry. The combination of concanavalin A (Con A), tetradecanoylphorbol acetate (TPA), and IL-2 was shown to be the most reliable stimulus for the proliferation of CD3-CD1- thymocytes for up to 15 days in a culture system in vitro. Flow cytometric analysis for the phenotypic change of CD3-CD1- thymocytes revealed a steady increase of CD3 antigen after a 3-day cultivation, whereas there was no change in CD1 antigen intensity. A combination of Con A and IL-2 was both sufficient and necessary to induce growth of CD3+CD1- thymocytes. The major population of immature cortical thymocytes (CD3-CD1+ or CD3+CD1+), which are considered to be the most unresponsive dead-end cells, could not be maintained or stimulated with any combination used in this experiment, even in the presence of thymic accessory cells.

APPLICATION OF COLLOIDAL GOLD-TECHNIQUE FOR LOCALIZATION OF CON A RECEPTORS IN HUMAN SPERM / 解剖学报

Intact and gossypol-treated spermatozoa from healthy man were labeled withCon A-HRP-G two-step method.Comparative observations of these sperms were doneunder LM,TEM and SEM.Results show that there are gold particles on spermsurface membrane.Their distribution is more dens on plasma membrane of spermhead and middle piece.On the intracellular membrane structures of sperm which plasmamembrane was damaged by gossypol the gold particles also can be seen,especially onthe membranes of acrosome and mitochondria.It was proved that Con A receptorsare present not only on plasma membrane of sperm,but also on the membrane ofacrosome and mitochondrion.The significance of Con A receptors on sperm struc-tures,advantages of Con A-HRP-G two-step labeled method and comparativeobservations under LM,TEM and SEM were discussed.