No abstract available.
Adult ; Autoantibodies/*blood ; Gangliosides/blood/*immunology ; Humans ; Male ; Ophthalmoplegia/blood/*diagnosis/immunology ; Recurrence ; Syndrome

OBJECTIVETo study the effect of endogeneous gangliosides (Gls) on integrin alpha2beta1-mediated adhesion of neuroblastoma cells to collagen (Col).

METHODSNeuroblastoma SK-N-SH cell line was cultured in the modified eagle's medium with the presence of 10 mum D-threo-1-phenyl-2-decanolamino-3-morphinolin-1-propanol (D-PDMP), an inhibitor of glucosylceramide synthase. Flow cytometry was used to detect the expression of integrin alpha2beta1 in the cell line. The effects of Mg2(+) and monoclonal antibodies to integrin alpha2beta1 on the adhesion of the cell line to immobilized Col were observed. The adhesion cell number was measured with the BCA method and presented with absorptance A570.

RESULTSThere was a high expression of integrin alpha2beta1 in the SK-N-SH cell line without D-PDMP treatment. Endogenous Gls in the cells were almost depleted after 6-day exposure to D-PDMP, but the integrin alpha2beta1 expression was not significantly changed. 1 mmoL/L Mg2(+) treatment increased significantly the number of adhesion cells in the SK-N-SH cell line. The adhesion to Col of the SK-N-SH cells exposed to D-PDMP which Gls was depleted was significantly reduced compared with the control SK-N-SH cells treated with 1 mmoL/L Mg2(+) (A570: 0.33 +/- 0.016 vs 0.57 +/- 0.033; P < 0.01). After endogeneous Gls was added into the Gls-depleted SK-N-SH cells, the adhesion of the cells was restored (A570: 0.52 +/- 0.035). The adhesion of SK-N-SH cells was significantly blocked by anti-alpha2 and anti-beta1 monoclonal antibodies, with A570 of 0.31 +/- 0.018 and 0.36 +/- 0.021 respectively.

CONCLUSIONSEndogenous tumor Gls increases neuroblastoma cell adhesion to Col by regulating the function of integrin alpha2beta1, but has no effects on the integrin expression. It is suggested that tumor Gls may play a role in migration, invasion and metastasis of tumor cells.

Antibodies, Monoclonal ; immunology ; Cell Adhesion ; Cell Line, Tumor ; Collagen ; physiology ; Gangliosides ; physiology ; Humans ; Integrin alpha2beta1 ; physiology ; Magnesium ; pharmacology ; Morpholines ; pharmacology ; Neuroblastoma ; pathology

This study sought to construct a recombinant vector that expresses anti-GD2/anti-CD16 bispecific single-chain antibody(sc-BsAb), and to assess its biological activities. The anti-GD2 gene and the anti-CD16 gene (NM3E2) were obtained using PCR amplification technique, and then the fusion gene was constructed by overlapping PCR. The sc-BsAb gene was subcloned into the pET-22b(+) plasmid from the pMD18-T easy vector by digestion with NcoI, Hind III restriction endonucleases, whose sites exist in both the vectors. Then the combinant plasmids were transferred into E. coli BL21 (DE3). The expression product in the periplasmic was analyzed by both SDS-PAGE and Western blot technique, then was purified with Ni2+ -NTA superflow affinity chromatography. It was demonstrated that the linker in the sc-BsAb fusion protein is SerGly4Ser. and the molecular is 53 KD.
Antibodies, Bispecific ; biosynthesis ; genetics ; Antibodies, Neoplasm ; biosynthesis ; genetics ; Base Sequence ; Cell Line, Tumor ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gangliosides ; immunology ; HeLa Cells ; Humans ; Melanoma ; pathology ; Molecular Sequence Data ; Receptors, IgG ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics

By combining interleukin2 (IL-2) with a tumor specific antibody, immunotherapy of tumors may become more effective in the future. Anti-GD2 single chain antibody directed to the extracellular domain of GD2 disialoganglioside can result in an antitumor response in some pateins with tumors expressing GD2. In this study, the fusion protein consisting of GD2 single chain antibody (ScFv) and IL-2(Ala125) was constructed. Anti-GD2 ScFv and IL-2 genes were obtained by PCR, then the ScFv-IL-2 gene was constructed by over lap PCR. The gene was inserted into the pMD18-T easy vector. Genes from pMD18-T -vector were inserted into expression vector pSE380. Recombinant expression vector was identified by restriction enzyme-cutting and then was transformed into BL21. SDS-PAGE and Western blot analysis confirmed that the transformed E. Coli BL21 could express ScFv-IL-2 fusion-proteins and the molecular weight is 43 kDa. The fusion protein was purified by affinity chromatograph and Sephacryl S-200HR then was identified through ELISA. The results show that the fusion protein retains the activities of both antigen binding and IL-2.
Antibodies ; genetics ; metabolism ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gangliosides ; immunology ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Interleukin-2 ; biosynthesis ; genetics ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics

The human colorectal carcinoma-associated GA733 antigen epithelial cell adhesion molecule (EpCAM) was initially described as a cell surface protein selectively expressed in some myeloid cancers. Gangliosides are sialic acid-containing glycosphingolipids involved in inflammation and oncogenesis. We have demonstrated that treatment with anti-EpCAM mAb and RAW264.7 cells significant inhibited the cell growth in SW620 cancer cells, but neither anti-EpCAM mAb nor RAW264.7 cells alone induced cytotoxicity. The relationship between ganglioside expression and the anti-cancer effects of anti-EpCAM mAb and RAW264.7 was investigated by high-performance thin-layer chromatography. The results demonstrated that expression of GM1 and GD1a significantly increased in the ability of anti-EpCAM to inhibit cell growth in SW620 cells. Anti-EpCAM mAb treatment increased the expression of anti-apoptotic proteins such as Bcl-2, but the expression of pro-apoptotic proteins Bax, TNF-alpha, caspase-3, cleaved caspase-3, and cleaved caspase-8 were unaltered. We observed that anti-EpCAM mAb significantly inhibited the growth of colon tumors, as determined by a decrease in tumor volume and weight. The expression of anti-apoptotic protein was inhibited by treatment with anti-EpCAM mAb, whereas the expression of pro-apoptotic proteins was increased. These results suggest that GD1a and GM1 were closely related to anticancer effects of anti-EpCAM mAb. In light of these results, further clinical investigation should be conducted on anti-EpCAM mAb to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer.
Animals ; Antibodies, Monoclonal/*immunology/*therapeutic use ; Antigens, Neoplasm/*immunology ; Apoptosis/drug effects ; Cell Adhesion Molecules/*immunology ; Cell Line ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Colon/drug effects/immunology/metabolism/pathology ; Colonic Neoplasms/*drug therapy/genetics/*immunology/pathology ; Gangliosides/genetics/*immunology ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Male ; Mice ; Mice, Inbred BALB C

Reported herein is an adult case of Fisher syndrome (FS) that occurred as a complication during the course of community-acquired pneumonia caused by Mycoplasma pneumoniae. A 38-yr-old man who had been treated with antibiotics for serologically proven M. pneumoniae pneumonia presented with a sudden onset of diplopia, ataxic gait, and areflexia. A thorough evaluation including brain imaging, cerebrospinal fluid examination, a nerve conduction study, and detection of serum anti-ganglioside GQ1b antibody titers led to the diagnosis of FS. Antibiotic treatment of the underlying M. pneumoniae pneumonia was maintained without additional immunomodulatory agents. A complete and spontaneous resolution of neurologic abnormalities was observed within 1 month, accompanied by resolution of lung lesions.
Adult ; Anti-Bacterial Agents/therapeutic use ; Antibodies/blood ; Diplopia/etiology ; Erythrocyte Count ; Gangliosides/immunology ; Humans ; Lung/radiography ; Male ; Miller Fisher Syndrome/*diagnosis/etiology ; Pneumonia, Mycoplasma/complications/*diagnosis/drug therapy ; Tomography, X-Ray Computed

In the injured brain, microglia is known to be activated and produce proinflammatory mediators such as interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). We investigated the role of protein kinase A (PKA) in microglial activation by both plasminogen and gangliosides in rat primary microglia and in the BV2 immortalized murine microglial cell line. Both plasminogen and gangliosides induced IL-1beta, TNF-alpha and iNOS mRNA expression, and that this expression was inhibited by the addition of the PKA inhibitors, KT5720 and H89. Both plasminogen and gangliosides activated PKA and increased the DNA binding activity of the cAMP response element- binding protein (CREB). Furthermore, KT5720 and H89 reduced the DNA binding activities of CREB and NF-kappaB in plasminogen-treated cells. These results suggest that PKA plays an important role in plasminogen and gangliosides- induced microglial activation.
Animals ; Carbazoles/pharmacology ; Cell Line ; Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors/*physiology ; DNA-Binding Protein, Cyclic AMP-Responsive/metabolism ; DNA-Binding Proteins/metabolism ; Gangliosides/pharmacology/*physiology ; Gene Expression Regulation ; Indoles/pharmacology ; Interleukin-1/genetics ; Isoquinolines/pharmacology ; Mice ; Microglia/drug effects/*enzymology/*immunology ; NF-kappa B/metabolism ; Nitric-Oxide Synthase/genetics ; Plasminogen/pharmacology/*physiology ; Pyrroles/pharmacology ; RNA, Messenger/analysis/metabolism ; Rats ; Research Support, Non-U.S. Gov't ; Sulfonamides/pharmacology ; Tumor Necrosis Factor-alpha/genetics