Multiple phenotypic changes in mice after knockout of the B3gnt5 gene, encoding Lc3 synthase--a key enzyme in lacto-neolacto ganglioside synthesis.
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2010-11-18
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BACKGROUND: Ganglioside biosynthesis occurs through a multi-enzymatic pathway which at the lactosylceramide step is branched into several biosynthetic series. Lc3 synthase utilizes a variety of galactose-terminated glycolipids as acceptors by establishing a glycosidic bond in the beta-1,3-linkage to GlcNaAc to extend the lacto- and neolacto-series gangliosides. In order to examine the lacto-series ganglioside functions in mice, we used gene knockout technology to generate Lc3 synthase gene B3gnt5-deficient mice by two different strategies and compared the phenotypes of the two null mouse groups with each other and with their wild-type counterparts. RESULTS: B3gnt5 gene knockout mutant mice appeared normal in the embryonic stage and, if they survived delivery, remained normal during early life. However, about 9% developed early-stage growth retardation, 11% died postnatally in less than 2 months, and adults tended to die in 5-15 months, demonstrating splenomegaly and notably enlarged lymph nodes. Without lacto-neolacto series gangliosides, both homozygous and heterozygous mice gradually displayed fur loss or obesity, and breeding mice demonstrated reproductive defects. Furthermore, B3gnt5 gene knockout disrupted the functional integrity of B cells, as manifested by a decrease in B-cell numbers in the spleen, germinal center disappearance, and less efficiency to proliferate in hybridoma fusion. CONCLUSIONS: These novel results demonstrate unequivocally that lacto-neolacto series gangliosides are essential to multiple physiological functions, especially the control of reproductive output, and spleen B-cell abnormality. We also report the generation of anti-IgG response against the lacto-series gangliosides 3'-isoLM1 and 3',6'-isoLD1.
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Kuan, Chien-Tsun, Jinli Chang, Jan-Eric Mansson, Jianjun Li, Charles Pegram, Pam Fredman, Roger E McLendon, Darell D Bigner, et al. (2010). Multiple phenotypic changes in mice after knockout of the B3gnt5 gene, encoding Lc3 synthase--a key enzyme in lacto-neolacto ganglioside synthesis. BMC Dev Biol, 10. p. 114. 10.1186/1471-213X-10-114 Retrieved from https://hdl.handle.net/10161/4341.
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Scholars@Duke
Chien-Tsun Kuan
Research Interests:
Conventional therapy for malignant brain tumors is ineffective. Targeted therapy using tumor-specific antibodies (MAb) alone or MAbs armed with radionuclides or toxins is a promising alternative approach for increasing therapeutic efficacy and decreasing toxicity to normal tissue. The major factors that influence antibody-targeted therapy for cancer treatment, including glioma therapy, are specificity, affinity, tumor penetration, toxicity and immunogenicity. The effective use of radioimmunotherapy (RAIT) for the treatment of solid malignancies has been limited by inadequate tumor penetration and non-targeted myelotoxicity resulting from the presence of radioimmunoconjugates in circulation. We believe that these limitations to direct RAIT can be overcome by using smaller engineered antibody-based molecules as vehicles and by selecting therapeutic radioisotopes with physical properties that complement the pharmacokinetics and pharmacodynamics of the antibody.
Our research is focused upon exploiting engineered antibody fragments to treat brain tumors by targeting to glioma-associated, oncofetal epitopes such as tenascin, glioma variant epidermal growth factor variant III (EGFRvIII), medulloblastoma-associated developmental markers, as well as the newly identified glioma-associated antigens, GPNMB and MRP3, by serial analysis of gene expression (SAGE). Projects performed in the current years have: 1) produced and evaluated the monovalent single-chain Fv (scFv) against EGRvIII in athymic mice bearing human glioma xenografts; 2) begun the development of divalent form of scFv, including diabody and minibody, to increase the efficacy of therapeutic agents in vivo; 3) generated CH2 domain-deleted Ch81C6 vs tenascin and evaluated the pharmacokinetics in mice and canines; 4) begun an extensive analysis of GPNMB and MRP3 protein expression correlated with measurement of RNA transcript levels and degree of DNA amplification.
Unarmed antibody can be effective against both subcutaneous and intracranial tumor models. The unarmed antibody approach with Mab Y10 vs EGFRvIII is very similar to the successful use of HerceptinTM. The mechanism is most likely both a direct antiproliferative effect with the induction of apoptosis and an indirect effect through the mobilization of antibody-mediated immune effector functions, such as complement and antibody-dependent cell-mediated cytotoxicity (ADCC). We also have begun to construct human/mouse chimeric Y10 to reduce immunogenicity of the Mab reagent and possibly enhance ADCC.
Our objectives for the coming years are to continue the optimization of engineered-antibody systems for in vivo application, namely; a) development of human/mouse chimeric anti-EGFRvIII murine Y10 with the same affinity and specificity but reduced immunogenicity and enhanced ADCC for in vivo application; b) to generate a totally human scFv specific to EGFRvIII but with anti-proliferative activity via screening from human phage libraries; c) generation of monomeric and dimeric anti-GPNMB/MRP3 scFvs and construction of immunoconjugate toxins or radiolabeled to determine the efficacy of therapeutic reagents in athymic rodent in athymic rodent in vivo models of intracranial glioma.
Roger Edwin McLendon
Brain tumors are diagnosed in more than 20,000 Americans annually. The most malignant neoplasm, glioblastoma, is also the most common. Similarly, brain tumors constitute the most common solid neoplasm in children and include astrocytomas of the cerebellum, brain stem and cerebrum as well as medulloblastomas of the cerebellum. My colleagues and I have endeavored to translate the bench discoveries of genetic mutations and aberrant protein expressions found in brain tumors to better understand the processes involved in the etiology, pathogenesis, and treatment of brain tumors. Using the resources of the Preston Robert Brain Tumor Biorepository at Duke, our team, consisting of Henry Friedman, Allan Friedman, and Hai Yan and lead by Darell Bigner, have helped to identify mutations in Isocitrate Dehydrogenase (IDH1 and IDH2) as a marker of good prognosis in gliomas of adults. This test is now offered at Duke as a clinical test. Working with the Molecular Pathology Laboratory at Duke, we have also brought testing for TERT promoter region mutations as another major test for classifying gliomas in adults. Our collaboration with the Toronto Sick Kids Hospital has resulted in prognostic testing for childhood medulloblastomas, primitive neuroectodermal tumors, and ependymomas at Duke.
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