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Rengifo C.E.,Manuel Fajardo General Hospital | Hernandez T.,Research and Development Direction
Acta Microscopica | Year: 2014

N-glycolyl GM3 ganglioside (NeuGcGM3) expression in lung carcinomas using formalin-fixed and paraffin-embedded (FFPE) samples has been recently demonstrated. In the present work, it was confirmed the tissue expression of NeuGcGM3 in lung carcinomas by mean of two different mAbs, P3 (anti-NeuGc-containing gangliosides and sulfated glycolipids) and 14F7 (a highly specific for NeuGcGM3) using FFPE samples. In addition, it was reported the tissue expression of NeuGcGM3 in frozen lung carcinoma sections, also supported by the chemical extraction of this ganglioside with organic solvents such as: ethanol, methanol and chloroform/methanol. Moreover, the murine, chimeric and humanized versions of 14F7 mAb showed a similar pattern of immunostaining in this kind of samples. It was also demonstrated that formalin fixation prevent the damage and/or extraction of the antigenic determinant recognized by 14F7 mAb. Consequently, the detection of NeuGcGM3 in frozen samples and their FFPE counterparts was comparable. Our data seems to be in agreement with the potential use of chimeric and/or humanized versions of 14F7 mAb for the passive immunotherapy of lung carcinoma expressing NeuGcGM3. The reactivity of 14F7 murine mAb in FFPE tissues permits to consider it as a useful tool in the selection of patients for specific therapies. © 2014, Interamerican Society for Electron Microscopy (CIASEM). All rights reserved.

Blanco D.,National Institute of Oncology and Radiobiology | Escobar X.,National Institute of Oncology and Radiobiology | Rengifo C.E.,Manuel Fajardo General Hospital | Osorio M.,National Institute of Oncology and Radiobiology | And 3 more authors.
Pathology Research International | Year: 2013

The reactivity of the 14F7 Mab, a highly specific IgG1 against N-glycolyl GM3 ganglioside (NeuGcGM3) in normal tissues, lymphomas, lymph node metastasis, and other metastatic sites was assessed by immunohistochemistry. In addition, the effect of chemical fixation on the 14F7 Mab staining using monolayers of P3X63Ag.653 cells was also evaluated. Moreover, the ability of 14F7 to bind NeuGcGM3 ganglioside inducing complement-independent cytotoxicity by a flow cytometry-based assay was measured. The 14F7 Mab was reactive in unfixed, 4% paraformaldehyde, 4% formaldehyde, and acetone fixed cells. Postfixation with acetone did not alter the localization of NeuGcGM3, while the staining with 14F7 Mab was significantly eliminated in both cells fixed and postfixed with methanol but only partially reduced with ethanol. The staining with 14F7 Mab was evidenced in the 89.2%, 89.4%, and 88.9% of lymphomas, lymph node metastasis, and other metastatic sites, respectively, but not in normal tissues. The treatment with 14F7 Mab affected both morphology and membrane integrity of P3X63Ag.653 cells. This cytotoxic activity was dose-dependent and ranged from 24.0 to 84.7% (10-1000 g/mL) as compared to the negative control. Our data could support the possible use of NeuGcGM3 as target for both active and passive immunotherapy against malignancies expressing this molecule. © 2013 Rancés Blanco et al.

Blanco D.,National Institute of Oncology and Radiobiology | Rengifo C.E.,Manuel Fajardo General Hospital | Carr A.,Research and Development Direction
Pathology Research International | Year: 2015

The prognostic role of N-glycolyl GM3 ganglioside (NeuGcGM3) expression in non-small cell lung carcinoma (NSCLC) still remains controversial. In this study, the NeuGcGM3 expression was reevaluated using an increased number of NSCLC cases and the 14F7 Mab (a highly specific IgG1 raised against NeuGcGM3). An immunohistochemical score integrating the percentage of 14F7-positive cells and the intensity of reaction was applied to reassess the relationship between NeuGcGM3 expression, some clinicopathological features, and the overall survival (OS) of NSCLC patients. The double and the triple expression of NeuGcGM3 with the epidermal growth factor receptor (EGFR) and/or its ligand, the epidermal growth factor (EGF), were also evaluated. NeuGcGM3 expression correlates with both S-Phase fraction (p = 0.006) and proliferation index (p = 0.000). Additionally, NeuGcGM3 expression was associated with a poor OS of patients in both univariate (p = 0.020) and multivariate (p = 0.010) analysis. Moreover, the double and/or the triple positivity of tumors to NeuGcGM3, EGFR, and/or EGF permitted us to identify phenotypes of NSCLC with a more aggressive biological behavior. Our results are in agreement with the negative prognostic significance of NeuGcGM3 expression in NSCLC patients. However, standardization of techniques to determine the expression of NeuGcGM3 in NSCLC as well as the implementation of a universal scoring system is recommended. © 2015 Rancés Blanco et al.

Perez R.,Research and Development Direction
Molecular Immunology | Year: 2010

Detailed information on the immunological relevance of α-type anti-idiotypic antibodies is lacking after more than 30 years since Jerne postulated his Idiotypic Network Theory. The B7Y33 mutant is a mouse-human chimeric version of the B7 MAb, a polyreactive α-type anti-idiotypic antibody, generated against an anti-GM2 ganglioside IgM Ab1 antibody. It retained the unusual self-binding activity and multispecificity of the parental murine antibody, being able to recognize several anti-ganglioside IgM antibodies as well as non-immunoglobulin antigens. Previous work with the murine B7 MAb suggested that this antibody might have immunoregulatory properties, and therefore we investigated the possible interaction of B7Y33 with immune cells. We found that B7Y33 binds to human and murine B lymphocytes. Inhibition assays using flow cytometry indicated that this antibody is capable of binding the Fc γ receptor II (FcγRII). The recognition of FcγRII-expressing K562, Raji and Daudi human cell lines, together with the capability of inhibiting the binding of an anti-human FcγRII antibody to these cells, suggest that B7Y33 interacts with both the FcγRIIa and FcγRIIb isoforms. We evaluated the contribution to the binding of different surface-exposed residues at the top of the heavy chain variable region (VH) CDR loops through the construction of mutants with substitutions in the three conventional VH CDRs (HCDRs) and the " HCDR4" , located in the framework 3 (HFR3). In addition, we assessed the involvement of the Fc region by performing key mutations in the CH2 domain. Furthermore, chimeric hybrid molecules were obtained by combining the B7Y33 heavy chain with unrelated light chains. Our results indicate that the multispecificity and self-binding properties of B7Y33 are not linked to its recognition of B lineage cells, and that this phenomenon occurs in a non-classical way with the participation of both the variable and constant regions of the antibody. Two possible models for this interaction are proposed, with B7Y33 binding to two FcγRIIb molecules through the Fc and Fv regions, or simultaneously to FcγRIIb and another unknown antigen on B cells. The FcγRIIb has recently received great attention as an attractive target for therapies directed to B lymphocytes. The recognition of peripheral B lymphocytes from B cell chronic lymphocytic leukemia (B-CLL) patients by B7Y33 suggests its potential application for the treatment of B cell malignancies. © 2010 Elsevier Ltd.

Rengifo C.E.,Manuel Fajardo General Hospital | Carr A.,Research and Development Direction
Pathology Research International | Year: 2012

Lung carcinoma is the leading cause of cancer-related mortality worldwide. Therefore, numerous studies are focusing on the assessment of other biological and molecular prognostic factors in these tumors. We evaluated the relationship between 14F7 Mab reactivity, pathological features, DNA-content and S-phase fraction (SPF), and their impact in the survival of NSCLC patients. Hematoxylin and eosin staining and immunohistochemistry optical microscopy assays as well as DNA content and SPF measuring using flow cytometry were performed. The 14F7 reactivity was widely observed in NSCLC sections, no depending of the clinicopathological characteristics. We also obtained differences in the intensity of reaction with 14F7 as well as in the SPF between diploid and aneuploid carcinomas. Patients with diploid tumors showing higher SPF and 14F7 reaction joint to a low mitotic index displayed higher survival rates. Our results are in agreement with the assumption of the possible positive prognostic value of 14F7 staining in NSCLC. © 2012 Rancés Blanco et al.

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