New anti-tumor vaccine and immunotherapeutic strategies based on optimal stimulation of CD4+ T Helper cells.

Vaccination with a poorly or not immunogenic tumor fails to protect the host from a subsequent challenge with the same tumor. The mechanisms underlying the failure of these tumors to sensitize therapeutic T cells are not clearly understood, but the inability of host T cells to recognize tumor has been implicated. Previous works from our laboratory have shown that the highly aggressive BALB/c mammary adenocarcinoma TS/A, can be rejected if genetically modified to express MHC class II genes upon transfection of the MHC class II transactivator CIITA. MHC class II molecules are required for presenting antigenic peptides to T helper cell and thus activate the cascade of events leading to immune effector functions such as antibody production and cytolytic T cell activity. The purpose of my thesis was to investigate whether the above approach could be generalized and thus extended to tumors of distinct histological origin. Moreover, it was assessed whether anti-tumor lymphocytes generated by this approach could be used as an immunotherapeutic tool for established cancers. Beside the previously described TS/A-CIITA breast carcinoma cells, stable CIITA-transfectants of colon adenocarcinoma C51, renal adenocarcinoma RENCA, and sarcoma WEHI-164, were generated. Tumor cells transfectants were injected in vivo and their grow kinetics and recipient\u2019s immune response were analyzed. Tumor rejection and/or retardation of growth was found for CIITA-transfected C51 and RENCA adenocarcinomas, as well as WEHI-164 sarcoma. As for TS/A, this tumor rejection correlated mostly with the stability and the amount of CIITA-induced MHC class II expression. Interestingly, mice rejecting CIITA-transfected tumors acquired specific immunological memory, as demonstrated by resistance to challenge with untransfected parental tumors. Adoptive cell transfer experiments demonstrated that tumor immunity correlates with CD4+ and CD8+ T lymphocytes. In this thesis, it is also shown that CIITA-induced MHC class II expression on tumor cells is able to generate antitumor therapeutic T cells. Indeed, T cells from TS/A-vaccinated mice were used in an adoptive cell immunotherapy (ACT) model of established tumors. The results showed the cure at early stages and a significantly prolonged survival at later stages of tumor progression. Importantly, CD4+ T cells were clearly superior to CD8+ T cells in anti-tumor protective function. Interestingly, the protective phenotype was associated to both a Th1 and Th2 polarization of the immune effectors. Of great importance, the T cells obtained from vaccinated mice were therapeutic also without other in vitro activation passages, like treatment with anti-CD3 and IL-2, differently to some other approaches of vaccination and ACT. Furthermore, ACT induced proliferation of tumor-specific immune splenocytes in receiving mice, leading to rejection of subsequent tumor challenge or to protection from metastasis in animals having established tumors. These data support our original idea that CIITA-expressing tumor cells can act as antigen-presenting cells (APCs) for their own tumor-associated antigens to CD4+ T cells, to induce specific and potent anti-tumor responses. These results establish the general application of our tumor vaccine model. This approach let us to envisage additional applications of this strategy for producing better lymphocyte effectors for adoptive anti-tumor cell immunotherapy, particularly in those cases of tumors non-responsive to existing therapies

New anti-tumor vaccine and immunotherapeutic strategies based on optimal stimulation of CD4+ T Helper cells.

Vaccination with a poorly or not immunogenic tumor fails to protect the host from a subsequent challenge with the same tumor. The mechanisms underlying the failure of these tumors to sensitize therapeutic T cells are not clearly understood, but the inability of host T cells to recognize tumor has been implicated. Previous works from our laboratory have shown that the highly aggressive BALB/c mammary adenocarcinoma TS/A, can be rejected if genetically modified to express MHC class II genes upon transfection of the MHC class II transactivator CIITA. MHC class II molecules are required for presenting antigenic peptides to T helper cell and thus activate the cascade of events leading to immune effector functions such as antibody production and cytolytic T cell activity. The purpose of my thesis was to investigate whether the above approach could be generalized and thus extended to tumors of distinct histological origin. Moreover, it was assessed whether anti-tumor lymphocytes generated by this approach could be used as an immunotherapeutic tool for established cancers. Beside the previously described TS/A-CIITA breast carcinoma cells, stable CIITA-transfectants of colon adenocarcinoma C51, renal adenocarcinoma RENCA, and sarcoma WEHI-164, were generated. Tumor cells transfectants were injected in vivo and their grow kinetics and recipient’s immune response were analyzed. Tumor rejection and/or retardation of growth was found for CIITA-transfected C51 and RENCA adenocarcinomas, as well as WEHI-164 sarcoma. As for TS/A, this tumor rejection correlated mostly with the stability and the amount of CIITA-induced MHC class II expression. Interestingly, mice rejecting CIITA-transfected tumors acquired specific immunological memory, as demonstrated by resistance to challenge with untransfected parental tumors. Adoptive cell transfer experiments demonstrated that tumor immunity correlates with CD4+ and CD8+ T lymphocytes. In this thesis, it is also shown that CIITA-induced MHC class II expression on tumor cells is able to generate antitumor therapeutic T cells. Indeed, T cells from TS/A-vaccinated mice were used in an adoptive cell immunotherapy (ACT) model of established tumors. The results showed the cure at early stages and a significantly prolonged survival at later stages of tumor progression. Importantly, CD4+ T cells were clearly superior to CD8+ T cells in anti-tumor protective function. Interestingly, the protective phenotype was associated to both a Th1 and Th2 polarization of the immune effectors. Of great importance, the T cells obtained from vaccinated mice were therapeutic also without other in vitro activation passages, like treatment with anti-CD3 and IL-2, differently to some other approaches of vaccination and ACT. Furthermore, ACT induced proliferation of tumor-specific immune splenocytes in receiving mice, leading to rejection of subsequent tumor challenge or to protection from metastasis in animals having established tumors. These data support our original idea that CIITA-expressing tumor cells can act as antigen-presenting cells (APCs) for their own tumor-associated antigens to CD4+ T cells, to induce specific and potent anti-tumor responses. These results establish the general application of our tumor vaccine model. This approach let us to envisage additional applications of this strategy for producing better lymphocyte effectors for adoptive anti-tumor cell immunotherapy, particularly in those cases of tumors non-responsive to existing therapies

An investigation of the applicability of facilities-location techniques to an electronic engineering problem: the locating of components within electronic equipment to minimize wiring costs

This thesis investigated Operations Research developed techniques for the facilities-location problem for application to this electronic engineering problem. One technique stood out as the most promising and it was applied to this problem. The technique chosen was CRAFT developed in 1963

γ Heavy Chain Disease in Man: cDNA Sequence Supports Partial Gene Deletion Model

Human gamma heavy chain disease (HCD) is characterized by the presence in serum of a short monoclonal Ig gamma chain unattached to light chains. Although most HCD proteins have internal deletions, in some the defect is NH2-terminal. The OMM gamma 3 HCD serum protein is of the latter type, having undergone an extensive NH2-terminal deletion with a sequence starting within the hinge. A cell line synthesizing the OMM protein has enabled us to study the biogenesis of the abnormal molecule. In vitro translation of isolated mRNA yields a protein containing a hydrophobic NH2-terminal leader sequence. In the intact cell, the precursor molecule is processed normally to yield a protein with an NH2-terminal sequence homologous to the beginning of the variable (V) region. The nucleotide sequence of cDNA prepared from the OMM mRNA encodes a 19-amino acid leader followed by the first 15 residues of the V region. An extensive internal deletion encompasses the remainder of the V and the entire CHl domain. Immediately following the short V region, there is information in the cDNA for the entire normal hinge. The primary synthetic product is thus an internally deleted molecule that undergoes postsynthetic degradation to yield the NH2-terminally deleted serum protein. The structure of the OMM mRNA suggests that the protein abnormality results from a partial gene deletion rather than defective splicing

Multi-step approach for automated scaling of photogrammetric micro-measurements

[EN] Photogrammetry can be used for the measurement of small objects with micro-features, with good results, low costs, and the possible addition of texture information to the 3D models. The performance of this technique is strongly affected by the scaling method, since it retrieves a model that must be scaled after its elaboration. In this paper, a fully automated multi-step scaling system is presented, which is based on machine vision algorithms for retrieving blurred areas. This method allows researchers to find the correct scale factor for a photogrammetric micro model and is experimentally compared to the existing manual method basing on the German guideline VDI/VDE 2634, Part 3. The experimental tests are performed on millimeter-sized certified workpieces, finding micrometric errors, when referred to reference measurements. As a consequence, the method is candidate to be used for measurements of micro-features. The proposed tool improves the performance of the manual method by eliminating operator-dependent procedures. The software tool is available online as supplementary material and represents a powerful tool to face scaling issues of micro-photogrammetric activities.Frangione, A.; Sánchez Salmerón, AJ.; Modica, F.; Percoco, G. (2019). Multi-step approach for automated scaling of photogrammetric micro-measurements. The International Journal of Advanced Manufacturing Technology. 102(1-4):747-757. https://doi.org/10.1007/s00170-018-03258-w7477571021-

Differential Degradation of Amyloid β Genetic Variants Associated with Hereditary Dementia or Stroke by Insulin-degrading Enzyme

Inherited amino acid substitutions at position 21, 22, or 23 of amyloid beta (Abeta) lead to presenile dementia or stroke. Insulin-degrading enzyme (IDE) can hydrolyze Abeta wild type, yet whether IDE is capable of degrading Abeta bearing pathogenic substitutions is not known. We studied the degradation of all of the published Abeta genetic variants by recombinant rat IDE (rIDE). Monomeric Abeta wild type, Flemish (A21G), Italian (E22K), and Iowa (D23N) variants were readily degraded by rIDE with a similar efficiency. However, proteolysis of Abeta Dutch (E22Q) and Arctic (E22G) was significantly lower as compared with Abeta wild type and the rest of the mutant peptides. In the case of Abeta Dutch, inefficient proteolysis was related to a high content of beta structure as assessed by circular dichroism. All of the Abeta variants were cleaved at Glu3-Phe4 and Phe4-Arg5 in addition to the previously described major sites within positions 13-15 and 18-21. SDS-stable Abeta dimers were highly resistant to proteolysis by rIDE regardless of the variant, suggesting that IDE recognizes a conformation that is available for interaction only in monomeric Abeta. These results raise the possibility that upregulation of IDE may promote the clearance of soluble Abeta in hereditary forms of Abeta diseases.Fil: Morelli, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Llovera, Ramiro Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gonzalez, Silvia Adriana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Affranchino, Jose Luis. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Prelli, Frances. University of New York; Estados UnidosFil: Frangione, Blas. University of New York; Estados UnidosFil: Ghiso, Jorge. University of New York; Estados UnidosFil: Castaño, Eduardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin