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S Casares

Publications and source records attributed to S Casares.

At least 19 recordsLinked to original sources
PubMed1999

Antineoplastic efficacy of doxorubicin enzymatically assembled on galactose residues of a monoclonal antibody specific for the carcinoembryonic antigen.

We have developed a novel procedure to couple enzymatically the antineoplastic agent doxorubicin (Dox) on the galactose residues of a monoclonal antibody specific for the tumor-associated carcinoembryonic antigen. The synthesis of the immunoconjugate consists of covalent attachment of the NH2 terminus of Dox to oxidized galactose residues of desialylated monoclonal antibody, followed by concurrent stabilization of Schiff bases by mild reduction with pyridine borane. The immunoconjugate preserved both antibody specificity and drug cytotoxicity. At equimolar concentrations, the immunoconjugate was 8 times more cytotoxic against two carcinoembryonic antigen-expressing carcinoma cell lines, LoVo and SW-480, than Dox alone. The intracellular drug accumulation was 8-8.5 times higher than that obtained with free Dox, and >50% of the drug delivered by the conjugate was retained for 24 h in the tumor cells. Only 4 days after treatment with a single dose of immunoconjugate carrying 2.5 ng of Dox, LoVo and SW-480 tumor transplants on the chorioallantoic membrane of embryonated hen eggs showed reduced tumor-induced angiogenesis and tumor progression by half, with no detectable damage to surrounding tissues. In contrast, the same amount of free drug induced insignificant changes in tumor progression and tumor-induced angiogenesis. Enzymatically mediated, glycosidic coupling of antineoplastic agents to antibodies specific for tumor-associated antigens may represent a novel platform for the development of more efficient anticancer agents with reduced side effects.

Animals↗
PubMed1998

Cellular mechanisms involved in protection against influenza virus infection in transgenic mice expressing a TCR receptor specific for class II hemagglutinin peptide in CD4+ and CD8+ T cells.

Mice transgenic for a TCR that recognizes peptide110-120 of hemagglutinin of PR8 influenza virus in the context of MHC class II I-Ed molecules express the transgenes in both CD4+ and CD8+ T cells. We have found that these TCR-hemagglutinin (TCR-HA) transgenic mice display a significantly increased resistance to the primary infection with PR8 virus compared with the wild-type mice. The TCR-HA transgenic mice mounted significant MHC type II and enhanced MHC type I-restricted cytotoxicity as well as increased cytokine responses in both spleen and lungs after infection with PR8 virus. In contrast, the primary humoral response against PR8 virus was not significantly different from that of the wild-type mice. In vivo depletion and adoptive cell transfer experiments demonstrated that both CD4+ and CD8+ TCR-HA+ T cell subsets were required for the complete clearance of pulmonary virus following infection with a dose that is 100% lethal in wild-type mice. Whereas CD4+ TCR-HA+ T cells were necessary for effective activation and local recruitment of CD8+ T cells, CD8+ TCR-HA+ T cells showed a Th1-biased pattern and MHC type II-restricted cytotoxicity. However, in the absence of in vivo expression of MHC type I molecules on the infected cells, the protection conferred by the TCR-HA+ T cells was impaired, indicating that the enhanced MHC class I-restricted cytotoxicity due to TCR-HA+ CD4+ Th cells was a critical element for clearance of the pulmonary virus by the transgenic mice.

Animals↗
PubMed1998

Towards development of T-cell vaccines.

Recent studies on the recognition of antigens by CD4+ and CD8+ T cells have revealed new ways of preparing efficient T-cell vaccines. Here, Constantin Bona and colleagues discuss several approaches for the development of T-cell vaccines, with applications ranging from the induction of protective immunity against intracellular parasites to the development of therapeutic agents against autoimmune disorders, allergic diseases and cancer.

Animals↗
PubMed1997

Antigen presentation by dendritic cells after immunization with DNA encoding a major histocompatibility complex class II-restricted viral epitope.

Intramuscular and intracutaneous immunization with naked DNA can vaccinate animals to the encoded proteins, but the underlying mechanisms of antigen presentation are unclear. We used DNA that encodes an A/PR/8/34 influenza peptide for CD4 T cells and that elicits protective antiviral immunity. DNA-transfected, cultured muscle cells released the influenza polypeptide, which then could be presented on the major histocompatibility complex class II molecules of dendritic cells. When DNA was injected into muscles or skin, and antigen-presenting cells were isolated from either the draining lymph nodes or the skin, dendritic, but not B, cells presented antigen to T cells and carried plasmid DNA. We suggest that the uptake of DNA and/or the protein expressed by dendritic cells triggers immune responses to DNA vaccines.

Animals↗
PubMed1997

Structure of the mutant fibrillin-1 gene in the tight skin (TSK) mouse.

Mice carrying the tight skin (TSK) mutation harbors a 3.0-kb genomic duplication (exons 17-40) of the fibrillin-1 gene (Fbn-1) located on band F of chromosome 2 as TSK mutation. We cloned and sequenced the mutated Fbn-1 gene, since it is believed to be responsible for TSK syndrome. Sequence analysis showed numerous amino acid differences in the 5' and 3' segments between the TSK mutation and wild-type fbn-1 gene, but any amino acid difference between the TSK mutation and C57BL/6 mice. (TSK and C57B1/6 mice are genetically similar, differing only by TSK mutation.) Four amino acid differences were observed between two copies of TSK's fbn-1 gene encoded by exons 17-40. Our results suggest that the majority of structural differences occurred in the N and C termini segments during strain divergence and only a few after the duplication event.

Animals↗
PubMed1997

Presentation of a viral peptide assembled on the carbohydrate moieties of immunoglobulin does not require processing.

We have previously demonstrated that an immunodominant CD4 T cell epitope, HA110-120 of the hemagglutinin (HA) of the A/PR/8/34 influenza virus, enzymatically assembled on the carbohydrate moieties of self immunoglobulins (Ig) primed the precursors of peptide-specific T cells and induced efficient proliferation in vivo of naive lymphocytes from transgenic mice expressing the peptide-specific T cell receptor. Here, we show that an immuno-galacto-peptide construct, IgG-gal-HA, does not require intracellular or extracellular processing to present the peptide to the specific T cells. The presentation occurs following the binding of the IgG-gal-HA construct to Fc gamma receptor on the surface of antigen-presenting cells (APC), with concurrent interaction of the peptides to their neighboring major histocompatibility complex class II molecules. This mechanism of peptide presentation may harness the immune response in vivo by the engagement of APC with a low capacity of antigen processing, such as neonatal B cells. In addition, the enzymatic method of assembling various aminated compounds on the sugar moieties of Ig may offer novel perspectives on immuno-targeting of antagonist peptides, cytostatic drugs, and biologically active ligands of therapeutic use.

Animals↗
PubMed1997

Repopulation of circulating T, B and NK lymphocytes following bone marrow and blood stem cell transplantation.

A variety of T, B and natural killer (NK) cell subsets defined by surface markers were analyzed by double immunofluorescence flow cytometry in the peripheral blood of patients following autologous bone marrow transplantation (ABMT, n = 14), autologous peripheral blood stem cell transplantation (PBSCT, n = 10) and allogeneic bone marrow transplantation (allo-BMT, n = 6). Patients following ABMT were divided in 2 groups, those who did not received G-CSF post-transplant (ABMT, n = 6) and those who did (ABMT + G, n = 8). All patients following PBSCT or allo BMT received G-CSF. In all the groups prolonged significant decreases with respect to normal numbers were observed for the T CD3+, CD2+ and CD25+ subsets, more profound for the CD4+ subset but less for the CD8+ subset, especially following PBSCT (only decreased at 1 month). A significant expansion of the CD3+CD57+ and CD8+CD57+ phenotypes was noticed between 9 and 12 months following ABMT, the group of longer follow-up. Long-lasting expansion of the NK-like CD3+CD56+ and CD3+CD16+ subsets was also observed. The B CD19+ and CD20+ subsets had a significant overexpression from 4 months after ABMT, showing a normally balanced Igk+:Ig1+ ratio. Concordantly, the HLA-DR+ and HLA-DQ+ subsets showed significant increases. The NK CD56+ and CD16+ subsets had a faster recovery than the T or B subsets in all the groups. However, the CD3-CD56+, CD3-CD16+, CD16+CD56+, CD3-CD8+, and especially the CD3-CD57+, CD16+CD57+, and CD56+CD57+ subsets had a slower recovery than the global CD56+, CD16+, or CD57+ subsets. The biological and clinical implications of these findings are discussed.

B-Lymphocyte Subsets↗
PubMed1997

Protective immunity elicited by vaccination with DNA encoding for a B cell and a T cell epitope of the A/PR/8/34 influenza virus.

Numerous reports have demonstrated that immunization with plasmids bearing influenza virus hemagglutinin (HA) or nucleoprotein (NP) genes elicits humoral and cellular protective responses. Herein we describe the generation of a plasmid (pVH-TB) encoding for a VH region of a self-Ig in which both the major B cell epitope HA150-159 and the immunodominant CD4 T cell epitope HA110-120 of HA of the A/PR/8/34 influenza virus were genetically inserted in the CDR2 and CDR3 loops, respectively. Our results demonstrate unequivocally that i.m. injection of pVH-TB plasmid in BALB/c mice elicited specific cellular and humoral immune responses able to protect against infection with lethal doses of A/PR/8/34 influenza virus.

Animals↗
PubMed1997

Engineering and characterization of a murine MHC class II-immunoglobulin chimera expressing an immunodominant CD4 T viral epitope.

T cells recognize peptides derived from the processing of proteins by antigen presenting cells (APCs) in association with the major histocompatibility complex (MHC) molecules. We have engineered a murine MHC class II antigen presenting molecule consisting of the extracellular domains of I-E(d)alpha and I-E(d)beta chains to which the CD4 T cell immunodominant epitope HA110-120 of the hemagglutinin (HA) of the A/PR/8/34 influenza virus was covalently linked at the N-terminus of the I-E(d)beta chain. The HA110-120-I-E(d)alphabeta complex was dimerized by the Fc portion of an IgG2a linked at the C-terminus of the I-E(d)beta chain. SF9 insect cells infected with baculovirus carrying both I-E(d)alpha and HA110-120-I-E(d)beta-Fcgamma2a genes, secreted a disulfide-stabilized dimer of the HA110-120-I-E(d)alphabeta-Fcgamma2a molecule, designated as DEF. The chimeric molecule preserved the structural integrity of both MHC-peptide complex and Fc portion of IgG2a, and was able to: (i) bind specifically to the cognate T cell receptors (TCRs) and to the immunoglobulin FcgammaRII receptor (FcR), (ii) induce complement-mediated cell cytotoxicity, and (iii) trigger early production of IL-2 in cognate T cells. Chimeric antigen presenting molecules with these characteristics may represent a novel platform for the development of immunomodulatory agents of therapeutic use.

Amino Acid Sequence↗
PubMed1997

Immunogenicity of a contiguous T-B synthetic epitope of the A/PR/8/34 influenza virus.

A contiguously linked T-B synthetic viral epitope (110HA120-150HA159,T-B) was investigated for its potency in inducing humoral and cellular immune responses in vivo. The T-cell epitope 110HA120 corresponds to the site 1 hemagglutinin (HA) of the A/PR/8/34 (PR8) influenza virus and is recognized by CD4 T cells in association with I-Ed class II major histocompatibility complex molecules. The 150HA159 represents a major B-cell epitope of the HA protein. T-B dipeptide emulsified in Freund's complete adjuvant was able to induce strong antiviral antibody titers and a high frequency of specific T-cell precursors after a single inoculation in BALB/c mice. In contrast, immunization under identical conditions with equimolar mixtures of T and B peptides did not elicit antibody titers or a cellular immune response. As indicated by the isotypes of antiviral antibodies, the T-B dipeptide preferentially induced a Th1-like immune response. Challenge with T-B dipeptide, but not with T or B peptide alone, stimulated peptide-specific T memory cells in mice previously primed with PR8 virus or with T-B dipeptide. As a consequence, 71 and 57% of these mice, respectively, survived infection with two 100% lethal doses of PR8 virus. Our results suggest that, inasmuch as contiguity between T- and B-cell epitopes provides enough signaling capacity to trigger the mechanisms of T-B-cell cooperation in vivo, a T-B contiguous epitope may well represent a minimal built-in subunit vaccine. Aside from their potential bioavailability, the T-B contiguous epitopes may also represent attractive tools for investigating the molecular mechanisms of T-B-cell cooperation responsible for antiviral protection.

Amino Acid Sequence↗
PubMed1996

Immunopotency of a viral peptide assembled on the carbohydrate moieties of self immunoglobulins.

The T-cell receptor recognizes peptides bound to the major histocompatibility complex antigens. Synthetic peptides corresponding to microbial epitopes can efficiently stimulate the in vitro proliferation of T-cell hybridoma or in vivo primed T cells. However, the in vivo immune responses elicited by synthetic peptides are weak because of their short half-life and poor immunogenicity. We previously showed that a genetically engineered immunoglobulin (Ig-HA), in which the CDR3 region of VH gene was replaced with a viral peptide recognized by CD4+ T cells, was able to deliver this epitope in the correct frame to antigen-processing cells that efficiently presented the peptide to T cells. Recently, we developed an enzymatic method to assemble viral peptides on the sugar moieties of immunoglobulins without alteration of the biological functions of either molecule. The viral peptide carried by these conjugates was twenty times more efficient in activating a T-cell hybridoma than the free peptide as calculated on a molar basis. We show that such conjugates are able to prime in vivo the precursors of peptide-specific T cells and to induce proliferation of naive lymphocytes from transgenic mice expressing a peptide-specific T-cell receptor in both CD4 and CD8 T-cell subsets. Our results suggest that peptides enzymatically linked to the carbohydrate moieties of immunoglobulins, using galactose residues as peptide acceptor, can be used as a safe and efficient delivery system of protective epitopes for the prevention of infectious diseases. The enzymatic engineering of immunoglobulins may also allow the development of immunotherapeutic agents to deliver antagonist peptides to autoreactive T cells or to direct immunomodulatory agents such as interleukins or cytolytic drugs to tumor cells.

Animals↗
PubMed1995

The microsatellite mutator phenotype of colon cancer cells is often recessive.

A new mutator mechanism for tumorigenesis, characterized by somatic genomic instability (SGI) at simple repeated sequences (SRS) or microsatellites, underlies hereditary nonpolyposis colorectal cancer (HNPCC) and some sporadic tumors of the colon and other types. To determine whether the microsatellite mutator phenotype (MMP) is dominant or recessive, we generate somatic cell hybrids between a tumor cell line without SGI at SRS (D98OR) and colon carcinoma cell lines with relative low (HCT-15) and high (LS174-T) SGI at SRS. The normal fidelity of replication of these unstable sequences was observed in each of these cell hybrids. Fusion of HCT-15/DLD-1 low instability cells, with LS174-T, HCT116 and LoVo cell lines, all exhibiting relative high instability, also restored the replication fidelity of SRS in all of the hybrids. Hybrids between the high instability cell lines did not grow possibly because of senescence or apoptosis. These results indicate that, in the cell lines analysed, the characterized mutator phenotype of the mismatch repair system resulting in high SGI at SRS, and the uncharacterized mutator phenotype underlying low SGI at SRS, are both recessive. The results also suggest that different tumor cells of the MMP harbor distinct altered growth-related genes.

Colonic Neoplasms↗
PubMed1994

Natural killer cytotoxicity and lymphocyte subpopulations in patients with acute leukemia.

We have studied lymphocyte subpopulations and the NK cytotoxicity of the PBMC from acute leukemia patients in complete remission after chemotherapy or ABMT and from normal donors. We have found a positive linear correlation between the percentage of subpopulations with CD3-CD16+, CD3-CD56+ and CD3-CD8+ phenotypes and the percentage of NK cytotoxicity. The slopes of the regression lines in the two groups of patients were lower than in normal donors, indicating a decreased ability of these cells to operate. The percentages of these subpopulations represent parameters for estimating the percentage of NK cytotoxicity both in normal donors and in patients with acute leukemia.

Acute Disease↗
PubMed1994

Lymphokine-activated killer cytotoxicity and lymphocyte subpopulations in patients with acute leukemia.

In this report we investigated lymphokine-activated killer (LAK) cytotoxicity and lymphocyte subpopulations of peripheral blood mononuclear cells (PBMCs) of patients with acute leukemia in complete remission after chemotherapy or autologous bone marrow transplantation (ABMT) and of normal donors. A positive linear correlation was found between the percentage of spontaneous LAK activity and that of lymphocyte subpopulations with phenotypes CD56+, CD3-CD8+ CD3-CD56+ and CD3-CD57+ in both groups of patients. A 3-day culture with IL-2 produced an up-regulation in the expression of the CD25, CD69, and HLA-DR markers proportional to the LAK cytotoxicity levels generated in the culture. Determination of percentages of spontaneous and in vitro generated LAK activity as well as of the above mentioned phenotypic markers contribute to the analysis of the process of LAK activation in patients with acute leukemia and may also be useful in those cases in which immunotherapy with IL-2 and/or LAK cells is anticipated.

Acute Disease↗
PubMed1994

T-cell receptor gene rearrangements in lymphoid and non-lymphoid leukaemias.

Rearrangements of delta-, tau-, and beta- T-cell receptor (TcR) chain genes were analysed in 64 haematologic malignancies comprising T- and B-lineage acute lymphoblastic leukaemias (ALL), B-chronic lymphocytic leukaemias (CLL), acute myeloid leukaemias (AML) and acute undifferentiated leukaemias AUL). The TcR genes were rearranged in 5/6 T-ALL. In non-T-leukaemias the frequency of TcR gene rearrangements was higher in B-lineage ALL (8/11), although they were all detected in B-CLL (5/29), AML (1/16) and AUL (2/4). Immunoglobulin (Ig) gene rearrangements were observed in 1/6 T-ALL and 2/14 AML. The analysis of these gene configurations has a diagnostic application since it allows the definition of the clonality of malignant proliferation and although they are not lineage specific such configurations represent a further parameter to evaluate, together with the immunophenotype and morphology, in the assignment or exclusion of the differentiation lineage of the haematologic malignancies.

Gene Rearrangement, T-Lymphocyte↗
PubMed1994

[Immunotherapy with interleukin-2 and allogenic LAK cells in a patient with acute myeloblastic leukemia].

At the present time the effectiveness of interleukin-2 (IL-2) administered together with IL-2 in vitro activated lymphocytes (LAK) in the treatment of malignant neoplasias is being assessed. We report the case of an 8 -year- old patient suffering from acute myeloid leukemia (AML) who had relapsed from an autologous bone marrow transplant (ABMT) and received a second ABMT in partial remission. After the second ABMT, the patient, who entered into a phase of complete remission (CR) but with a severe thrombocytopenia, was treated with IL-2 and allogeneic LAK cells obtained from his father. During treatment the lymphocyte subpopulations and cytolytic activity were assayed. Increases in the patient's lymphocytes with CD3- CD4- CD8- CD16+, CD3- CD4- CD8- CD56+, CD3- CD4- CD8+ CD56+ and CD3- CD4- CD8+ CD56+ phenotypes were observed and these were correlated with cytolytic activity measured against the K562 cell line. The patient remained in CR for longer (14 months) than after the first ABMT (5 months). The immune activation produced by this therapy could have had an antileukemic effect in the patient.

Child↗