PubMed Health⌕ Search

Biomedical subjects

C Bordignon

Publications and source records attributed to C Bordignon.

At least 19 recordsLinked to original sources
PubMed1999

Human proinsulin production in primary rat hepatocytes after retroviral vector gene transfer.

The development of autologous somatic cells, engineered for the synthesis and release of human insulin under physiological stimuli, would certainly represent a major breakthrough in the therapy of insulin-dependent diabetes mellitus. We generated a retroviral vector containing the human proinsulin cDNA and the gene coding for the human nerve growth factor receptor for quantitative analysis of transduced cells. Primary rat hepatocytes were selected as target cells because of the constitutive expression of the pancreatic beta-cell glucose transporter GLUT-2 and the glycolitic enzyme glucokinase. Appropriate conditions for culture and retroviral transduction are described. The highest transduction efficiency, evaluated as percentage of LNGFr expressing cells was obtained by repeated infection cycles (40+/-10%). Human proinsulin accumulated in the culture medium of transduced rat hepatocytes (mean+/-SD): 18.1+/-7.9 (range 8.7-36.4) ng/24h/10(6) cells. Primary rat hepatocytes can be efficiently transduced by a retroviral vector and the de novo synthesis of human proinsulin can be induced. Primary cultured hepatocytes represent an useful model to test retroviral constructs engineered for the glucose-inducible expression of insulin under the control of liver-specific promoters.

Animals↗
PubMed1998

Herpes simplex virus thymidine kinase gene transfer for controlled graft-versus-host disease and graft-versus-leukemia: clinical follow-up and improved new vectors.

We previously demonstrated that severe graft-versus-host disease (GvHD), associated with the therapeutic infusion of donor lymphocytes after allogeneic marrow transplantation (BMT), can be efficiently controlled by the SFCMM-2-mediated expression of the herpes simplex virus thymidine kinase (HSV-tk) suicide gene into the allogeneic lymphocytes. This was achieved by selective elimination of transduced lymphocytes by ganciclovir (GCV) infusion. Despite the positive results of the pilot clinical trial, two vector-related limitations were observed. The induction of a strong immune response against genetically modified cells was observed in two patients. In addition, the only patient who developed chronic GvHD showed only partial response to ganciclovir treatment. In an attempt to overcome these limitations, we developed a new generation of vectors. The neomycin resistance (neoR) gene was removed from the SFCMM-3 and SFCMM-4 retroviral vectors. These vectors are less immunogenic and able to confer higher ganciclovir sensitivity to transduced human lymphocytes. All the vectors carry a modified form of the low-affinity nerve growth factor receptor cDNA, as cell surface selectable marker (deltaLNGFR). The vectors were compared in terms of gene transfer efficiency, and ability to confer high and specific sensitivity to ganciclovir. The SFCMM-3 vector, carrying the entire HSV-tk gene driven by the LTR promoter, allows efficient transduction of human T lymphocytes and confers the highest GCV sensitivity to transduced lymphocytes with a high and a low proliferation index. The expression of the deltaLNGFR marker allows an easier in vitro manipulation and a faster immune selection of transduced cells compared with neoR selection. Finally, the elimination of the neoR gene removes a potent immunogen from transduced lymphocytes.

Anti-Bacterial Agents↗
PubMed1998

Translation of a retained intron in tyrosinase-related protein (TRP) 2 mRNA generates a new cytotoxic T lymphocyte (CTL)-defined and shared human melanoma antigen not expressed in normal cells of the melanocytic lineage.

We report here the identification of a new shared human melanoma antigen recognized by a human leukocyte antigen (HLA)-A*68011-restricted cytotoxic T lymphocyte clone (CTL 128). The cDNA encoding this antigen is composed of a partially spliced form of the melanocyte differentiation antigen tyrosinase-related protein (TRP)-2, containing exons 1-4 with retention of intron 2 and part of intron 4 (TRP-2-INT2). The sequence coding for the antigenic epitope is located at the 5' end of intron 2 and is available for translation in the same open reading frame of the fully spliced TRP-2 mRNA. This peptide is also recognized by CTL 128 when presented by the HLA-A*3301, a member of the HLA-A3-like supertype that includes the HLA-A*68011. Quantitative reverse transcription PCR analysis carried out on total and/or cytoplasmic mRNA demonstrated that, in contrast to the fully spliced TRP-2 mRNA expressed in melanomas, normal skin melanocytes, and retina, the TRP-2-INT2 mRNA could be detected at significant levels in melanomas but not in normal cells of the melanocytic lineage. Instead, in these normal samples, both the spliced and the unspliced transcript of gp100 were expressed at high levels. Absence of endogenous TRP-2-INT2 expression in melanocytes was also confirmed by lack of recognition of HLA-A*68011-transduced, TRP-2(+) melanocyte lines by CTL 128. These results indicate that a partially spliced form of a differentiation antigen mRNA, present in the cytoplasmic compartment of neoplastic but not normal cells of the melanocytic lineage, can be the source of a melanoma-restricted T cell epitope.

Alleles↗
PubMed1998

Transduced fibroblasts and metachromatic leukodystrophy lymphocytes transfer arylsulfatase A to myelinating glia and deficient cells in vitro.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease, caused by deficiency of arylsulfatase A (ASA), that manifests primarily in the white matter of the nervous system. Currently, no specific treatment exists that will reverse its fatal outcome. Replacement therapy has been hampered by the blood-brain barrier (BBB). To circumvent this problem we designed an ex vivo gene therapy strategy that includes the retrovirus-mediated ASA transduction of cells, such as activated lymphocytes, that are able to traverse the BBB or other membranes of the CNS. For this purpose, two recombinant retroviruses based on the pLXSN vector were produced, containing the wild-type ASA cDNA or a pseudodeficiency ASA cDNA, which encodes a smaller enzyme with normal activity. After transduction, ASA activity increased more than 100-fold in fibroblasts from an MLD patient. Furthermore, ASA-transduced MLD PBLs expressed 30 times higher ASA activity when compared with control PBLs. Moreover, cell culture experiments demonstrated that transduced fibroblasts could efficiently transfer ASA to deficient cells across a transwell barrier, whereas transduced MLD lymphocytes could transfer ASA to deficient fibroblasts only by direct cell-to-cell contact. Finally, ASA was taken up by normal oligodendrocytes and Schwann cells, the target myelinating glial cells for therapy in MLD. These data suggest possible short-term strategies for transfer of ASA into the CNS via transduced autologous cells while long-term strategies, related to autologous transduced bone marrow transplant, take effect in patients.

Cerebroside-Sulfatase↗
PubMed1998

High homogeneity of MAGE, BAGE, GAGE, tyrosinase and Melan-A/MART-1 gene expression in clusters of multiple simultaneous metastases of human melanoma: implications for protocol design of therapeutic antigen-specific vaccination strategies.

Human melanoma cells express several antigens which are recognized by autologous and specific CTL clones in association with HLA-class-I molecules. Many of these antigens represent suitable targets for tumor immunotherapy, since their expression in human melanoma cells is common and highly specific. In order to achieve real clinical success with therapeutic vaccination strategies, one important requirement is the expression of the target antigen by all the tumor lesions of a patient. We have studied this issue by assessing, through an RT-PCR approach, the expression of MAGE-1, MAGE-2, MAGE-3, BAGE, GAGE-1/2, Tyrosinase and Melan-A/MART-1 genes in 17 clusters of simultaneous in-transit or regional lymph-node metastases collected from 15 stage-III and 1 stage-IV (AJCC/UICC pTNM system) melanoma patients. In 14 out of 17 clusters of simultaneous metastatic lesions (82%), the homogeneity in the pattern of gene expression within the cluster was complete. Heterogeneity within the same cluster was observed in only 3 out of 17 clusters (18%) and represented only minor features. Our data reveal that, in AJCC-stage-III melanoma patients, different but simultaneous metastatic lesions express the same pattern of antigen-coding genes. These observations have 2 main clinical implications: (i) the antigenic characterization of one single and easily accessible lesion allows identification of optimal targets for an active antigen-specific immunotherapy treatment; (ii) almost all the metastatic lesions are expected to be hit by the immune response eventually induced against the tumor antigen. Moreover, these data suggest that active specific immunotherapy directed against MAGE-1, MAGE-3, BAGE, GAGE-1/2, Melan-A/MART-1 and Tyrosinase antigens could be exploited as an adjuvant treatment to surgery in high-risk AJCC-stage-III-melanoma patients.

Antigens, Neoplasm↗
PubMed1998

The DAM gene family encodes a new group of tumor-specific antigens recognized by human leukocyte antigen A2-restricted cytotoxic T lymphocytes.

The DAM family of genes has a high degree of homology with MAGE, both in nucleotide sequence and in neoplastic tissue-specific expression. This study describes, for the first time, the identification of CTLs specific for a peptide epitope encoded by DAM genes. A human leukocyte antigen (HLA)-A2-restricted CTL clone was raised against a peptide, D10/6-271, encoded by codons 271-279 in the DAM cDNA. The corresponding peptide in the MAGE-3 sequence, M3-271, has been shown previously to be a natural T-cell epitope for HLA-A2-restricted CTLs recognizing the MAGE-3 protein. The D10/6-271-specific CTL clone required approximately 3 nM exogenous peptide for half-maximal lysis of target cells and was able to specifically recognize endogenous DAM antigen on HLA-A2+ melanoma cells infected with a vaccinia vector recombinant for gene DAM-6. These data suggest that DAM genes might encode a new group of tumor-specific antigens useful for the design of specific antitumor vaccines.

Antigens, Neoplasm↗
PubMed1998

Suicide-gene-transduced T-cells for the regulation of the graft-versus-leukemia effect.

In allogeneic marrow transplantation (BMT), donor lymphocytes play a central therapeutic role in both graft-versus-leukemia and immune reconstitution. However, their use is limited by the risk of severe graft-vs-host disease (GvHD). Different strategies have been investigated to obtain all the benefits derived from donor lymphocytes while avoiding the risk of GvHD. In the first pilot study, infusions of donor lymphocytes transduced with the Herpes Simplex virus thymidine kinase (HSV-tk) suicide resulted in anti-tumor activity in 50% of patients. Acute GvHD could be effectively controlled by ganciclovir-induced elimination of the transduced cells. If these results will be confirmed in larger studies, genetic manipulation of donor lymphocytes will increase efficacy and safety of allogeneic marrow transplantation.

Aged↗
PubMed1997

Cell-surface marking of CD(34+)-restricted phenotypes of human hematopoietic progenitor cells by retrovirus-mediated gene transfer.

Human CD34+ cells lacking detectable levels of HLA-DR antigens (CD34+ DR-) are highly enriched in hematopoietic pluripotent progenitors with long-term marrow repopulating ability. We investigated the feasibility of transducing and marking CD34+ DR- progenitor cells from bone marrow (BM) or mobilized peripheral blood samples (MPB) of 13 patients undergoing BM transplantation with the purpose of developing a protocol for a large-scale clinical application. A new retroviral vector coding for the truncated form (delta) of the low-affinity nerve growth factor receptor (LNGFR) was used to quantitate the level of gene transfer into CD34+ cells and their progeny by multiparameter cytofluorimetry and immunocytochemistry. Light-density mononuclear cells as well as purified CD34+ cells were transduced either by direct incubation with retroviral supernatants or prestimulated in vitro with various combinations of growth factors prior to transduction. Transduction efficiency, assessed as G418-resistant growth of granulocyte-macrophage colony-forming units (CFU-GM) progenitors from MPB, was 1.7-fold higher (14.9% +/- 4.5%) than those from BM (8.5% +/- 3.9%) and it was further improved (26.9% +/- 3.1%) using a purified CD34+ population as target cells. Three-color fluorescence-activated cell sorting (FACS) analysis demonstrated the presence of transduced delta LNGFR+ cells within the CD34+ DR- subpopulation. In the absence of growth factors, gene transfer into BM or MPB CD34+ DR- cells was generally poor, but following a 72-hr prestimulation it peaked at 38% of total CD34+ DR- bone marrow (BM) cells in the presence of the c-kit ligand (KL) and at 31% in the presence of IL-3. Furthermore, KL gave, compared to the other cytokines, the highest absolute yield of BM delta LNGFR+ CD34+ DR- cells recovered after transduction (p = 0.05 compared to 24 hr). Gene transfer into in vitro primitive progenitor cells was further confirmed by expression of the delta LNGFR marker on CD34+ cells and CFU-GM derived from 5-week long-term culture on stroma.

Adult↗
PubMed1997

Functional heterogeneity of HLA-A*02 subtypes revealed by presentation of a MAGE-3-encoded peptide to cytotoxic T cell clones.

The peptide-binding and presentation characteristics of seven naturally occurring HLA-A2 subtypes were studied using M3(271), a peptide derived from the tumor-specific Ag encoded by gene MAGE-3, which has been shown to be processed and presented by A*0201+ melanoma lines. Three independent M3(271)-specific CTL clones were obtained from two unrelated A*0201+ donors. B lymphoblastoid cell lines (BLCLs) expressing A*0201, A*0207, or A*0209 could be sensitized to lysis by all three clones upon incubation with the relevant peptide. Furthermore, the same BLCLs were able to present endogenous M3(271) in IFN-gamma release assays. These findings demonstrate, for the first time, the existence of a functional overlap between A*0207 and other A*02 subtypes. One of the CTL clones also lysed M3(271)-pulsed BLCLs expressing A*0204 and A*0206, while the other two clones recognized M3(271) only in the context of either of these two subtypes. Peptide-pulsed BLCLs expressing A*0202 or A*0205 were not lysed, although A*0205 and, with lower affinity, A*0202 molecules were shown to bind peptide M3(271). These findings have implications for the selection of cancer patients for specific immunotherapy with peptide M3(271).

Antigen Presentation↗
PubMed1997

HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia.

In allogeneic bone marrow transplantation (allo-BMT), donor lymphocytes play a central therapeutic role in both graft-versus-leukemia (GvL) and immune reconstitution. However, their use is limited by the risk of severe graft-versus-host disease (GvHD). Eight patients who relapsed or developed Epstein-Barr virus-induced lymphoma after T cell-depleted BMT were then treated with donor lymphocytes transduced with the herpes simplex virus thymidine kinase (HSV-TK) suicide gene. The transduced lymphocytes survived for up to 12 months, resulting in antitumor activity in five patients. Three patients developed GvHD, which could be effectively controlled by ganciclovir-induced elimination of the transduced cells. These data show that genetic manipulation of donor lymphocytes may increase the efficacy and safety of allo-BMT and expand its application to a larger number of patients.

Bone Marrow Transplantation↗
PubMed1996

Characterization of antigenic peptides presented by HLA-B44 molecules on tumor cells expressing the gene MAGE-3.

The amino acid sequence of the protein encoded by the gene MAGE-3 was screened for peptides containing the binding motif for HLA-B44. Nine peptides were synthesized, and their binding affinity for HLA-B*4402 and -B*4403 was analyzed in an HLA class I alpha-chain refolding assay. Four peptides with binding affinity for HLA-B*4403 were chosen for in vitro cytotoxic T-lymphocyte induction assays using as antigen-presenting cells peptide-pulsed, autologous activated B lymphoblasts from a healthy, B*4403+ donor. Peptide-specific effectors could be raised only against one peptide, M3-167. Cytotoxic T lymphocytes specific for this peptide were also able to recognize melanoma cell lines expressing HLA-B44 and the gene MAGE-3, strongly suggesting that M3-167 is a naturally processed MAGE-3-encoded epitope presented by HLA-B44. M3-167 is a I amino acid N-terminal extension of M3-168, a naturally processed epitope MAGE-3-encoded epitope presented by HLA-A1 that has been previously described. TAP binding studies of these 2 peptides revealed that the TAP affinity of M3-167 is about 9-fold higher than that of M3-168. M3-167 or a longer precursor could be transported into the endoplasmatic reticulum, where it could be trimmed for presentation by HLA-A1 or -B44 molecules. Taken together, our data suggest that M3-167 could be an immunodominant peptide encoded by the gene MAGE-3.

Amino Acid Sequence↗