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Biomedical subjects

T Velu

Publications and source records attributed to T Velu.

At least 19 recordsLinked to original sources
PubMed2001

Cloning and sequencing of defective particles derived from the autonomous parvovirus minute virus of mice for the construction of vectors with minimal cis-acting sequences.

The production of wild-type-free stocks of recombinant parvovirus minute virus of mice [MVM(p)] is difficult due to the presence of homologous sequences in vector and helper genomes that cannot easily be eliminated from the overlapping coding sequences. We have therefore cloned and sequenced spontaneously occurring defective particles of MVM(p) with very small genomes to identify the minimal cis-acting sequences required for DNA amplification and virus production. One of them has lost all capsid-coding sequences but is still able to replicate in permissive cells when nonstructural proteins are provided in trans by a helper plasmid. Vectors derived from this particle produce stocks with no detectable wild-type MVM after cotransfection with new, matched, helper plasmids that present no homology downstream from the transgene.

Base Sequence↗
PubMed2001

Chorioretinal post-transplant lymphoproliferative disorder induced by the Epstein-Barr virus.

BACKGROUND: The Epstein-Barr virus (EBV) is responsible for the lymphoproliferative disorders observed in transplanted patients. METHODS: The case history is described of a 59 year old man with a chorioretinal lesion who had received a single lung transplant and was on immunosuppressive treatment. Immunoglobulin gene rearrangement and EBV detection by polymerase chain reaction (PCR) with semiquantification were used on the vitreous material. RESULTS: A proliferation of B lymphocytes with a monoclonal subpopulation was found by PCR on the vitreous sample. The large amounts of EBV genomes found in the vitreous suggest that EBV was the cause of the lymphoproliferation. Healing of the lesion was achieved by a decrease in immunosuppressive treatment and the use of nucleotide analogues. CONCLUSION: The diagnosis of ocular post-transplant lymphoproliferative disorder (PTLD) can be made by PCR on vitreous material. Early diagnosis and treatment can lead to regression of limited monoclonal lesions.

Choroid Diseases↗
PubMed2001

Transient expansion of peptide-specific lymphocytes producing IFN-gamma after vaccination with dendritic cells pulsed with MAGE peptides in patients with mage-A1/A3-positive tumors.

Assessment of T-cell activation is pivotal for evaluation of cancer immunotherapy. We initiated a clinical trial in patients with MAGE-A1 and/or -A3 tumors using autologous DC pulsed with MAGE peptides aimed at analyzing T-cell-derived, IFN-gamma secretion by cytokine flow cytometry and ELISPOT. We also tested whether further KLH addition could influence this response favorably. Monocyte-derived DC were generated from leukapheresis products. They were pulsed with the relevant MAGE peptide(s) alone in group A (n=10 pts) and additionally with KLH in group B (n=16 pts). A specific but transient increase in the number of peripheral blood T lymphocytes secreting IFN-gamma in response to the vaccine peptide(s) was observed in 6/8 patients of group A and in 6/16 patients of group B. We conclude that anti-tumor vaccination using DC pulsed with MAGE peptides induces a potent but transient anti-MAGE, IFN-gamma secretion that is not influenced by the additional delivery of a nonspecific, T-cell help.

Adult↗
PubMed2001

[Anti-tumor immunotherapy based on dendritic cells].

In cancer immunotherapy, the use of dendritic cells (DC) loaded with tumor-associated antigens (TAA) emerged as a promising strategy. We initiated 3 pilot clinical trials with immunological endpoints using TAA loaded autologous DC. These trials showed that this approach was safe and associated with the induction of potent TAA specific IFN-gamma responses, which were transient despite the providing a further help through KLH presentation. Subcutaneous (s.c.) IL-2 administration was associated with long-lasting TAA specific IL-5 production. Clinical responses were observed in about 1/3 of the patients. Further improvements will take advantage of the use of a new type of DC cells (IL-3/IFN-beta DC) and of tumor cell-DC hybrids.

Antigen Presentation↗
PubMed2000

Tropism of AAV-2 vectors for neurons of the globus pallidus.

A recombinant AAV-2 vector encoding the green fluorescent protein (gfp) under the control of the cytomegalovirus (CMV) promoter was injected into the striatum at varying antero-posterior coordinates. When the virus was delivered to the anterior part of the striatum, transduction efficiency was low and limited to the vicinity of the needle tract. In contrast, after injection into the posterior part of the striatum, in addition to a localized transduced area in the striatum, efficient and widespread transduction was observed at distance from the injection site, in the globus pallidus. In the latter case, labelled cells were also detected in the internal capsule and in the stria terminalis. The number of transduced cells in the striatum increased up to I month and then decreased whereas in the globus pallidus, transduction was maximal as early as 2 weeks post-injection. In the striatum and in the globus pallidus, the labelled cells had a neuron-like morphology. In contrast, in the internal capsule, labelled cells had a glial-like morphology.

Animals↗
PubMed2000

Comparative in vitro and in vivo cytotoxic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and its arabinosyl derivative, (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU), against tumor cells expressing either the Varicella zoster or the Herpes simplex virus thymidine kinase.

The inhibitory effects of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and its arabinosyl derivative (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU) on the growth of both MDA-MB-435 human breast carcinoma and 9L rat gliosarcoma cells expressing the thymidine kinase (tk)-encoding gene of the Varicella zoster virus (VZV) or the Herpes simplex virus (HSV) were evaluated. In vitro, BVDU and BVaraU effectively killed both cell types expressing VZVtk, with 50% inhibitory concentration values ranging from 0.06 to 0.4 microM, whereas ganciclovir (GCV) lacked activity. On HSVtk+ cells, BVDU had high cytotoxic activity, with 50% inhibitory concentration values that were similar to those of GCV, whereas BVaraU was inactive. In vivo, BVDU applied intraperitoneally caused a 50% tumor growth inhibition in nude mice inoculated subcutaneously with VZVtk+ as well as HSVtk+ mammary tumor cells. In mice and at variance with the in vitro results, BVaraU had very little activity against the VZVtk+ mammary cells; GCV had the highest activity on the HSVtk+ cells, resulting in a 50% eradication of the tumors. With the 9L rat gliosarcoma model, the VZVtk/BVDU system completely failed to inhibit the development of VZVtk+ glioma tumors induced subcutaneously in syngeneic rats, although BVDU had a similar 45-minute half-life in both rats and mice. Factors other than degradation of the prodrug and related to the mode of action of these analogs are possibly involved in the observed discrepancies between the in vitro and in vivo results.

Animals↗
PubMed2000

The role of cellular- and prodrug-associated factors in the bystander effect induced by the Varicella zoster and Herpes simplex viral thymidine kinases in suicide gene therapy.

To investigate the factors influencing the bystander effect--a key element in the efficacy of suicide gene therapy against cancer--we compared the effect triggered by four extremely efficient gene/prodrug combinations, i.e., VZVtk/BVDU, the thymidine kinase of Varicella zoster virus associated with (E)-5-(2-bromovinyl)-2'-deoxyuridine; VZVtk/BVaraU, the same enzyme associated with (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil; HSVtk/BVDU, the association of the Herpes simplex virus thymidine kinase with BVDU; and the classical HSVtk/GCV (ganciclovir) paradigm. The cells used, the human MDA-MB-435 breast cancer, and the rat 9L glioblastoma lines were equally sensitive in vitro to these four associations. In both cell types, the combinations involving pyrimidine analogues (BVDU, BVaraU) displayed a smaller bystander killing than the combination involving the purine analogue (GCV). In addition, the bystander effect induced by all the tk/prodrug systems was reduced in MDA-MB-435 cells in comparison to 9L cells; albeit, the viral kinases were produced at a higher level in the breast cancer cells. All systems induced apoptotic death in the two cell types, but the MDA-MB-435 cells, deprived of connexin 43, were noncommunicating in striking contrast with the 9L cells. That functional gap junctions have to be increased in order to improve the breast cancer cell response to suicide gene therapy was demonstrated by transducing the Cx43 gene: this modification enhanced the bystander effect associated in vitro with GCV treatment and, by itself, decreased the tumorigenicity of the untreated cells. However, the noncommunicating MDA-MB-435 cells triggered a significant bystander effect both in vitro and in vivo with the HSVtk/GCV system, showing that communication through gap junctions is not the only mechanism involved.

Animals↗
PubMed2000

Role and expression of CD40 on human retinal pigment epithelial cells.

PURPOSE: To examine the CD40 costimulatory molecule expression on normal resting or activated adult human retinal pigment epithelium (hRPE) cells and to evaluate its role as an activation molecule considering the potential antigen presentation functions of hRPE cells. METHODS: Expression of HLA-DR and costimulatory (CD40, B7.1, B7.2, CD54, and CD58) molecules on hRPE cells was analyzed by flow cytometry. CD40 triggering was performed using soluble CD40L or cocultures with CD40L transfected fibroblasts. Interleukin (IL)-6, -8, -10, and -12 secretions were measured by enzyme-linked immunosorbent assay. Antigen presentation function of hRPE cells was assessed by coculturing hRPE cells with allogeneic T cells. T-cell proliferation was measured by [(3)H]-thymidine incorporation, and T-cell apoptosis by measurement of caspase-3 activity. RESULTS: Interferon (IFN)gamma-activated hRPE cells expressed CD40, but not B7.1 or B7.2. Although interferongamma enhanced IL-6 and IL-8 production, CD40 triggering of IFNgamma-activated hRPE cells did not induce IL-12 secretion. hRPE cells did not stimulate allogeneic resting T cells and downregulated phytohemagglutinin-activated allogeneic T cells via a cell-to-cell contact-dependent mechanism. Some induction of apoptosis was detected. CONCLUSIONS: CD40 is expressed on IFNgamma-activated hRPE cells. Its ligation leads to an increased production of IL-6 and IL-8 but fails to induce B7.1 or B7. 2 expression, or to induce IL-12 secretion. Accordingly, hRPE cells do not activate allogenic T cells but inhibit T-cell proliferation, partly through induction of apoptosis. These results suggest that hRPE cells could be implicated more in a deviant antigen presentation. If the exact molecular mechanisms are unclear, it is likely that CD40-CD40L interaction could play a role in this process.

Animals↗
PubMed1999

Influence of sequence and size of DNA on packaging efficiency of parvovirus MVM-based vectors.

We have derived a vector from the autonomous parvovirus MVM(p), which expresses human IL-2 specifically in transformed cells (Russell et al., J. Virol 1992;66:2821-2828). Testing the therapeutic potential of these vectors in vivo requires high-titer stocks. Stocks with a titer of 10(9) can be obtained after concentration and purification (Avalosse et al., J. Virol. Methods 1996;62:179-183), but this method requires large culture volumes and cannot easily be scaled up. We wanted to increase the production of recombinant virus at the initial transfection step. Poor vector titers could be due to inadequate genome amplification or to inefficient packaging. Here we show that intracellular amplification of MVM vector genomes is not the limiting factor for vector production. Several vector genomes of different size and/or structure were amplified to an equal extent. Their amplification was also equivalent to that of a cotransfected wild-type genome. We did not observe any interference between vector and wild-type genomes at the level of DNA amplification. Despite equivalent genome amplification, vector titers varied greatly between the different genomes, presumably owing to differences in packaging efficiency. Genomes with a size close to 100% that of wild type were packaged most efficiently with loss of efficiency at lower and higher sizes. However, certain genomes of identical size showed different packaging efficiencies, illustrating the importance of the DNA sequence, and probably its structure.

Bacteriophage lambda↗
PubMed1999

Cellular contaminants of adeno-associated virus vector stocks can enhance transduction.

Transduction efficiency of different types of recombinant (r)AAV-2 based vectors preparations markedly differed, with apparently no correlation with the replicative titers. Using HeLa cells as target for transduction, 105 and 30 infectious units were necessary to observe one transductant using respectively cesium-chloride-purified rAAV and crude lysates of producer cells obtained by sonication. The purified vectors were however able to transduce HEK-193 cells efficiently, but transgene expression was detected with some delay compared with crude lysates. The unexpected high transduction efficiency of sonicated crude lysates was due to virally mediated gene transfer, since similar sonicated crude lysates, but with no AAV rep and cap genes, did not lead to detection of transgene products after incubation with HeLa cells. Furthermore, sonicated cellular extracts of 293 or 293/T cells given in trans stimulate transduction of HeLa cells by purified rAAV. In contrast, neither extracts from the adenovirus E1-transformed 911 cell line, nor from other cell lines not harboring any adenovirus gene, had enhancing effect on rAAV-mediated transduction. These data suggest that 293 sonicated extracts contain factors which stimulate rAAV-mediated transduction of cells that are normally poorly transduced and offer a system to identify such factors and to characterize further the steps limiting the transfer of gene by AAV vectors.

Dependovirus↗
PubMed1999

Sentinel lymph node mapping in the management of high risk malignant melanoma.

In patients with malignant melanoma, the selective biopsy of the first draining lymph node, so-called the sentinel lymph node, allows to identify, with a low morbidity, the patients with nodal metastasis that require radical lymphadenectomy and adjuvant systemic chemotherapy. Herein, we report our initial experience in sentinel lymph node biopsy in 16 patients with malignant melanoma. The sentinel lymph node was localised using preoperative lymphoscintigraphy and injection of dye blue. Intraoperatively, the dissection was guided with a gamma probe and by the recognition of the blue nodes. In the 16 cases the sentinel lymph node was localised. In 50% of the cases, multiple sentinel nodes were demonstrated at lymphoscintigraphy and found during surgery. A limited postoperative morbidity was observed in three cases. Three patients presented nodal metastasis and underwent further radical lymphadenectomy. We conclude that sentinel lymph node mapping is a feasible and reproductive procedure. The preoperative lymphoscintigraphy is essential to identify multiple sentinel nodes and guide surgical dissection. The impact of this approach on the overall survival of patients with high-risk melanoma has still to be demonstrated in studies with a long follow-up.

Adult↗
PubMed1998

Relative contribution of Ki-ras gene analysis and brush cytology during ERCP for the diagnosis of biliary and pancreatic diseases.

BACKGROUND: Ki-ras mutation analysis from material collected during ERCP has been claimed to improve the diagnosis of pancreatic and bile duct carcinomas as compared with conventional cytology. Our aim was to study the relative contribution of both Ki-ras analysis and brush cytology in patients with a significant stricture at ERCP. METHODS: Brushings were collected in duplicate for both analyses in 142 patients in whom a definitive diagnosis was obtained by histology or a minimal follow-up of 6 months. RESULTS: For pancreatic strictures, sensitivity, specificity, and accuracy of Ki-ras analysis vs. cytology in detecting malignancy were 81% vs. 66%, 72% vs. 100%, and 70% vs. 74%, respectively. For biliary strictures, they were 25% vs. 42%, 100% vs. 100%, and 35% vs. 43%, respectively. The combination of the two methods only marginally increased their sensitivity and accuracy in both types of strictures. CONCLUSION: Ki-ras analysis is a sensitive method for diagnosing pancreatic but not biliary carcinoma. However, its specificity is lowered by a high frequency of Ki-ras mutations in patients with chronic pancreatitis (25%) who did not manifest cancer development within a 6-month follow-up period. In pancreatic duct strictures, brush cytology appears to be more specific in detecting malignancy; specificity for Ki-ras and cytology are equivalent for the diagnosis of malignant bile duct strictures. Therefore, making a clinical decision on the sole basis of Ki-ras analysis is probably not justified in the majority of the cases.

Adult↗
PubMed1997

Disseminated angiosarcoma presenting as a hemophagocytic syndrome in a renal allograft recipient.

A case of angiosarcoma arising in the setting of transplantation is reported. This rare and malignant tumor of the endothelial system is seldom observed in allograft recipients, with only seven cases having been previously reported. What is interesting about the present observation is that the tumor is thought to have developed in the vicinity of a Dacron graft and that it showed prominent erythrophagocyte-like activity. This activity was associated with a particular clinical syndrome that shared some attributes with infection-associated hemophagocytic syndrome.

Diagnosis, Differential↗
PubMed1997

Morphologic and phenotypic changes of the leukemic cells in a case of marginal zone B-cell lymphoma.

A particular case of marginal zone B-cell lymphoma (MZBCL) presenting with leukemic lymphocytes is reported. In the present observation, the leukemic cells not only displayed a remarkable morphological fluctuation but also had an unusual phenotype, changing with time. These phenotypic features, which have been functionaly investigated by in vitro assays, might simply reflect an activation state depending on the microenvironment. Because of its disconcerting similarities with hairy cell leukemia (HCL) and splenic lymphoma with villous lymphocytes (SLVL), this case relaunches the debate about whether close relationships might exist between the splenic marginal zone, SLVL and HCL.

Antigens, CD19↗
PubMed1997

Interleukin-10 modulates susceptibility in experimental cerebral malaria.

In this study, we examined the effects of interleukin-10 (IL-10) on the outcome of experimental cerebral malaria (CM), a lethal neurological syndrome that occurs in susceptible strains of mice after infection with Plasmodium berghei ANKA (PbA). Constitutive IL-10 mRNA levels were significantly higher in the spleen and brain of resistant animals. In vivo neutralization of endogenous IL-10 in CM-resistant mice induced the neurological syndrome in 35.7% of these mice, as opposed to 7.7% in controls. IL-10 inhibited PbA antigen-specific interferon-gamma (IFN-gamma) production in vitro but not tumour necrosis factor (TNF) serum levels in vivo. Susceptible mice, on the other hand, were significantly protected against CM when injected with recombinant IL-10. Overall, our findings suggest that IL-10 plays a protective role against experimental cerebral malaria.

Animals↗