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Biomedical subjects

V Ramakrishna

Publications and source records attributed to V Ramakrishna.

At least 19 recordsLinked to original sources
PubMed1999

Preparation and functional properties of monoclonal antibodies to human, mouse and rat OX-2.

We have prepared mouse and rat hybridomas to a 43-kDa molecule expressed in the thymus, on a subpopulation of dendritic cells, and in the brain, in mammalian tissue derived from mouse, rat and human. Using CHO cells transiently transfected with adenovirus vector(s) expressing a cDNA construct for the relevant OX-2 gene, we show these monoclonal antibodies (Mabs) detect a molecule encoded by this construct (rat OX-2 (rOX-2), mouse OX-2 (mOX-2) and human OX-2 (huOX-2), respectively). Furthermore, at least some of the anti-rat Mabs detect determinants expressed on the murine OX-2 molecule, as we predicted in an earlier publication. Previous studies have implied that this molecule might serve an important role in regulation of cell signaling for cytokine production. Using one-way mixed leukocyte reactions we show that when cells are cultured in the presence of the species-specific Mab, cytokine production becomes polarized 'away from' type-2 cytokine production, with preferentially increased expression of type-1 cytokine production.

Animals↗
PubMed1999

Evidence that an OX-2-positive cell can inhibit the stimulation of type 1 cytokine production by bone marrow-derived B7-1 (and B7-2)-positive dendritic cells.

We reported that hepatic mononuclear, nonparenchymal cells (NPC) can inhibit the immune response seen when allogeneic C57BL/6 dendritic cells (DC) are incubated with C3H spleen responder cells. Cells derived from these cultures transfer increased survival of C57BL/6 renal allografts in C3H mice. We also found that increased expression of OX-2 on DC was associated with inhibition of cytokine production and renal allograft rejection. We explored whether inhibition by hepatic NPC was a function of OX-2 expression by these cells. Fresh C57BL/6 spleen-derived DC were cultured with C3H spleen responder cells and other putative coregulatory cells. The latter were derived from fresh C3H or C57BL/6 liver NPC, or from C3H or C57BL/6 mice treated for 10 days by i.v. infusion of human Flt3 ligand. Different populations of murine bone marrow-derived DC from cultures of bone marrow with IL-4 plus granulocyte-macrophage-CSF were also used as a source of putative regulator cells. Supernatants of all stimulated cultures were examined for functional expression of different cytokines (IL-2, IL-4, IFN-gamma, and TGFbeta). We found that fresh C57BL/6 splenic DC induced IL-2, not IL-4, production. Cells from the sources indicated inhibited IL-2 and IFN-gamma production and promoted IL-4 and TGFbeta production. Inhibition was associated with increased expression of OX-2 on these cells, as defined by semiquantitative PCR and FACS analysis. By size fractionation, cells expressing OX-2 were a subpopulation of NLDC145+ cells. Our data imply a role for cells expressing OX-2 in the regulation of induction of cytokine production by conventional allostimulatory DC.

Adjuvants, Immunologic↗
PubMed1999

Alterations in chemokine mRNA expression in animals receiving portal vein immunization and renal allo- or xenotransplantation precede altered cytokine production.

We have analyzed chemokine mRNA expression in graft tissue of C3H/HEJ mice receiving allogeneic (C57BL/6) or xenogeneic [Lewis (LEW) rat donors] kidney grafts and correlated this with graft survival. Since donor-specific portal vein (pv) immunization is known to increase allo- and xenograft survival, in some cases recipients also received pretransplant pv or intravenous (iv) immunization; other animals received the antioxidant N-acetylcysteine (NAc) to examine the role of ischemia/reperfusion injury in the changes observed. Graft tissue and lymph nodes draining the respective grafts were obtained at various times posttransplantation and used for quantitative polymerase chain reaction analysis of mRNAs for different chemokines. In addition, lymphocytes were restimulated in culture with donor antigen and supernatants assayed for different cytokines. We observed that early increases in mRNA for MCP-1 preceded a polarization to type 2 cytokine production. Infusion of NAc twice daily for 4 days following transplantation further altered chemokine mRNA expression (increased MCP-1 and RANTES; decreased CINC); led to more enhanced type 2 cytokine production relative to control animals; and further increased xenograft survival. By use of heteroantibodies to different chemokines, anti-MCP-1 alone, but not antibodies to MIP-1alpha or RANTES, abolished this early polarization in cytokine production, implying a causal link between MCP-1 production and polarization in cytokine production. We conclude that manipulation of chemokine production early after transplantation might indirectly modify graft outcome by modifying cytokine production.

Acetylcysteine↗
PubMed1998

Targeting of interleukin 2 to human ovarian carcinoma by fusion with a single-chain Fv of antifolate receptor antibody.

To provide a new tool for the immunotherapy of human ovarian carcinoma, we constructed a fusion protein between interleukin-2 (IL-2) and the single-chain Fv (scFv) of MOV19, a monoclonal antibody directed against alpha-folate receptor (alpha-FR), known to be overexpressed on human nonmucinous ovarian carcinoma. This was accomplished by fusing the coding sequences in a single open reading frame and expressing the IL-2/MOV19 scFv chimera under the control of the murine immunoglobulin K promoter in J558L plasmacytoma cells. The design allowed the construction of a small molecule combining the specificity of MOV19 with the immunostimulatory activity of IL-2. This might improve the tissue penetration and distribution of the fusion protein within the tumor, reduce its immunogenicity, and avoid the toxicity related to the systemic administration of IL-2. The IL-2/MOV19 fusion protein was stable on purification from the cell supernatant and was biologically active. Importantly, this construct was able to target IL-2 onto the surface of alpha-FR-overexpressing tumor cells and stimulated the proliferation of the IL-2-dependent CTLL-2 cell line as well as that of human resting peripheral blood lymphocytes. In a syngeneic mouse model, IL-2/MOV19 scFv specifically targeted a-FR gene-transduced metastatic tumor cells without accumulating in normal tissues, due to its fast clearance from the body. Prolonged release of IL-2/MOV19 scFv by in vivo transplanted J558-EF6.1 producer cells protected 60% of mice from the development of lung metastases caused by an i.v. injection of a-FR gene-transduced tumor cells. Moreover, treatment with IL-2/MOV19 scFv, but not with recombinant IL-2, significantly reduced the volume of s.c. tumors. The pharmacokinetics and biological characteristics of IL-2/NMOV19 scFv might allow us to combine the systemic administration of this molecule with the adoptive transfer of in vitro retargeted T lymphocytes for the treatment of ovarian cancer, thereby providing local delivery of IL-2 without toxicity.

Animals↗
PubMed1998

A fetal sheep liver extract reverses age-related increments in spontaneous and induced cytokine production by indirect environmental effects.

BALB/c, DBA/2 and C57BL/6 mice of different ages (ranging from 8 to 110 weeks of age) were used as spleen cell donors to assay cytokine production from ConA activated spleen and Peyer's Patch (PP) lymphocytes. As reported in an earlier publication, there was an age-related decline in IL-2 production in all strains, with a general increase in IL-4 and IL-10 production with age, this being particularly marked for PP cell preparations. Similar conclusions were reached from independent analysis of CD44hi and CD44lo cell populations in these groups (memory vs. naive cells, respectively). Interestingly, IL-6 production was dramatically increased (some 4-5-fold in the different strains) and significantly increased levels of IL-6 were detected in the serum of aged mice. A previously described sheep fetal liver extract was able to reverse, to varying degrees, these cytokine changes associated with aging. Interestingly, when cells from aged mice were adoptively transferred to lethally irradiated young (8 week) recipients, the cytokine production phenotype of cells harvested from recipient mice 3 weeks later was that of the aged donor, unless recipients were treated continually with extract. Treatment of the donor alone produced minimal changes in cytokine production 3 weeks following adoptive transfer. The effect of extract was reversed in treated aged mice by concomitant daily intravenous infusion of the competitive inhibitor of nitric oxide synthesis (NG-monomethyl-L-arginine (NMMA)), which also decreased the increased serum nitrate levels in mice treated with extract. Our data suggest an important role for reactive nitrogen products, themselves induced by fetal liver extract, in age-associated changes in cytokine production.

Adoptive Transfer↗
PubMed1998

Conformational features of a peptide model Ac-DTVKLMYKGQPMTFR-NH2, corresponding to an early folding beta hairpin region of staphylococcal nuclease.

Recent H-D exchange 1H NMR studies of the refolding of Staphylococcal nuclease (P117G) variant suggest that, a region of the protein corresponding to a beta hairpin in the native structure folded early in the refolding process. In order to investigate whether the formation of beta hairpin is an early folding event, we investigated the conformational features of the beta hairpin peptide model Ac-DTVKLMYKGQPMTFR-NH2 from Staphylococcal nuclease with 1H NMR techniques. It appears that the peptide aggregates even at a low concentration. However, based on the observation of weak dnn(i, i + 1) NOEs between K8-G9, G9-Q10, an upfield shift of Gly9 NH and a low temperature coefficient (-d delta/dT) for Gly9 NH, we suggest that the sequence YKGQP as part of the beta hairpin peptide model samples conformational forms with reduced conformational entropy and turn potential. The presence of aggregation could be restricting the population of folded conformational forms and formation of beta hairpin at detectable concentrations. We suggest that, formation of beta hairpin could be an early event in the folding of Staphylococcal nuclease and this observation correlates with H-D exchange 1H NMR results and also with the prediction of a protein folding model proposed in literature.

Amino Acid Sequence↗
PubMed1997

Generation and phenotypic characterization of new human ovarian cancer cell lines with the identification of antigens potentially recognizable by HLA-restricted cytotoxic T cells.

This study describes a simple method for long-term establishment of human ovarian tumor lines and prediction of T-cell epitopes that could be potentially useful in the generation of tumor-specific cytotoxic T lymphocytes (CTLs). Nine ovarian tumor lines (INT.Ov) were generated from solid primary or metastatic tumors as well as from ascitic fluid. Notably all lines expressed HLA class I, intercellular adhesion molecule-1 (ICAM-1), polymorphic epithelial mucin (PEM) and cytokeratin (CK), but not HLA class II, B7.1 (CD80) or BAGE. While of the 9 lines tested 4 (INT.Ov1, 2, 5 and 6) expressed the folate receptor (FR-alpha) and 6 (INT.Ov1, 2, 5, 6, 7 and 9) expressed the epidermal growth factor receptor (EGFR); MAGE-1 and p185HER-2/neu were only found in 2 lines (INT.Ov1 and 2) and GAGE-1 expression in 1 line (INT.Ov2). The identification of class I MHC ligands and T-cell epitopes within protein antigens was achieved by applying several theoretical methods including: 1) similarity or homology searches to MHCPEP; 2) BIMAS and 3) artificial neural network-based predictions of proteins MAGE, GAGE, EGFR, p185HER-2/neu and FR-alpha expressed in INT.Ov lines. Because of the high frequency of expression of some of these proteins in ovarian cancer and the ability to determine HLA binding peptides efficiently, it is expected that after appropriate screening, a large cohort of ovarian cancer patients may become candidates to receive peptide-based vaccines.

Antigens, Neoplasm↗
PubMed1997

Upregulation of actin gene expression in cells expressing exogenous beta-amyloid precursor protein.

We investigated the effects of beta-amyloid peptide precursor (APP) overexpression upon the levels of other mRNAs. Using quantitative slot-blot hybridization and immunoblot analysis we observed that enhanced levels of APP elevated the levels of beta-actin and beta-actin mRNA. Our results also suggest that the cytoplasmic domain of APP is crucial for the elevation in beta-actin gene expression.

Actins↗
PubMed1997

A pentapeptide model for an early folding step in the refolding of staphylococcal nuclease: the role of its turn propensity.

Recently the folding of a staphylococcal nuclease (P117G) variant was examined with the hydrogen-deuterium (H-D) exchange technique. Many of the residues that showed significant protection are located in beta-sheet regions. About half the residues protected belong to an antiparallel beta-hairpin structure (residues 21-35) in the native structure. The beta-hairpin structure is formed by strands 2 and 3 of sheet 2 connected by the sequence 27Y KGQP31 in a type 1' reverse turn conformation with a 4-->1 hydrogen bonding between Q30 NH and Y27 C = O. We have targeted the conformational characterization of the peptide model Ac-YKGQP-NH2 with 1H two-dimensional nmr techniques in aqueous solution with a view to assessing its propensity to sample turn conformational forms and thus initiate the formation of beta-hairpin structure. Based upon the observed d alpha n (i, i + 1), d alpha n (i, i + 3), and dnn (i, i + 1) nuclear Overhauser effect connectivities, temperature coefficients for amide protons and conformational analysis with quantum mechanical perturbative configuration interaction over localized orbitals method, we conclude that the model peptide samples turn conformational forms with reduced conformational entropy. We suggest that the turn can nucleate the formation of the beta-hairpin structure in the refolding of nuclease. Observation of turn propensity for this sequence is consistent with the folding mechanism of the Greek key motif (present in Staphylococcal nuclease) proposed in the literature.

Micrococcal Nuclease↗
PubMed1997

Comparison of three different methods for radiolabelling human activated T lymphocytes.

One approach in the treatment of ovarian cancer patients involves the infusion of autologous T lymphocytes coupled with a bispecific monoclonal antibody MOv18/anti-CD3 (biMAb OC/TR), which recognizes a 38-kDa glycoprotein expressed on ovarian carcinomas and the CD3 T cell receptor. However, little is known about the in vivo biodistribution of injected activated lymphocytes, information that could be obtained by scintigraphic imaging of radiolabelled T cells in order to visualize the migratory pattern. We compared the efficiency, stability and toxicity of technetium-99m hexamethylpropylene amine oxime (HMPAO), indium-111 oxine and fluorine-18 2-fluoro-2-deoxy-d-glucose (FDG) in radiolabelling activated lymphocytes targeted with biMAb OC/TR. The mean labelling efficiencies of 111In-oxine and 18F-FDG using 2.5x10(8) lymphocytes (68% and 64%, respectively) were more than twice that of 99mTc-HMPAO (31%). Retention of the radionuclide in the cell was highest in the case of 111In-oxine labelling (less than 25% of the initial cell-bound activity released after 240 min, as compared with 44% of the 99mTc label in the same period and 45% of 18F radionuclide released after 150 min). None of the three radiolabelling reagents induced any significant alteration in cell viability or immunophenotype. However, both 111In-oxine and 18F-FDG induced a loss of cytotoxic activity of lymphocytes against the ovarian carcinoma cell line IGROV1, and all three radiolabelling reagents caused a significant reduction in the proliferative ability of labelled lymphocytes compared to controls, with cell death occurring after 8-9 days. Radiolabelling with the more stable 111In-oxine reagent using a higher number of lymphocytes (1.4x10(9)) but the same total activity (around 55.5 MBq) resulted in improved labelled T cell viability and proliferative ability, although the mean labelling efficiency decreased (35.8%). Together the data suggest that 111In-oxine at low activity per cell is the most appropriate reagent for radiolabelling activated retargeted T lymphocytes useful for in vivo biodistribution studies.

Cell Death↗
PubMed1997

An antibody specific for interleukin-6 reverses age-associated changes in spontaneous and induced cytokine production in mice.

A number of quantitative and qualitative changes in the pattern of cytokine production have been reported to accompany the process of ageing in laboratory animals and in human populations, including an increase in serum levels of interleukin-1 (IL-1) and IL-6, as well as increased concanavalin A (ConA)-stimulated production of IL-4, IL-10 and transforming growth factor-beta (TGF-beta), and decreased production of IL-2 from cultured spleen cells. Increased IL-1 and IL-6 production is a feature of splenic adherent cells and peritoneal exudate cells taken from aged mice and stimulated with lipopolysaccharide in vitro. We have asked whether the altered production of lymphocyte-derived cytokines (IL-4, IL-2, TGF-beta) is itself a function of a primary alteration in IL-1/IL-6 production (from macrophage/monocytes) by infusing monoclonal antibodies to these cytokines prior to harvesting cells from aged mice and stimulating the cells in vitro. Anti-IL-6, but not anti-IL-1, reversed the age-associated alteration in lymphocyte cytokine production. The general pattern of cytokine production in aged mice is of a type-2 cytokine type, and thus these data are consistent with the idea that increased production of IL-6 in aged animals is causally implicated in this age-associated polarization to type-2 cytokine production.

Aging↗
PubMed1996

Cytokine-based gene therapy of human tumors. An overview.

This review first summarizes the different strategies of gene therapy of cancer and then focuses on the immunological approach. Several studies in animal models with cytokine gene-transduced tumor cells indicate that local cytokine release usually results in tumor growth inhibition. Moreover, in a number of cases vaccination with such cells can reduce growth of established tumors or even cure the tumor-bearing animals. Translation of such a principle in human clinical setting is reported. We have transduced human melanoma cells with genes coding for interleukin (IL)-2, IL-4 or B7-1 and characterized such lines. The phenotype did not change after gene insertion but the functional, immunostimulatory activity of IL-2 or B7-1 gene-transduced melanoma cells was significantly increased compared to that of parental lines. These-lines were then used to vaccinate melanoma patients. Preliminary results of trials with IL-2 gene-transduced cells are presented which indicate a weak clinical response and the activation of a melanoma-specific cytotoxic T lymphocyte response in a low percentage of patients.

Animals↗
PubMed1995

Signalling pathways involved in the mitogenic action of lysophosphatidylinositol.

Lysophosphatidylinositol has been previously shown to stimulate cell proliferation in differentiated and in K-ras transformed thyroid cells. Increased levels of lysophosphatidylinositol, but not lysophosphatidylcholine or lysophosphatidylethanolamine, are present in thyroid as well as in other ras-transformed cell lines. We have now investigated the mechanism of action of this lysolipid by analysing its effects in a differentiated thyroid cell line. Lysophosphatidylinositol did not increase the levels of cAMP, the main stimulator of cell proliferation in the thyroid, whereas it stimulated phosphoinositide breakdown, mobilization of cytosolic Ca2+ and arachidonic acid release, suggesting that it activates both phospholipases C and A2. None of the effects of lysophosphatidylinositol were prevented by pretreatment of cells with pertussis toxin. Instead, the tyrosine kinase inhibitors, tyrphostins AG18 and AG561, completely blocked its mitogenic action. The effects of lysophosphatidylinositol were distinguishable from those of the well known mitogen lysophosphatidic acid, which affected differently the signalling pathways analysed and was not mitogenic in ras-transformed cells. These results suggest that the mitogenic activity of lysophosphatidylinositol is associated with the activation of phospholipase C and phospholipase A2 and is relatively specific for ras-transformed cells.

Animals↗
PubMed1995

Bispecific antibody targeted T cell therapy of ovarian cancer: clinical results and future directions.

The high frequency of relapse after induction chemotherapy in advanced ovarian carcinoma patients calls for new therapeutic modalities. Retargeted T cell-mediated lysis can be achieved using the bispecific antibody (BsmAb) OCTR, directed to CD3 on T cells and to the folate receptor on ovarian carcinoma cells. Twenty-eight patients with limited intraperitoneal disease after first-line therapy entered a phase II study. They received two i.p. 5 day cycles of activated PBMC retargeted with OCTR. Despite unfavorable tumor characteristics, 7 of 26 patients (27%) showed complete or partial intraperitoneal responses with strict surgicopathologic evaluation. In most cases, the disease relapsed outside the peritoneal cavity, and in 1 case complete intraperitoneal response was accompanied by progression in retroperitoneal lymph nodes. The morbidity was mild to moderate and transient. Combination of i.v. and i.p. administration of OCTR-retargeted lymphocytes will possibly lead to extraperitoneal cure. Ongoing clinical studies indicate that the i.v. infusion of up to 8 x 10(8) OCTR-retargeted T lymphocytes does not induce a higher toxicity than the i.p. treatment. To avoid PBMC preactivation, new approaches for delivering accessory signals are under investigation. Preliminary results indicate that nonactivated PBMC retargeted by OCTR in the presence of an anti-CD28 monoclonal antibody (mAb) are able to significantly inhibit tumor growth.

Antibodies, Bispecific↗
PubMed1993

Increased projection of MHC and tumor antigens in murine B16-BL6 melanoma induced by hydrostatic pressure and chemical crosslinking.

The B16-BL6 melanoma, like most spontaneously arising tumors, is poorly immunogenic and expresses low levels of major histocompatibility complex (MHC) antigens. Treatment of cells of this tumor in vitro by hydrostatic pressure in the presence of adenosine 2',3'-dialdehyde (oxAdo), a membrane-impermeant crosslinker, caused elevated projection of MHC and a specific tumor antigen as demonstrated by flow-cytometric analysis. Maximum projection of both the MHC and the tumor antigens could be reached by application of 1200 atm for 15 min in the presence of 20 mM oxAdo. It is not yet clear whether this passive increase in availability of antigens on the cell surface originated from a dormant pool of antigens in the plasma membrane or from pressure-induced fusion of antigen-rich intracellular organelles (e.g. the endoplasmic reticulum). The immunogenic properties of the antigen-enriched B16-BL6 cells are described in the following paper.

Animals↗