Biomedical subjects

V Umansky

Publications and source records attributed to V Umansky.

At least 37 records · Page 2Linked to original sources

PubMed2002

Generation of dendritic cells from human bone marrow mononuclear cells: advantages for clinical application in comparison to peripheral blood monocyte derived cells.

Dendritic cells (DCs) currently used for vaccination in clinical studies to induce immunity against malignant cells are normally generated from peripheral blood-derived monocytes. Here we studied conditions for the generation of DCs from unseparated human bone marrow (BM) mononuclear cells and compared them functionally with DCs from blood. The two types of DCs, from bone marrow (BM-DC) and peripheral blood (BL-DC), were generated in parallel from the same normal healthy donors by culturing in serum-free X-VIVO 20 medium containing GM-CSF and IL-4, and then the phenotypes and functions were compared. BM-DC generation occurred in 14 days and involved proliferative expansion from CD34 stem cells and differentiation while BL-DC generation occurred in 7 days from CD14 monocytes and involved only differentiation. A 7- to 25-fold higher number of DCs could be obtained from BM than from blood. BM-DC had similar phenotypes as BL-DC. The capacity to stimulate MLR reactivity in allogeneic T lymphocytes was higher with BM-DC than that with BL-DC. Also, the capacity to stimulate autologous memory T cell responses to tetanus toxoid (TT) or tuberculin (PPD) was higher with BM-DC than with BL-DC. These results suggest that BM-DC as produced here may be a very economic and useful source of professional antigen-presenting cells for anti-tumor immunotherapeutic protocols.

Antigen Presentation↗

PubMed2001

Inhibition of nitric-oxide-mediated apoptosis in Jurkat leukemia cells despite cytochrome c release.

We have recently shown that nitric-oxide (NO)-induced apoptosis in Jurkat human leukemia cells requires degradation of mitochondria phospholipid cardiolipin, cytochrome c release, and activation of caspase-9 and caspase-3. Moreover, an inhibitor of lipid peroxidation, Trolox, suppressed apoptosis in Jurkat cells induced by NO donor glycerol trinitrate. Here we demonstrate that this antiapoptotic effect of Trolox occurred despite massive release of the mitochondrial protein cytochrome c into the cytosol and mitochondrial damage. Incubation with Trolox caused a profound reduction of intracellular ATP concentration in Jurkat cells treated by NO. Trolox prevented cardiolipin degradation and caused its accumulation in Jurkat cells. Furthermore, Trolox markedly downregulated the NO-mediated activation of caspase-9 and caspase-3. Caspase-9 is known to be activated by released cytochrome c and together with caspase-3 is considered the most proximal to mitochondria. Our results suggest that the targets of the antiapoptotic effect of Trolox are located downstream of the mitochondria and that caspase activation and subsequent apoptosis could be blocked even in the presence of cytochrome c released from the mitochondria.

Adenosine Triphosphate↗

PubMed2001

Enrichment of memory T cells and other profound immunological changes in the bone marrow from untreated breast cancer patients.

Previous studies with animal tumors showed that bone marrow (BM) is a privileged site where potentially lethal tumor cells are controlled in a dormant state by the immune system. Here, we investigated BM of breast cancer patients with respect to tumor cell content, immune activation status and memory T-cell content. BM-derived cells from primary operated breast cancer patients (n = 90) were compared with those from healthy donors (n = 10) and also with cells from respective blood samples. Cytokeratin 19-positive tumor cells were detected by nested polymerase chain reaction. Three-color flow cytometry was used to identify numbers and activation state of T cells, natural killer (NK) cells, monocytes/macrophages and subsets by a panel of monoclonal antibodies (mAbs). The proportion of memory T cells among the CD4 and CD8 T cells was much higher in BM of cancer patients than in healthy donors (p < 0.001). The extent of memory T-cell increase was related to the size of the primary tumor. Patient-derived BM memory CD8 T cells could be shown to contain specific HLA-A2/Her-2/neu(369-377) tetramer binding cells. Patients with disseminated tumor cells in their BM had more memory CD4 T cells and more CD56(+) CD8(+) cells than patients with tumor cell-negative BM. Only some of the immunological changes seen in BM samples of cancer patients were also detectable in peripheral blood samples. Our hypothesis that BM is a special compartment for immunological memory and tumor dormancy is supported by the above findings. The overall results reveal that BM is a valuable additional compartment for immune diagnosis in pathological conditions and possibly for follow-up treatment strategies.

Adult↗

PubMed2001

Evidence of Hofstadter's Fractal Energy Spectrum in the Quantized Hall Conductance.

The energy spectrum of a two-dimensional electron system in a perpendicular homogeneous magnetic field and a weak lateral superlattice potential with square symmetry is composed of Landau bands with recursive internal subband structure. The Hall conductance in the minigaps is anticipated to be quantized in integer multiples of e(2)/h that vary erratically from minigap to minigap in accordance with a Diophantine equation. Hall measurements on samples with the requisite properties uncover this long searched for evidence of Hofstadter's butterflylike energy spectrum.

Journal Article↗

PubMed2001

Nitric oxide-induced apoptosis in tumor cells.

Nitric oxide (NO), an important molecule involved in neurotransmission, vascular homeostasis, immune regulation, and host defense, is generated from a guanido nitrogen of L-arginine by the family of NO synthase enzymes. Large amounts of NO produced for relatively long periods of time (days to weeks) by inducible NO synthase in macrophages and vascular endothelial cells after challenge with lipopolysaccharide or cytokines (such as interferons, tumor necrosis factor-alpha, and interleukin-1), are cytotoxic for various pathogens and tumor cells. This cytotoxic effect against tumor cells was found to be associated with apoptosis (programmed cell death). The mechanism of NO-mediated apoptosis involves accumulation of the tumor suppressor protein p53, damage of different mitochondrial functions, alterations in the expression of members of the Bcl-2 family, activation of the caspase cascade, and DNA fragmentation. Depending on the amount, duration, and the site of NO production, this molecule may not only mediate apoptosis in target cells but also protect cells from apoptosis induced by other apoptotic stimuli. In this review, we will concentrate on the current knowledge about the role of NO as an effector of apoptosis in tumor cells and discuss the mechanisms of NO-mediated apoptosis.

Animals↗

PubMed2001

Therapy of human tumors in NOD/SCID mice with patient-derived reactivated memory T cells from bone marrow.

In an analysis of 84 primary-operated breast cancer patients and 11 healthy donors, we found that the bone marrow of most patients contained memory T cells with specificity for tumor-associated antigens. Patients' bone marrow and peripheral blood contained CD8+ T cells that specifically bound HLA/peptide tetramers. In short-term culture with autologous dendritic cells pre-pulsed with tumor lysates, patients' memory T cells from bone marrow (but not peripheral blood) could be specifically reactivated to interferon-gamma-producing and cytotoxic effector cells. A single transfer of restimulated bone-marrow T cells into NOD/SCID mice caused regression of autologous tumor xenotransplants associated with infiltration by human T cells and tumor-cell apoptosis and necrosis. T cells from peripheral blood showed much lower anti-tumor reactivity. Our findings reveal an innate, specific recognition of breast cancer antigens and point to a possible novel cancer therapy using patients' bone-marrow-derived memory T cells.

Amino Acid Sequence↗

PubMed2000

Evolution of quasiparticle charge in the fractional quantum hall regime

The charge of quasiparticles in a fractional quantum Hall (FQH) liquid, tunneling through a partly reflecting constriction with transmission t, was determined via shot noise measurements. In the nu = 1/3 FQH state, a charge smoothly evolving from e(*) = e/3 for t(1/3) congruent with 1 to e(*) = e for t(1/3)<<1 was determined, agreeing with chiral Luttinger liquid theory. In the nu = 2/5 FQH state the quasiparticle charge evolves smoothly from e(*) = e/5 at t(2/5) congruent with 1 to a maximum charge less than e(*) = e/3 at t(2/5)<<1. Thus it appears that quasiparticles with an approximate charge e/5 pass a barrier they see as almost opaque.

Journal Article↗

PubMed2000

Breaking tolerance to a tumor-associated viral superantigen as a basis for graft-versus-leukemia reactivity.

A major goal in tumor immunotherapy consists of breaking potential tumor-specific T-cell unresponsiveness (tolerance), which may explain tumor growth in cancer patients. We report that immunological tolerance to a tumor-associated viral superantigen (SAg) is overcome in a mouse lymphoma model by transfer of allogeneic T cells expressing SAg-reactive Vbeta6 T-cell receptor chains. Surprisingly, upon contact with SAg-expressing lymphoma cells, Vbeta6 T cells became activated rather than tolerized (as reported previously). They also developed SAg-specific cytotoxic T-lymphocyte activity and secreted IL-2 and IFN-gamma. The grafted T cells infiltrated liver metastases, formed close contact with SAg-expressing tumor cells, and caused significant graft-vs. -leukemia (GvL) effects. Selection for tumor resistance among the progeny from a cross between SAg-negative donor and SAg- positive recipient strains revealed a strict correlation between loss of the endogenous SAg tolerogen, rescue of Vbeta6 T cells from SAg-mediated deletion, and leukemia resistance. These findings suggest that immune responses to SAg can be exploited to break tolerance and augment immune responses to tumors.

Adoptive Transfer↗

PubMed2000

Glutathione is a factor of resistance of Jurkat leukemia cells to nitric oxide-mediated apoptosis.

We have previously reported that nitric oxide (NO) stimulates apoptosis in different human neoplastic lymphoid cell lines through mitochondrial damage (including degradation of cardiolipin, a major mitochondrial lipid) followed by activation of caspases. Here we demonstrate that Jurkat human leukemia cells which survive after 24 h treatment with NO form subpopulations with higher and lower cardiolipin content (designated as NAO(high) and NAO(low), respectively). Sorted NAO(high) cells were found to survive in culture whereas sorted NAO(low) cells died. Moreover, NAO(high) cells acquired an increased resistance to the exposure to NO donors which remained unchanged during long-term culture. These cells showed a similar cardiolipin content and expressed the same level of anti-apoptotic proteins Bcl-2 and Bcl-x(L) as APO-S unsorted cells but contained significantly higher concentration of the antioxidant glutathione. Depletion of glutathione in these cells with buthionine-sulfoximine (BSO) correlated with a significant stimulation of NO-mediated apoptosis whereas the exposure of NO-sensitive APO-S cells to the glutathione precursor N-acetylcysteine (NAC) resulted in a substantial suppression of this effect. Our data suggest a complex mechanism of the resistence to NO-induced apoptosis in Jurkat human leukemia cells in which glutathione plays an important role.

Acridine Orange↗

PubMed2000

Nitric oxide-mediated apoptosis in human breast cancer cells requires changes in mitochondrial functions and is independent of CD95 (APO-1/Fas).

We have previously shown that nitric oxide (NO) induces apoptosis in different human neoplastic lymphoid cells through caspase activation. Here we studied the NO-mediated apoptosis in human breast cancer cell lines derived from primary tumor (BT-20) or from metastasis (MCF-7). NO donor glycerol trinitrate (GTN) induced apoptosis in both cell lines which was completely abrogated after pretreatment with the broad spectrum caspase inhibitor zVAD-fmk. NO triggered also a time-dependent activation of caspase-1, caspase-3, and caspase-6 in these cells. Moreover, NO caused a release of mitochondrial protein cytochrome c into the cytosol, an increase in the number of cells with low mitochondrial transmembrane potential and with high level of reactive oxygen species production. However, NO did not induce mRNA expression of CD95 (APO-1/Fas) ligand. FAS-associated phosphatase-1 (FAP-1) molecule was constitutively expressed at the mRNA level and did not show any changes upon NO treatment in both breast cancer cell lines. The expression of the pro-apoptotic protein Bax and of the anti-apoptotic protein Bcl-2 remained unchanged in MCF-7 and BT-20 cells upon GTN treatment. We suggest that the mechanism of NO-mediated activation of the caspase cascade and subsequent apoptosis in human breast cancer cells required mitochondrial damage (in particular, cytochrome c release, disruption of mitochondrial transmembrane potential and generation of reactive oxygen species) but not the activation of the CD95/CD95L pathway.

Apoptosis↗

PubMed2000

Newcastle disease virus activates macrophages for anti-tumor activity.

Newcastle Disease Virus (NDV), an agent with interesting immune stimulatory and anti-tumor activity, was investigated for its capacity to activate anti-tumor activity in murine macrophages in vitro and in vivo. Direct macrophage activation was seen under a variety of experimental conditions using two different strains of NDV, different sources of macrophages (spleen and peritoneum) and different strains of mice (DBA/2, C57BL/6, 615). Various macrophage enzymes (ADA, iNOS, lysozyme, acid phosphatase) became upregulated and anti-tumor effector molecules such as nitric oxide (NO) and TNF-alpha were found in the supernatant. NDV activated macrophages performed anti-tumor activity in vitro such as anti-tumor cytostasis and anti-tumor cytotoxicity. The cytotoxic anti-tumor activity was broad and active against all tumor lines tested including mammary carcinoma, lung carcinoma, mastocytoma and immune escape variants (lymphoma). Macrophage activation via BCG/LPS also caused a broad range anti-tumor cytotoxic activity while activation via mixed lymphocyte culture conditioned medium had restricted anti-tumor activity. Anti-tumor activity of NDV activated macrophages could be transfered in vivo. Transfer of macrophages which had not been appropriately activated exerted either no effect or a tumor growth augmenting effect. Repeated intravenous transfer of NDV activated macrophages exerted a significant suppressive effect on pulmonary metastases in a mammary carcinoma tumor model as well as in a lung carcinoma model. Taken together these results demonstrate that NDV can strongly activate macrophages to perform anti-tumor activities in vitro and in vivo.

Animals↗

PubMed2000

Functional and in situ evidence for nitric oxide production driven by CD40-CD40L interactions in graft-versus-leukemia reactivity.

In a murine tumor model, complete tumor remission is achievable at even advanced metastasized stages by transfer of immune T cells from donor B10.D2 (H-2d, Mls(b)) into tumor-bearing DBA/2 (H-2d, Mls(a)) mice. We showed previously that this graft-versus-leukemia (GvL) effect is dependent on synergistic interactions of transferred CD4+ and CD8+ T cells with host sialoadhesin (SER)-positive macrophages. We now show that the CD40-CD40L (CD154) interaction is involved in the induction of inducible nitric oxide synthase (iNOS) expression during adoptive immunotherapy (ADI). We demonstrate that during ADI, the level of CD40 expression in the liver becomes significantly augmented in comparison to livers of tumor-bearing, untreated animals. CD40 expression is found mostly on SER+ macrophages and to a lesser extent on dendritic cells (DCs). In GvL animals, more SER+ macrophages express iNOS than untreated animals. iNOS expressing cells are found in close proximity to apoptotic cells, at early time points of the therapy in areas of metastasis, and at late stages around portal veins, where CD4+ and CD8+ T lymphocytes form clusters with SER+ macrophages. Blocking of CD40L in vivo at days 5 and 20, when all iNOS+ cells express CD40, leads to significantly reduced CD40 and iNOS expression as well as to a marked inhibition of the therapeutic effect. These data provide functional and in situ evidence that the increased CD40 and iNOS expression observed during ADI contribute to the eradication of liver metastases and to the clearance of donor lymphocytes from the liver.

Animals↗

PubMed1999

Sialoadhesin-positive host macrophages play an essential role in graft-versus-leukemia reactivity in mice.

We recently established an effective immune T-cell-mediated graft-versus-leukemia (GVL) murine model system in which complete tumor remissions were achievable even in advanced metastasized cancer. We now describe that this T-cell-mediated therapy is dependent on host macrophages expressing the lymphocyte adhesion molecule sialoadhesin (Sn). Depletion of Kupffer cells in tumor-bearing mice during adoptive immunotherapy (ADI) or the treatment of these animals with anti-Sn monoclonal antibodies led to complete or partial inhibition of the immune T-cell-mediated therapeutic effect. Furthermore, Sn+ host macrophages in livers formed clusters during ADI with donor CD8 T cells. To test for a possible antigen presentation function of these macrophages, we used as an in vitro model the antigen beta-galactosidase for which a dominant major histocompatibility complex (MHC) class I Ld-restricted peptide epitope is known to be recognized by specific CD8 cytotoxic T lymphocytes (CTL). We demonstrate that purified Sn+ macrophages can process exogenous beta-galactosidase and stimulate MHC class I peptide-restricted CTL responses. Thus, Sn+ macrophages, which are significantly increased in the liver after ADI, may process tumor-derived proteins via the MHC class I pathway as well as via the MHC class II pathway, as shown previously, and present respective peptide epitopes to CD8 as well as to CD4 immune T cells, respectively. The synergistic interactions observed before between immune CD4 and CD8 T cells during ADI could thus occur in the observed clusters with Sn+ host macrophages.

Animals↗

PubMed1999

Nitric-oxide-induced apoptosis in human leukemic lines requires mitochondrial lipid degradation and cytochrome C release.

We have previously shown that nitric oxide (NO) stimulates apoptosis in different human neoplastic lymphoid cell lines through activation of caspases not only via CD95/CD95L interaction, but also independently of such death receptors. Here we investigated mitochondria-dependent mechanisms of NO-induced apoptosis in Jurkat leukemic cells. NO donor glycerol trinitrate (at the concentration, which induces apoptotic cell death) caused (1) a significant decrease in the concentration of cardiolipin, a major mitochondrial lipid; (2) a downregulation in respiratory chain complex activities; (3) a release of the mitochondrial protein cytochrome c into the cytosol; and (4) an activation of caspase-9 and caspase-3. These changes were accompanied by an increase in the number of cells with low mitochondrial transmembrane potential and with a high level of reactive oxygen species production. Higher resistance of the CD95-resistant Jurkat subclone (APO-R) cells to NO-mediated apoptosis correlated with the absence of cytochrome c release and with less alterations in other mitochondrial parameters. An inhibitor of lipid peroxidation, trolox, significantly suppressed NO-mediated apoptosis in APO-S Jurkat cells, whereas bongkrekic acid (BA), which blocks mitochondrial permeability transition, provided only a moderate antiapoptotic effect. Transfection of Jurkat cells with bcl-2 led to a complete block of apoptosis due to the prevention of changes in mitochondrial functions. We suggest that the mitochondrial damage (in particular, cardiolipin degradation and cytochrome c release) induced by NO in human leukemia cells plays a crucial role in the subsequent activation of caspase and apoptosis.

Apoptosis↗

PubMed1998

Identification of four genes in endothelial cells whose expression is affected by tumor cells and host immune status--a study in ex vivo-isolated endothelial cells.

A spontaneously metastasizing, well-defined mouse lymphoma was chosen as an in vivo model to study the effect of tumor-host interaction on gene expression in liver sinusoidal endothelial cells. Forty-nine bovine aortic endothelial cell (BAEC) genes, recently isolated by a differential screening approach of a cDNA library enriched for tumor necrosis factor-alpha (TNF-alpha) suppressed genes, were investigated. Four of these genes were finally selected because they were affected differentially by host immuno-competence, TNF-alpha, and tumor cells. Sequence analysis showed them to encode the bovine polyubiquitin (A4), elongation factor 1alpha (B2), the acidic ribosomal phosphoprotein PO (C3), and the ribosomal protein S2 (E10). Gene expression was analyzed by dot-blot or Northern blot analysis. TNF-alpha and tumor cell conditioned supernatant suppressed the genes additive in BAEC but not in other endothelial cells except for bovine capillary endothelial cells. Ex vivo-isolated liver endothelial cells of tumor-bearing syngeneic DBA/2 mice showed strong downregulation of these four genes in comparison to normal control values. In contrast, endothelial cells of tumor-bearing immuno-incompetent Balb/c (nu/nu) mice showed no downregulation but upregulation of these genes. Consistently, all four genes were also downregulated when BAEC were incubated with supernatants derived from ex vivo-isolated liver metastases from immuno-competent but not from -incompetent mice. Thus, the expression of a group of genes involved in protein translation and processing was more profoundly altered in endothelial cells in vivo than in vitro, suggesting that microenviromental factors and cell-cell and cell-matrix interactions play an important role.

Amino Acid Sequence↗

PubMed1998

Loss of endogenous mouse mammary tumor virus superantigen increases tumor resistance.

From a cross between a tumor-susceptible mouse strain (DBA/2; D) and a tumor-resistant MHC-identical strain (B10.D2; D2) new recombinant inbred mouse strains were established over many generations of inbreeding and tumor resistance selection. Since resistance to the highly metastatic DBA/2 lymphoma variant ESb had an immunologic basis, and the two parental strains differed in endogenous viral superantigens (vSAGs), DNA of three D2 x D recombinant inbred mouse lines was typed for endogenous mouse mammary tumor viruses using mouse mammary tumor virus long terminal repeat- and env gene-specific probes. The resistant D2 x D mice were very similar to the susceptible parental strain D in their Mtv Southern blots, except for the lack of a single band corresponding to Mtv-7, the provirus coding for the strong DBA/2 superantigen Mls-1a. A backcross analysis revealed that Mtv-7-negative F2 mice were significantly more resistant than Mtv-7-positive F2 mice. When Mtv-7 was reintroduced into the resistant lines by crossing them with either CBA/J or BALB/D2.Mls-1a, the mice became again more tumor susceptible. Finally, we demonstrate the ability to transfer immunoresistance and graft-vs-leukemia reactivity from tumor-resistant to tumor-susceptible mice.

Animals↗

PubMed1998

Caspase activation is required for nitric oxide-mediated, CD95(APO-1/Fas)-dependent and independent apoptosis in human neoplastic lymphoid cells.

Nitric oxide (NO), an important effector molecule involved in immune regulation and host defense, was shown to induce apoptosis in lymphoma cells. In the present report the NO donor glycerol trinitrate was found to induce apoptosis in Jurkat cells that are sensitive to CD95-mediated kill. In contrast, a CD95-resistant Jurkat subclone showed substantial protection from apoptosis after exposure to NO. NO induced mRNA expression of CD95 (APO-1/Fas) and TRAIL/APO-2 ligands. Moreover, NO triggered apoptosis in freshly isolated human leukemic lymphocytes which were also sensitive to anti-CD95 treatment. The ability of NO to induce apoptosis was completely blocked by a broad-spectrum ICE (interleukin-1beta converting enzyme)-protease/caspase inhibitor and correlated with FLICE/caspase-8 activation. This activation was abrogated in some neoplastic lymphoid cells but not in others by the inhibitor of protein synthesis cycloheximide. Our results were confirmed using an in vitro experimental model of coculture of human lymphoid target cells with activated bovine endothelial cells generating NO as effectors. Furthermore, the inhibition of endogenous NO production with the inducible NO synthase inhibitor NG-monomethyl-L-arginine caused a complete abrogation of the apoptotic effect. Our data provide evidence that NO-induced apoptosis in human neoplastic lymphoid cells strictly requires activation of caspases, in particular FLICE, the most CD95 receptor-proximal caspase. Depending on the cell line tested this activation required or was independent of the CD95 receptor/ligand system.

Animals↗

PubMed1998

Co-stimulatory effect of nitric oxide on endothelial NF-kappaB implies a physiological self-amplifying mechanism.

Here we investigated the effects of the second messenger molecule NO at various concentrations on the activation of transcription factor NF-kappaB, IkappaB-alpha kinase (IKK-alpha), Jun N-terminal kinase (JNK) and apoptosis in murine endothelial cells. Low concentrations of NO alone failed to activate NF-kappaB, IKK-alpha and JNK. When NF-kappaB was prestimulated by TNF-alpha or phorbol 12-myristate 13-acetate, the addition of NO at low concentrations enhanced the activation of NF-kappaB. This provides a mechanism for a self-amplifying signal in the inflammatory response, since the inducible NO synthase in endothelial cells is regulated by NF-kappaB. The co-stimulatory effect of NO on NF-kappaB activation was also evident from IKK-alpha kinase assays and reporter gene experiments in endothelial cells. High doses of NO impaired the TNF-alpha-induced DNA-binding activity of NF-kappaB. Accordingly, these high amounts of NO also repressed the TNF-alpha-induced transactivation by NF-kappaB as efficient as dexamethasone. The doses of NO required for the inhibition of NF-kappaB are not cytotoxic for the endothelial cells, enabling the establishment of an autoregulatory loop for NF-kappaB signaling.

Animals↗