PubMed Health⌕ Search

Biomedical subjects

R Tirosh

Publications and source records attributed to R Tirosh.

At least 19 recordsLinked to original sources
PubMed1999

Preparation of a diagnostic antigen of human melanoma based on lymphocyte activation as measured by intracellular fluorescein fluorescence polarization.

The intracellular fluorescein fluorescence polarization (IFFP) test indicates that peripheral blood lymphocytes (PBL) of cancer patients display stimulatory sensitivity to a short incubation with specific tumor protein extracts. In this work, a human lymphocyte activation melanoma antigen (LAMA) was purified from supernatant of a human melanoma cell line (L1M1), which could specifically stimulate lymphocytes of melanoma patients. The results showed a significant stimulation of lymphocytes from healthy donors after incubation with phytohaemagglutinin (PHA), while no stimulation was observed after incubation with LAMA. On the other hand, lymphocytes from melanoma patients showed a significant stimulation with LAMA, while generally showing minor or no stimulation with PHA. Melanoma specificity of LAMA was demonstrated by no response in lymphocytes from patients of lung, colon, or breast cancer. The purified fraction is therefore considered to be a shared tissue-specific antigen which may be useful in immunodiagnosis and immunotherapy of melanoma.

Antigens, Neoplasm↗
PubMed1997

Monitoring of effector and target cell stimulation during conjugation by fluorescence polarization.

The aim of the present study was to trace early intracellular changes induced in effector and target cells during their conjugation. This was performed by monitoring the intracellular fluorescein fluorescence polarization (IFFP), using the Cellscan apparatus. This apparatus permits the repetitive spectroscopic measurement of individual selected live cells within a population of many cells, while the location of each cell is known and preserved during the various cell manipulations and/or their suspending medium. Both natural killer (NK) and lymphocyte activated killer (LAK) cells were used as effector cells, while NK-sensitive K562 and NK-resistant Daudi cell lines were used as targets. In this study kinetic IFFP measurements were carried out for a period of approximately 4 h following cell attachment. Within minutes following effector-target conjugation, transient reduction of IFFP was observed consecutively, first in the effector and then in the target cells. A continuous reduction of IFFP occurring only in target cells was also found 50 min following conjugation. No reduction in IFFP was observed using NK- and LAK-resistant target cells. Good correlation was found between early stages of conjugation, as assessed by IFFP, and cytolytic efficiency as assessed by 51chromium release assay. When NK-resistant and LAK-resistant target cells were used, no reduction of IFFP was observed.

Cell Communication↗
PubMed1997

Cell activation influences cell staining kinetics.

Stimulation of cells has so far been observed, among other methods, by the decrease of the intracellular fluorescein fluorescence polarization (IFFP). It is shown that the rate constant of leakage of the fluorescent marker out of the cells increases with stimulation much more significantly than the polarization decreases; thus it might provide a more sensitive method to observe cells stimulation. It is also shown that due to negligible leakage of the marker out of the cells shortly after initiation of the staining of the cell suspension, the fluorescein fluorescence polarization (FFP) of the cell suspension, is very close to IFFP.

Fluoresceins↗
PubMed1997

Carboxyfluorescein as a fluorescent probe for cytoplasmic effects of lymphocyte stimulation.

The application of carboxyfluorescein (CF), as an impermeable fluorescent probe for lymphocyte stimulation with phytohaemagglutinin (PHA), is investigated by following a decrease in the degree of fluorescence polarization. Since CF does not enter the mitochondria, the present results indicate that the measured effect of stimulation occurs in the cytoplasm. The results also reveal that the fluorescence yield of intracellular CF is smaller than that of extracellular CF. Moreover, the degree of fluorescence polarization of intracellular CF is inversely related to its concentration. Following cell disruption, fluorescence intensity increases and polarization decreases. These effects might indicate a weak or reversible association of intracellular CF with cytoplasmic proteins.

Cytoplasm↗
PubMed1997

Influenza A virus affects the response of human peripheral blood mononuclear cells to phytohaemagglutinin A by altering the cytoskeleton.

In the present study, we demonstrate that the infection of human peripheral blood mononuclear cells (PBMC) with influenza A virus caused changes in intracellular fluorescein fluorescence polarization (IFFP) which, as previously described, reflect alterations in the polymerization of the cytoskeleton. Kinetic measurements revealed two cycles of an approximate 10% decrease in IFFP within 3.5 and 5 h after infection. Infection win influenza A virus also altered the response of PBMC to phytohaemagglutinin (PHA), which was manifested as changes of 5.3 and 4% in IFFP at 1 and 2 h after infection, respectively. the changes in IFFP correlated with DNA synthesis measured 72 h after exposure to PHA. These results show the ability of IFFP measurements to identify early intracellular metabolic events induced in virus-infected cells.

Cell Division↗
PubMed1997

Infection of K562 cells with influenza A virus increases their susceptibility to natural killer lysis.

Natural killer (NK) cells play a role in the natural immunity against tumor cells. In the present study, we demonstrate that infection of the NK-sensitive tumor cell line K562 with influenza A virus caused a substantial increase in lysis of up to sevenfold when compared to noninfected cells. Similar to NK cells, IL-2-activated killer cells exhibited higher lytic activity against virus-infected K562 cells. This effect of the virus correlated with the increase in the expression of intracellular adhesion molecule-1 (ICAM-1) on K562 cells. Changes in the susceptibility to NK lysis were accompanied by alterations, within minutes, in the cytoskeleton as detected by intracellular fluorescein fluorescence polarization measured on the Cellscan, a static cytometer. The possible role of iCAM-1 and the cytoskeleton in the cytotoxic response of NK cells is discussed.

Cytotoxicity, Immunologic↗
PubMed1997

Detection of melanoma by monitoring the intracellular fluorescein fluorescence polarization changes in lymphocytes.

The effectiveness of detecting melanoma by measuring the intracellular fluorescein fluorescent polarization (IFFP) of patients' SCM (structuredness of the cytoplasmic matrix)-responding lymphocytes was examined. SCM-responding lymphocytes from 46 melanoma patients and 32 healthy volunteers were labeled with fluorescein diacetate and challenged with different stimuli, and the resulting polarization was determined. The polarizations (P) obtained upon stimulation with nothing (P-0), encephalitogenic factor (P-EF), phytohaemagglutinin (P-PHA), or melanoma antigen (P-MEL), and the ratios RR(ef) (P-EF/P-PHA) and RR(mel) (P-MEL/P-PHA) were lower for SCM-responding lymphocytes from the patients as a group than for those of the controls. The specificity and sensitivity of the IFFP tests (using cutoff values) to detect melanoma were 90.6 and 73.9%, respectively. The IFFP tests may facilitate the discrimination between melanoma patients and healthy subjects, and may be used in follow-up of patients with melanoma.

Algorithms↗
PubMed1996

Glucose is essential for the initiation of fatty acid oxidation in ATP-depleted cultured ventricular myocytes.

Cultured cardiac myocytes were depleted of ATP by incubation with oligomycin (1 mg/ml). Then the ability of the cells to oxidize various substrates and to restore ATP levels was studied. Following ATP depletion, the cells were found to be able to oxidize glucose given alone, but not palmitate. However, with both substrates, palmitate was oxidized in the presence of glucose and ATP recovery was enhanced. Pyruvate had a minor effect on palmitate oxidation, while acetate given alone was oxidized, but did not enhance cellular ATP content. These results show that glucose is essential for restoration of mitochondrial function and the coupling between oxidation and ATP synthesis.

Adenosine Triphosphate↗
PubMed1996

Fourier analysis of differential light scattering for the quantitation of FSH response associated with structural changes in immortalized granulosa cells.

We have established granulosa cell lines which express constitutively the rat FSH receptors by cotransfection of primary granulosa cells obtained from preovulatory follicles with SV40 DNA, Ha-ras oncogene and a plasmid expressing FSH receptors. These cells respond specifically to ovine and human FSH by cell rounding, intracellular cAMP accumulation, and progesterone secretion in a dose-dependent manner. A new method for the demonstration and quantitation of changes in cell shape-Small Angle Laser Light Scattering (SALLS) analysis-has been utilized for measurement of cell rounding in response to FSH stimulation in these cells. When cells were incubated with increasing doses of either ovine or human FSH, partial rounding of cells was observed at FSH concentrations as low as 24 pM, while complete rounding of cells was observed at a range of 0.24-2.4 nM of FSH. Following aldehyde fixation, hormone-treated cells were examined using the method of SALLS analysis. Histograms obtained by applying SALLS analysis on FSH stimulated GFSHR-17 cells were a reflection of the structural changes induced by the hormone. FSH- and forskolin-incubated cells yielded structured distributions with defined mean size and standard deviations. Moreover, the increase in sharpness of dominant peak in the histogram was correlated with elevated concentration of FSH in a dose dependent manner. In conclusion, cellular response to FSH is correlated with a specific pattern of light scattered in immortalized granulosa cells expressing functional FSH receptors. Therefore, SALLS analysis may serve as a useful tool for in vitro bioassay of the gonadotropic hormone. Moreover, this method may lend itself to in vitro bioassay of any hormone that induces specific morphological changes in target cells.

Animals↗
PubMed1996

Lymphocyte fluorescence polarization measurements with the cellscan system: application to the SCM cancer test.

The SCM (Structuredness of Cytoplasmic Matrix) cancer test, a procedure based on detection of differences in lymphocyte activation between individuals with and without cancer, has remained controversial with inconsistent results reported by different authors. As originally described, the test includes two technically demanding steps, the first a lymphocyte separation procedure and the second a series of fluorescence polarization measurements. The Cellscan, a high-precision static cytometer system has been configured to perform the SCM test. The apparatus facilitates the polarization measurements and can analyze cells separated using simpler procedures than were originally described. Using methods and diagnostic criteria adapted for the Cellscan system, the SCM test correctly classified > 90% of patients with cancer and > 90% of individuals without cancer.

Adult↗
PubMed1996

Decrease of intracellular fluorescein fluorescence polarization (IFFP) in human peripheral blood lymphocytes undergoing stimulation with phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM) and anti-CD3 antibody.

In the present study we describe the induction of changes in intracellular fluorescein fluorescence polarization (IFFP) in lymphocytes undergoing activation with a variety of stimulants. These stimulants included the lectins phytohaemagglutinin (PHA), concanavalin (ConA), pokeweed mitogen (PWM) and anti-CD3 antibody. Changes in IFFP were detected in individual cells using the Cellscan apparatus. Our results show that by employing mitogenic concentrations of PHA, as revealed in a [3H]-thymidine incorporation assay, a decrease in the IFFP in human peripheral blood lymphocytes (PBL) occurred within 40 min. ConA and anti-CD3 affected similarly IFFP, whereas PWM, a B lymphocyte lectin, had no effect on IFFP at the concentrations employed. Kinetic analysis revealed that changes in IFFP occurred within 20-40 min after exposure to the stimulants and lasted for 24 h. Our results show that stimulants which activate CD3+ lymphocytes caused immediate changes in IFFP, in an enriched population of human PBL. The possible mechanisms involved in IFFP modulation following exposure to selected stimulants are discussed.

Antibody Specificity↗
PubMed1996

Adaptation of the cellscan technique for the SCM test in breast cancer.

The value of the SCM (Structuredness of Cytoplasmic Matrix) cancer test, a procedure based on the detection of differences in lymphocyte activation in the presence and absence of cancer, has remained controversial, with inconsistent results having been reported among investigators. The Cellscan, a high-precision static cytometer system, has been designed to perform the SCM test; the apparatus facilitates the polarisation measurements and can examine cells which have been separated by simpler procedures than were originally described. In this study, using methods and diagnostic criteria adapted for the Cellscan system in a hospital environment, the SCM test correctly classified over 90% (76/80) of patients with breast cancer and differentiated over 90% (72/73) of individuals without cancer.

Adult↗
PubMed1996

Role of iron in the potentiation of anthracycline cardiotoxicity: identification of heart cell mitochondria as a major site of iron-anthracycline interaction.

The role of iron in anthracycline toxicity was studied in rats in vivo in intact animals and in vitro in heart cell cultures. In animals treated with 8 mg/kg doxorubicin, iron loading resulted in severe weight loss and a twofold increase in rate of mortality. Studies in cultured heart cells aimed at defining the subcellular target of interaction between iron and anthracycline toxicity showed no evidence of anthracycline-induced damage to sarcolemmal thiolic enzymes represented by 5'-nucleotidase and only a limited increase in lysosomal fragility as monitored by an increase in beta-hexosaminidase activity in cell homogenates and its release into the culture medium. By contrast, doxorubicin treatment resulted in a marked inhibition of mitochondrial function as monitored by a decrease in carbon 14-labeled palmitate utilization, to 33% +/- 4% of controls, and prior iron loading resulted in a further decrease in palmitate utilization, to 18% +/- 3% of controls. Conversely, iron-chelation treatment by either deferoxamine or deferiprone (L1) eliminated the harmful effects of iron loading and resulted in a partial inhibition of doxorubicin toxicity in both normal and iron-loaded cells. Our studies represent the first demonstration in intact animals of the potentiation of anthracycline toxicity by iron overload. They also indicate that mitochondria represent an important target of combined iron-anthracycline toxicity. These observations provide new insights into the mechanism of anthracycline cardiotoxicity and may be useful in developing better strategies for tumor therapy.

Animals↗
PubMed1996

Oxygen deprivation and reoxygenation augment prostacyclin synthesis in cultured ventricular myocytes.

Prostacyclin production in cultured cardiomyocytes is not induced by cellular ATP depletion per se, suggesting that the mechanism of ischemic injury is more complex. In the present study we subjected cultured ventricular myocytes to 'simulated ischemia' followed by reoxygenation. A slight increase in 6-keto-PGF(1 alpha) (the stable metabolite of PGI(2)) was found during 'ischemia', which continued to increase markedly during reoxygenation. PGE(2) levels were pronouncedly enhanced during ischemia but decreased during reoxygenation, and TXB(2) levels remained undetectable throughout. These findings reflect a cardiomyocyte response to anoxic injury, suggesting that they act to protect against cardiac injury by producing the potent vasodilators PGI(2) and PGE(2) during ischemia and reoxygenation.

6-Ketoprostaglandin F1 alpha↗
PubMed1996

Intrathecal baclofen for severe torsion dystonia in a child.

The successful use of intrathecal baclofen, a structural analogue of gamma-aminobutyric acid, is described in the treatment of a 9-year-old boy with intractable torsion dystonia, not responding to conservative treatment. To our knowledge, this is the first reported case of continuous intrathecal baclofen for hereditary torsion dystonia. This case suggests that a continuous intrathecal infusion of baclofen may facilitate remission of intractable torsion dystonia, and provides a basis for further investigation of the treatment of intractable childhood dystonia.

Baclofen↗
PubMed1996

Effect of interleukin-1 alpha, interleukin-1 beta and tumor necrosis factor-alpha on the intracellular fluorescein fluorescence polarization of human lung fibroblasts.

In the present study we aimed to detect early intracellular changes in the cytoplasmic matrix induced in human, pulmonary-derived fibroblasts following exposure to interleukin (IL)-1 alpha, IL-1 beta and tumor necrosis factor-alpha. Such changes were detected by measuring intracellular fluorescein fluorescence polarization (IFFP) using the Cellscan apparatus. IFFP measurement was selected in our study since it has been shown to reflect the microviscosity of the cytoplasmic matrix. Significant reductions (> or = 5%) in the IFFP were induced in fibroblasts by all the cytokines employed. The effect of cytokines on IFFP was achieved at concentrations of 5-10 ng/ml of the cytokines. The reduction in IFFP, following stimulation with the cytokines, was detected as early as 20 min after exposure to the cytokines, lasted at least 40-60 min after exposure to IL-1 alpha and IL-1 beta, and was inhibited by vinblastine, an inhibitor of the polymerization of microtubules. Our results show that IFFP measurements by the Cellscan may reveal rapid intracellular changes occurring in the cytoskeleton components of activated cells.

Cytochalasin B↗
PubMed1996

Inhibition of mitogen-induced changes in intracellular fluorescein fluorescence polarization of human peripheral blood lymphocytes by colchicine, vinblastine and cytochalasin B.

We have previously reported that the exposure of human peripheral blood lymphocytes (PBL) to a variety of stimulants caused rapid changes in intracellular fluorescein fluorescence polarization (IFFP) in the activated cells. In the present study we further analyzed possible mechanisms responsible for the changes in IFFP in PBL exposed to phytohaemagglutinin (PHA) and anti-CD3 antibody. By employing several agents which are known to affect the polymerization of the cytoskeleton we showed that both cytochalasin B, which regulates the microfilaments structure, and vinblastine and colchicine, which affect the microtubules, completely abolished the changes induced in IFFP of human PBL by both PHA and anti-CD3. This effect was dose dependent and was noted at concentrations ranging from 10 to 100 microM of cytochalasin B and 10 microM of vinblastine and colchicine. The effect of these cytoskeleton modulators occurred within 20 minutes after the initiation of activation with PHA. Our results indicate that activation with PHA and anti-CD3 causes early changes in the microtubules and microfilaments components of the cytoskeleton. The possible application of IFFP measurement in analyzing early changes in the cytoskeleton following cell activation is discussed.

Colchicine↗