Michael Schlesinger

Time domain Dielectric Spectroscopy Study of Human Cells. II. Normal and Malignant White Blood Cells

The dielectric properties of human lymphocyte suspensions were studied by time domain dielectric spectroscopy (TDDS). Nine populations of malignant and normal lymphocytes were investigated. Analysis of the dielectric parameters of cell structural parts were performed in the framework of Maxwell-Wagner mixture formula and the double-shell model of cell. The specific capacitance of the cell membranes was estimated by the Hanai-Asami-Koisumi formula. It was shown that the dielectric permittivity, capacitance and conductivity values of cell membranes are higher for normal lymphocytes than for the malignant ones. The difference of the same parameters for normal B- and T-cells is also discussed.

The CD38 Lymphocyte Differentiation Marker: New Insight into Its Ectoenzymatic Activity and Its Role as a Signal Transducer

This work was supported in part by the Israel Ministry of Absorption and by grants from the Israel Cancer Association and the F. Goldhirsch Foundation.

Antigenic Changes in Lymph-Node Cells after Administration of Antiserum to Thymus Cells

Mice of the RIII and C57BL strains were treated with rabbit antiserum to thymus (ATS), and cells of their lymph nodes were analyzed serologi-cally at intervals after treatment. While lymph-node cells of untreated mice were sensitive to the cytotoxic effect of isoantibodies against the theta antigen, lymph-node cells of ATS-treated mice showed a significantly reduced sensitivity. Three days after ATS treatment lymph-node cells of most mice were completely refractory to the cytotoxic effect of theta antibodies. Administration of normal rabbit serum elicited only a slight reduction of the sensitivity of lymph-node cells to the cytotoxic effect of theta antibodies. The results support the hypothesis that ATS treatment selectively affects a population of thymus-dependent circulating lymphocytes.

TIME DOMAIN DIELECTRIC SPECTROSCOPY STUDY OF HUMAN CELLS. I. ERYTHROCYTES AND GHOSTS

A method, allowing correction of electrode polarization effect in case of cell suspensions of small volume fraction is proposed. The dielectric behavior of human erythrocytes and erythrocyte ghosts suspensions was studied by time domain dielectric spectroscopy (TDDS) and the estimation of the dielectric constants of cells structural parts (membrane, cytoplasm) on the basis of suitable models are presented. It was shown that in the case of small volume fraction of cells in suspension, the electrode polarization effect can be taken into account by the additional measurement of the corresponding supernatant. The optimal volume fraction of cells in suspensions in the TDDS measurements was found to be 3%-10%. The erythrocyte and erythrocyte ghost suspensions were found to demonstrate a single dispersion which can be described by the Debye equation. The membrane dielectric constant of different erythrocytes and ghosts was distributed near 5.

A Cannabinoid Quinone Inhibits Angiogenesis by Targeting Vascular Endothelial Cells

Recent findings on the inhibition of angiogenesis and vascular endothelial cell proliferation by anthracycline antibiotics, which contain a quinone moiety, make this type of compound a very promising lead in cancer research/therapy. We have reported that a new cannabinoid anticancer quinone, cannabidiol hydroxyquinone (HU-331), is highly effective against tumor xenografts in nude mice. For evaluation of the antiangiogenic action of cannabinoid quinones, collagen-embedded rat aortic ring assay was used. The ability of cannabinoids to cause endothelial cell apoptosis was assayed by TUNEL staining and flow cytometry analysis. To examine the genes and pathways targeted by HU-331 in vascular endothelial cells, human cDNA microarrays and polymerase chain reaction were used. Immunostaining with anti-CD31 of tumors grown in nude mice served to indicate inhibition of tumor angiogenesis. HU-331 was found to be strongly antiangiogenic, significantly inhibiting angiogenesis at concentrations as low as 300 nM. HU-331 inhibited angiogenesis by directly inducing apoptosis of vascular endothelial cells without changing the expression of pro- and antiangiogenic cytokines and their receptors. A significant decrease in the total area occupied by vessels in HU-331-treated tumors was also observed. These data lead us to consider HU-331 to have high potential as a new antiangiogenic and anticancer drug.

Age-associated changes in subpopulations of human lymphocytes.

Abstract Lymphocytes from cord bloods, from infants, and from young children were analyzed for their cell surface markers and for their responsiveness to various concentrations of lectins. The results obtained with lymphocytes from young individuals were compared with those obtained with lymphocytes from young adults (18 to 43 years of age) and with lymphocytes from elderly individuals (60 to 92 years of age). The proportion of lymphocytes forming E-rosettes with sheep red blood cells (SRBC) was significantly lower among cord blood lymphocytes than among peripheral blood lymphocytes (PBL) of adults. Cells forming E-rosettes reached adult levels during the third year of life. The proportion of cells forming EAC′-rosettes and stained by fluorescent anti-Ig serum was significantly higher among cord blood lymphocytes than among adult PBL but decreased to the level found in adults during the first year of life. The proportion of cells forming E-rosettes was lower among PBL of elderly individuals than among young adults, but there was no difference between PBL of adults and of elderly individuals in the proportion of cells forming EAC′-rosettes or stained by anti-Ig serum. Cells forming stable E-rosettes, resistant to prolonged incubation at 37°C, were found among cord blood lymphocytes and among PBL of elderly individuals but could not be detected in the blood of children over 1 year of age or among PBL of young adults examined in the present study. Cord blood lymphocytes responded to various concentrations of phytohemagglutinin and concanavalin A more vigorously than PBL of young adults. During the first year of life the response of PBL to lectins increased even further and remained higher than that of adult PBL throughout the first 5 years of life. Lymphocytes of 7- to 8-year-old children displayed a responsiveness similar to that of adults. The responsiveness of PBL of elderly individuals to various lectin concentrations did not differ significantly from that of young adult PBL, although as a result of an elevated “spontaneous proliferation,” the stimulation indices obtained with PBL of elderly individuals were lower than those obtained with PBL of young adults.

Increased expression of activation markers on CD8 lymphocytes in children with human immunodeficiency virus-1 infection.

ABSTRACT: The aims of the present study were to analyze the impact of perinatal human immunodeficiency virus (HIV)-1 infection on lymphocyte maturation in children, to determine the expression of activation markers on CD8+ cells, and to define predictors of survival in HIV-infected children. Seventy-one children presenting HIV-related symptoms were included in the study; 29 were less than 2 y old and 42 were 2 to 12 y of age. Results were compared with those obtained in normal children of a similar age. In HIV-infected children the proportion of CD4+ and CD8+ CD45RA+ cells was significantly decreased, whereas that of CD8+, CD8+CD38+, and CD8+CD45RO+ cells was strikingly increased compared with controls. In children less than 2 y old the absolute number of CD4+ and CD8+CD45RA+ cells decreased, and the number of CD8+CD45RO+ cells increased significantly, whereas the number of CD8+ and CD8+CD38+ cells did not change. The absolute number of CD4+ T cells declined with age both among controls and among HIV-infected children. In contrast, the absolute number of CD8+ cells and CD8 subsets decreased with age only in controls but not in infected children. In HIV-1-infected children the expression of the CD38 and CD45RO markers on CD8+ cells was significantly correlated, indicating that these were activated cells. The survival of less than 2-y-old children with AIDS symptoms was positively correlated with the total number of CD8 cells and CD8+CD38+ cells at time of entry into the study. Most of the children who died by the end of the study had a CD8 count of less than 750/mm3 and a CD38+CD8+ count of less than 600/mm3 when first seen, whereas most of those who were alive had higher counts.

HU-331, a novel cannabinoid-based anticancer topoisomerase II inhibitor

Anthracyclines, a large group of quinonoid compounds, are used to treat some forms of cancer. Although highly effective in cancer therapy, the mechanism of action of these compounds is not specific; they act on cancer and other cells by numerous mechanisms. A new anticancer quinone (HU-331) was synthesized from cannabidiol. It shows significant high efficacy against human cancer cell lines in vitro and against in vivo tumor grafts in nude mice. In this study, we investigated its mode of action and present evidence on its unique mechanism. HU-331 does not cause cancer cell cycle arrest, cell apoptosis, or caspase activation. HU-331–caused cell death of human cancer cell lines is not mediated by reactive oxygen intermediates/species, as exposure to HU-331 failed to elicit the generation of reactive oxygen species. HU-331 inhibits DNA topoisomerase II even at nanomolar concentrations but has only a slight nonsignificant effect on DNA topoisomerase I action. The cannabinoid quinone HU-331 is a highly specific inhibitor of topoisomerase II, compared with most known anticancer quinones. It might represent a new potent anticancer drug. [Mol Cancer Ther 2007;6(1):173–83]

Somatic mutations and intraclonal variations in the rearranged Vk genes of B-non-Hodgkin's lymphoma cell lines

Abstract:  Three established Burkitts lymphoma (BL) cell lines (Daudi, Raji and DG‐75) and three B‐non‐Hodgkins lymphoma (B‐NHL) of other types (Pfeiffer, Farage and Toledo) were analyzed with respect to the presence of somatic point mutations in their rearranged immunoglobulin Vk genes. Two of the Vk sequences of BL and two of those of the B‐NHL were heavily mutated (up to 11%), when compared with their closest germline variable region counterparts (“clonal mutations”). Only one of the six cell lines contained an unmutated germline Vk sequence. The clonal mutations have features characteristic of the mutation machinery operating in the course of the T‐dependent immune response, such as a preference of mutations in purine bases, more transitions than transversions and targeting to CDR and to known “hotspot” motifs. Sequence variations among different Vk PCR clones isolated from each of the cell lines (“intraclonal mutations”) showed that the Vk of Toledo exhibited about 5‐fold higher mutation frequency (MF) than the background level of Taq polymerase error (˜0.12% mut/bp). Similarly, the MF of Vk of two of the BL cell lines was 3–4‐fold higher than the Taq polymerase misincorporation rate. In contrast, the mutation frequencies of the Vk of DG‐75, Farage and Pfeiffer did not significantly exceed the level of Taq polymerase error.

A Cannabinoid Anticancer Quinone, HU-331, Is More Potent and Less Cardiotoxic Than Doxorubicin: A Comparative in Vivo Study

Several quinones have been found to be effective in the treatment of some forms of cancer; however, their cumulative heart toxicity limits their use. The cannabinoid quinone HU-331 [3S,4R-p-benzoquinone-3-hydroxy-2-p-mentha-(1,8)-dien-3-yl-5-pentyl] is highly effective against tumor xenografts in nude mice. We report now a comparison of the anticancer activity of HU-331 and its cardiotoxicity with those of doxorubicin in vivo. General toxicity was assayed in Sabra, nude and SCID-NOD mice. The anticancer activity in vivo was assessed by measurement of the tumors with an external caliper in HT-29 and Raji tumor-bearing mice and by weighing the excised tumors. Left ventricular function was evaluated with transthoracic echocardiography. Myelotoxicity was evaluated by blood cell count. Cardiac troponin T (cTnT) plasma levels were determined by immunoassay. HU-331 was found to be much less cardiotoxic than doxorubicin. The control and the HU-331-treated groups gained weight, whereas the doxorubicin-treated group lost weight during the study. In HT-29 colon carcinoma, the tumor weight in the HU-331-treated group was 54% smaller than in the control group and 30% smaller than in the doxorubicin-treated group. In Raji lymphoma, the tumor weight in the HU-331-treated group was 65% smaller than in the control group and 33% smaller than in the doxorubicin-treated group. In contrast to doxorubicin, HU-331 did not generate reactive oxygen species in mice hearts (measured by protein carbonylation levels and malondialdehyde levels). In vivo, HU-331 was more active and less toxic than doxorubicin and thus it has a high potential for development as a new anticancer drug.