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Biomedical subjects

R Levy

Publications and source records attributed to R Levy.

At least 19 recordsLinked to original sources
PubMed1999

Essential requirement of cytosolic phospholipase A(2) for activation of the H(+) channel in phagocyte-like cells.

The NADPH oxidase-producing superoxide is the major mechanism by which phagocytes kill invading pathogens. We previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient differentiated PLB-985 cells (PLB-D cells) and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for NADPH oxidase activation (Dana, R., Leto, T., Malech, H., and Levy, R. (1998) J. Biol. Chem. 273, 441-445). In the present study, we used this model to determine the physiological role of cPLA(2) in the regulation of both the H(+) channel and the Na(+)/H(+) antiporter and to study whether NADPH oxidase activation is regulated by either of these transporters. PLB-D cells and two controls: parent PLB-985 cells and PLB-985 cells transfected with the vector only (PLB cells) were differentiated using 1.25% Me(2)SO or 5 x 10(-8) M 1, 25-dihydroxyvitamin D(3). Activation of differentiated PLB cells resulted in a Zn(2+)-sensitive alkalization, indicating H(+) channel activity. In contrast, differentiated PLB-D cells failed to activate the H(+) channel, but the addition of exogenous AA fully restored this activity, indicating the role of cPLA(2) in H(+) channel activation. The presence of the H(+) channel inhibitor Zn(2+) caused significant inhibition of NADPH oxidase activity, suggesting a role of the H(+) channel in regulating oxidase activity. Na(+)/H(+) antiporter activity was stimulated in differentiated PLB-D cells, indicating that cPLA(2) does not participate in the regulation of this antiporter. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA for activation of the H(+) channel and suggest the participation of this channel in the regulation of NADPH oxidase activity.

Amiloride↗
PubMed1999

Association of storage and processing functions in the dorsolateral prefrontal cortex of the nonhuman primate.

The prominent role of the prefrontal cortex (PFC) in working memory (WM) is widely acknowledged both in nonhuman primates and in humans. However, less agreement exists on the issue of functional segregation within different subregions of the PFC with regard to the domains of spatial and nonspatial processing or involvement in simpler versus more complex aspects of WM, e.g., maintenance versus processing function. To address these issues, six monkeys were trained to perform four WM tasks that differed with respect to domain (spatial vs nonspatial) and level of WM demand (recall of one vs three items). The delayed response format was used to assess simple one-item memory, whereas self-ordering tasks were used to require the monkey to maintain and organize three items of information within WM. After training, the monkeys received bilateral PFC lesions in one of two different areas, Walker's areas 9 and 8B (dorsomedial convexity; n = 3) or areas 46 and 8A (dorsolateral cortex, n = 3) and then tested postoperatively on all tasks. Monkeys with lesions of the dorsomedial convexity were not impaired either on spatial or nonspatial WM tasks, whether the task required simple storage or sequential processing. By contrast, lesions of the dorsolateral cortex produced a significant and persistent impairment in both simple and complex spatial WM but no impairment in the two nonspatial WM tasks. These results support a functional segregation within the dorsolateral prefrontal cortex for WM: the dorsolateral prefrontal cortex (area 46/8A) is selectively involved in spatial WM, whereas the dorsomedial convexity (area 9/8B) is not critically engaged in either spatial or nonspatial working memory. Furthermore, the specific involvement of area 46/8A in spatial sequencing as well as in single-item storage WM tasks supports, in the nonhuman primate, an areal dissociation based on domain rather than on processing demand.

Animals↗
PubMed1999

Macrophage enrichment with the isoflavan glabridin inhibits NADPH oxidase-induced cell-mediated oxidation of low density lipoprotein. A possible role for protein kinase C.

Macrophage-mediated oxidation of low density lipoprotein (LDL) is considered to be of major importance in early atherogenesis; therefore, intervention means to inhibit this process are being extensively studied. In the present study, we questioned the ability of the isoflavan glabridin (from licorice) to accumulate in macrophages and to affect cell-mediated oxidation of LDL. We first performed in vitro studies, using mouse peritoneal macrophages (MPMs) and the J-774 A.1 macrophage-like cell line. Both cells accumulated up to 1.5 micrograms of glabridin/mg of cell protein after 2 h of incubation, and this process was time- and glabridin dose-dependent. In parallel, in glabridin-enriched cells, macrophage-mediated oxidation of LDL was inhibited by up to 80% in comparison with control cells. Glabridin inhibited superoxide release from MPMs in response to phorbol 12-myristate 13-acetate, or to LDL when added together with copper ions, by up to 60%. Translocation of P-47, a cytosolic component of NADPH oxidase to the plasma membrane was substantially inhibited. In glabridin-enriched macrophages, protein kinase C activity reduced by approximately 70%. All of the above effects of glabridin required the presence of the two hydroxyl groups on the flavonoid's B phenol ring. In order to assess the physiological significance of these results, we next performed in vivo studies, using the atherosclerotic apolipoprotein E-deficient (E0) mice. MPMs harvested from glabridin-treated E0 mice (20 micrograms/mouse/day for a period of 6 weeks) demonstrated reduced capability to oxidize LDL by 80% in comparison with placebo-treated mice. This latter phenomenon was associated with a reduction in the lesion oxysterols and a 50% reduction in the aortic lesion size. We thus conclude that glabridin accumulation in macrophages is associated with reduced cell-mediated oxidation of LDL and decreased activation of the NADPH oxidase system. These phenomena could be responsible for the attenuation of atherosclerosis in E0 mice, induced by glabridin.

Animals↗
PubMed1999

DNA vaccination against the idiotype of a murine B cell lymphoma: mechanism of tumor protection.

Several studies have shown that immunization with DNA, which encodes the idiotypic determinants of a B cell lymphoma, generates tumor-specific immunity. Although induction of antiidiotypic Abs has correlated with tumor protection, the effector mechanisms that contribute to tumor protection have not been clearly identified. This study evaluated the tumor protective effects of humoral and cellular immune mechanisms recruited by idiotype-directed DNA vaccines in the 38C13 murine B cell lymphoma model. Antiidiotypic Abs induced by DNA vaccination supported in vitro complement-mediated cytotoxicity of tumor cells, and simultaneous transfer of tumor cells and hyperimmune sera protected naive animals against tumor growth. However, in vitro stimulation of immune splenocytes with tumor cells failed to induce idiotype-specific cytotoxicity, and following vaccination, depletion of CD4 or CD8 T cell subsets did not compromise protection. Furthermore, protection of naive recipients against tumor challenge could not be demonstrated either by a Winn assay approach or by adoptive transfer of spleen and lymph node cells. Thus, in this experimental model, current evidence suggests that the tumor-protective effects of DNA vaccination can be largely attributed to idiotype-specific humoral immunity.

Adoptive Transfer↗
PubMed1999

Idiotype vaccination using dendritic cells after autologous peripheral blood stem cell transplantation for multiple myeloma--a feasibility study.

The idiotype (Id) determinant on the multiple myeloma (MM) protein can be regarded as a tumor-specific marker. Immunotherapy directed at the MM Id may stem the progression of this disease. We report here on the first 12 MM patients treated at our institution with high-dose therapy and peripheral blood stem cell transplantation (PBSCT) followed by Id immunizations. MM patients received PBSCT to eradicate the majority of the disease. PBSCT produced a complete response in 2 patients, a partial response in 9 patients and stable disease in 1 patient. Three to 7 months after high-dose therapy, patients received a series of monthly immunizations that consisted of two intravenous infusions of Id-pulsed autologous dendritic cells (DC) followed by five subcutaneous boosts of Id/keyhole limpet hemocyanin (KLH) administered with adjuvant. Between 1 and 11 x 10(6) DC were obtained by leukapheresis in all patients even after PBSCT. The administration of Id-pulsed DC and Id/KLH vaccines were well tolerated with patients experiencing only minor and transient side effects. Two of 12 patients developed an Id-specific, cellular proliferative immune response and one of three patients studied developed a transient but Id-specific cytotoxic T-cell (CTL) response. Eleven of the 12 patients generated strong KLH-specific cellular proliferative immune responses showing the patients' immunocompetence at the time of vaccination. The two patients who developed a cellular Id-specific immune response remain in complete remission. Of the 12 treated patients, 9 are currently alive after autologous transplantation with a minimum follow-up of 16 months, 2 patients died because of recurrent MM and 1 patient succumbed to acute leukemia. These studies show that patients make strong anti-KLH responses despite recent high-dose therapy and that DC-based Id vaccination is feasible after PBSCT and can induce Id-specific T-cell responses. Further vaccine development is necessary to increase the proportion of patients that make Id-specific immune responses. The clinical benefits of Id vaccination in MM remain to be determined.

Adjuvants, Immunologic↗
PubMed1999

TCR vaccines against T cell lymphoma: QS-21 and IL-12 adjuvants induce a protective CD8+ T cell response.

Tumor-specific TCR can serve as an effective target for active immunotherapy of T cell malignancies. Using the murine T cell tumor model C6VL, vaccination with C6VL TCR protected mice from a subsequent lethal dose of tumor cells. This study characterizes the immune mechanisms involved in the tumor protection, and the influence of immunologic adjuvants in inducing a protective immune response. Immune responses induced by TCR vaccines formulated with various adjuvants: QS-21, IL-12, SAF-1, CD40L, and GM-CSF were compared. QS-21, IL-12, and SAF-1 biased the humoral immune response toward Th1-type, reflected by the induction of IgG2a and IgG2b anti-C6VL TCR Abs. CD40L and GM-CSF exclusively produced IgG1 Abs, reflecting a Th2-type immune response. In our tumor model system, only vaccines containing adjuvants that induced a Th1-type immune response favored tumor protection. Furthermore, we demonstrated that CD8+ T cells were necessary and sufficient for tumor protection using anti-CD8 mAb depletion and adoptive cell transfer experiments. Transfer of hyperimmune serum containing anti-C6VL TCR Abs into na ive mice had modest anti-tumor effects and was not sufficient to prevent tumor growth. TCR-vaccinated B cell-deficient mice were not protected against C6VL tumor, and tumor protection was not completely restored after hyperimmune serum transfer. Thus, B cells may serve as important APCs in inducing a protective immune response. Based on these results future TCR vaccines should be designed to maintain native TCR conformation, as well as induce a strong Th1-type immune response.

Adjuvants, Immunologic↗
PubMed1999

Rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain Fv epitopes in tobacco plants.

Rapid production of protein-based tumor-specific vaccines for the treatment of malignancies is possible with the plant-based transient expression system described here. We created a modified tobamoviral vector that encodes the idiotype-specific single-chain Fv fragment (scFv) of the immunoglobulin from the 38C13 mouse B cell lymphoma. Infected Nicotiana benthamiana plants contain high levels of secreted scFv protein in the extracellular compartment. This material reacts with an anti-idiotype antibody by Western blotting, ELISA, and affinity chromatography, suggesting that the plant-produced 38C13 scFv protein is properly folded in solution. Mice vaccinated with the affinity-purified 38C13 scFv generate >10 micrograms/ml anti-idiotype immunoglobulins. These mice were protected from challenge by a lethal dose of the syngeneic 38C13 tumor, similar to mice immunized with the native 38C13 IgM-keyhole limpet hemocyanin conjugate vaccine. This rapid production system for generating tumor-specific protein vaccines may provide a viable strategy for the treatment of non-Hodgkin's lymphoma.

Amino Acid Sequence↗
PubMed1999

Tremor arrest with thalamic microinjections of muscimol in patients with essential tremor.

Six patients undergoing stereotactic procedures for essential tremor received microinjections of muscimol (a gamma-aminobutyric acid-A [GABA(A)] agonist) into the ventralis intermedius thalamus in areas where tremor-synchronous cells were identified electrophysiologically with microelectrode recordings and where tremor reduction occurred with electrical microstimulation. Injections of muscimol but not saline consistently reduced tremor in each patient. The effect had a mean latency of 7 minutes and lasted an average of 9 minutes. We propose that GABA-mediated thalamic neuronal inhibition may represent a mechanism underlying the effectiveness of surgery for tremor and that GABA analogues could potentially be used therapeutically.

Aged↗
PubMed1999

A cis proline turn linking two beta-hairpin strands in the solution structure of an antibody-bound HIV-1IIIB V3 peptide.

The refined solution structure of an 18-residue HIV-1IIIB V3 peptide in complex with the Fv fragment of an anti-gp120 antibody reveals an unexpected type VI beta-turn comprising residues RGPG at the center of a beta-hairpin. The central glycine and proline of this turn are linked by a cis peptide bond. The residues of the turn interact extensively with the antibody Fv. 15N[1H] NOE measurements show that the backbone of the peptide, including the central QRGPGR loop, is well ordered in the complex. The solution structure is significantly different from the X-ray structures of HIV-1MN V3 peptides bound to anti-peptide antibodies. These differences could be due to a two-residue (QR) insertion preceding the GPGR sequence in the HIV-1IIIB strain, and the much longer peptide epitope immobilized by the anti-gp120 antibody.

Amino Acid Sequence↗
PubMed1999

Screening for Chlamydia trachomatis and Ureaplasma urealyticum infection in semen from asymptomatic male partners of infertile couples prior to in vitro fertilization.

In a prospective study, the prevalence of infection with Chlamydia trachomatis and Ureaplasma urealyticum was evaluated in the semen of 92 asymptomatic male partners of infertile couples using polymerase chain reaction and culture, respectively. The results were compared with the detection of serologically specific antibodies. U. urealyticum and C. trachomatis were detected in 12 (13%) and 10 (10.8%) of the tested ejaculates, respectively. One mixed infection was detected. No correlation was found between detection of the pathogens in ejaculates and the presence of specific antibodies in serum. This study therefore confirms the limited diagnostic value of serological analysis to ascertain infection with C. trachomatis or U. urealyticum. The high frequency of detection of these pathogens among asymptomatic male partners of infertile couples emphasizes their potential role in the impairment of male fertility, and the need for sensitive and specific detection methods to prevent infection of the early embryo when using new reproductive techniques such as zona pellucida hatching or intracytoplasmic microinjection.

Adult↗
PubMed1999

Lycopene and 1,25-dihydroxyvitamin D3 cooperate in the inhibition of cell cycle progression and induction of differentiation in HL-60 leukemic cells.

Lycopene, the major tomato carotenoid, has been found to inhibit proliferation of several types of cancer cells, including those of breast, lung, and endometrium. By extending the work to the HL-60 promyelocytic leukemia cell line, we aimed to evaluate some mechanistic aspects of this effect. Particularly, the possibility was examined that the antiproliferative action of the carotenoid is associated with induction of cell differentiation. Lycopene treatment resulted in a concentration-dependent reduction in HL-60 cell growth as measured by [3H]thymidine incorporation and cell counting. This effect was accompanied by inhibition of cell cycle progression in the G0/G1 phase as measured by flow cytometry. Lycopene alone induced cell differentiation as measured by phorbol ester-dependent reduction of nitro blue tetrazolium and expression of the cell surface antigen CD14. Results of several recent intervention studies with beta-carotene, which have revealed no beneficial effects of this carotenoid, suggest that a single dietary component cannot explain the anticancer effect of diets rich in vegetables and fruits. Thus another goal of our study was to examine whether lycopene has the ability to synergize with other natural anticancer compounds, such as 1,25-dihydroxyvitamin D3, which when used alone are therapeutically active only at high and toxic concentrations. The combination of low concentrations of lycopene with 1,25-dihydroxyvitamin D3 exhibited a synergistic effect on cell proliferation and differentiation and an additive effect on cell cycle progression. Such synergistic antiproliferative and differentiating effects of lycopene and other compounds found in the diet and in plasma may suggest the inclusion of the carotenoid in the diet as a cancer-preventive measure.

Anticarcinogenic Agents↗
PubMed1999

Dendritic cell vaccines for cancer immunotherapy.

Human tumors express a number of protein antigens that can be recognized by T cells, thus providing potential targets for cancer immunotherapy. Dendritic cells (DCs) are rare leukocytes that are uniquely potent in their ability to present antigens to T cells, and this property has prompted their recent application to therapeutic cancer vaccines. Isolated DCs loaded with tumor antigen ex vivo and administered as a cellular vaccine have been found to induce protective and therapeutic anti-tumor immunity in experimental animals. In pilot clinical trials of DC vaccination for patients with non-Hodgkin's lymphoma and melanoma, induction of anti-tumor immune responses and tumor regressions have been observed. Additional trials of DC vaccination for a variety of human cancers are under way, and methods for targeting tumor antigens to DCs in vivo are also being explored. Exploitation of the antigen-presenting properties of DCs thus offers promise for the development of effective cancer immunotherapies.

Animals↗
PubMed1999

An individual bioequivalence approach to compare the intrasubject variability of two ciclosporin formulations, SangCya and Neoral.

A novel bioequivalence testing approach was used to determine intrasubject variability and switchability of two ciclosporin formulations, SangCya (test) and Neoral (reference). Twenty healthy volunteers were enrolled into a single-dose, randomized, open-label, 4-period, 2-sequence study with a crossover replicate design. Subject-by-formulation interaction variances were compared using a mixed effects linear model. Intrasubject variability for ln AUC(0-infinity) and ln C(max) of SangCya and Neoral were not significantly different. The 95% confidence intervals of the intrasubject variability of AUC(0-infinity) (0.94) and C(max) (1.28) as determined using the bootstrap nonparametric percentile method (n = 2,000) were below the individual bioequivalence limit estimated at 2.25. We concluded equivalent intrasubject variability of ciclosporin pharmacokinetics and switchability between SangCya and Neoral.

Adult↗
PubMed1999

Therapy of B-cell lymphoma with anti-CD20 antibodies can result in the loss of CD20 antigen expression.

Rituximab is a chimeric antibody with human gamma-1 and kappa constant regions and murine variable regions. It recognizes the CD20 antigen, a pan B-cell marker. Therapeutic trials in patients with B-cell non-Hodgkin's lymphoma (NHL) have shown significant efficacy with a primary response rate of 50%, and a secondary response rate of 44% after repeat treatments in prior responders. The selection for proliferating tumor cells that no longer express CD20 may compromise repeated treatment. We have identified a patient who developed a transformed NHL that lost CD20 protein expression after two courses of therapy with rituximab. In a pretreatment lymph node biopsy, 83% of B cells (as defined by CD19 and surface immunoglobulin) expressed surface CD20. A biopsy from the recurrent tumor after two courses of rituximab revealed a diffuse large cell NHL where 0% of B cells expressed CD20 with no evidence of bound rituximab. Cytoplasmic staining showed no CD20 protein. Sequencing of immunoglobulin heavy chain cDNA identified identical variable sequences in the initial and recurrent lymphomas, confirming the association between the two tumors. Literature and database review suggests that approximately 98% of diffuse large cell lymphomas express CD20, which suggests that these tumors rarely survive without CD20. This is the first identified case of loss of CD20 expression in a lymphoma that has relapsed after rituximab therapy, although several other cases have since been identified. Considering the significant number of patients treated with anti-CD20 antibodies, this may occur only rarely and is unlikely to preclude recurrent therapy with anti-CD20 antibodies in the majority of patients. However, because many patients have relapsed after anti-CD20 antibody therapy and have not been biopsied to identify clones with down-regulated CD20 antigen, we do not currently know the true frequency of this phenomenon. When possible, patients should undergo evaluation for CD20 expression before repeated courses of anti-CD20 therapy.

Antibodies, Monoclonal↗