Pregnancy in Hystricomorpha: gestational age and embryonic-fetal development of agouti (Dasyprocta prymnolopha, Wagler 1831) estimated by ultrasonography.

Thirty-one pregnant agoutis, between Days 9 and 103 of gestation (Day 1 = day of detection of sperm in the vaginal smear), underwent B-mode ultrasonography; gestational sac diameter (GSD), crown-rump length (CRL), embryonic-fetal diameter (EFD), and placenta diameter (PD) were measured. There were positive correlations (P < 0.05) between GSD and CRL (r = 0.98), GSD and PD (r = 0.88), CRL and PD (r = 0.86), days of gestation (DG) and CRL (r = 0.85), and DG and PD (r = 0.73). The gestational sac was first observed on Day 14. The embryo was first seen on Day 18 in 9/31 of pregnant agoutis and on Day 22 in 20/31 of pregnant agoutis. Heartbeats were detected from the Day 25 and placentas were observed in 100% of the animals from Day 25. Early limb bud and ossification of the fetal skull were identified on Days 27 (15/31) and 45 (24/31), respectively. Fetal orientation (head and body) was evident from Day 40, the stomach, liver and lungs were identified on Day 50, the kidneys were reliably seen only on Day 55, and the aorta and vena cava were seen on Day 70. The fetal bowel and the urinary bladder were the last structures to be observed (Day 85). Ultrasonography was effective for early pregnancy diagnosis in agouti and for obtaining information on embryonic and fetal structures that could be used to predict gestational age and birth, thereby contributing to their reproductive management in captivity.

Collection and evaluation of epididymal sperm in captive agoutis (Dasyprocta aguti).

The objective was to establish a protocol for the collection and evaluation of epididymal sperm in agoutis. Eight males (1-2 y old) underwent left orchidectomy and epididymal sperma were collected by retrograde flush. Average values were flush volume 32 µL, pH 6.9, sperm concentration 748 x 10(6) sperm/mL, with motility 86.5% and vigor 4.6. Viable sperm were present in all flush samples; 66% of sperm were alive, and 41.9% of sperm responded positively to the hypoosmotic test (using distilled water). There were 21.1% morphologically abnormal sperm, of which 2.0 and 19.1% were primary and secondary defects, respectively. The acrosome was intact in 99.5% of sperm. The sperm head was 4.89 ± 0.41 µm long and 3.13 ± 0.35 µm wide, with an area of 13.01 ± 2.01 µm(2). Midpieces were 5.33 ± 0.44 µm long and 0.98 ± 0.13 wide, sperm tails were 29.91 ± 2.29 µm, and overall sperm length was 40.12 ± 2.44 µm. In conclusion, epididymal sperm collection from agoutis was satisfactory; the collected sperm has the potential to be stored, facilitating development of other reproductive biotechnologies for this species.

A 140 mA cw deuteron electron cyclotron resonance source for the IFMIF-EVEDA project.

In the framework of the IFMIF-EVEDA phase (International Fusion Materials Irradiation Facility-Engineering Validation and Engineering Design Activities), the CEA-Saclay is in charged of the design and realization of the 140 mA cw deuteron source. The IFMIF EVEDA demonstrator will be installed in Japan in the next six years and will have to accelerate the deuteron beam up to 9 MeV. CEA will build the source and the low energy beam line (LEBT) and will test the cw high intensity deuteron production at Saclay. The SILHI source is an electron cyclotron resonance (ECR) source, operating at 2.45 GHz. In 2001, it produced more than 130 mA of deuteron beam in pulsed mode to minimize neutron production. Such a result pushes to develop a new ECR source based on the SILHI design and equipped with a specific extraction system. Several options of the accelerator column will be implemented in order to improve the reliability and the efficiency of the source. The IFMIF source and LEBT design will be reported.

BETSI, a new test bench for ion sources optimization at CEA SACLAY.

In the framework of several International HPPA projects (such as IFMIF, IPHI, and Spiral2) the CEA handles the design and the developments of several electron cyclotron resonance (ECR) ion sources. For the IFMIF EVEDA demonstrator, a 140 mA cw extracted deuteron beam will be required for high yield of neutron production. For radioactive ion production in the Spiral2 project, several milliamperes of deuterons will be delivered with a permanent magnet source. The optimization of the beam quality at the entrance of the radio frequency quadropole (RFQ) accelerator system triggered the need of a new test bench for ion source optimization and beam qualification. The BETSI ion source test bench will operate up to 50 kV and ignite cw or pulsed hydrogen plasma with a 2.45 GHz magnetron. Great care has already been taken to design electrostatic optics of the extraction system to minimize the emittance growth. Plasma diagnostics will be inserted in the source chamber and several beam diagnostics (emittance and current measurements, beam species analysis) will also be implemented on the low energy beam line transport (LEBT). These diagnostics allow the simultaneous analysis of the beam quality with the plasma parameters of the source. Regional funding request will also be needed to improve the LEBT for space charge compensation measurements. The design of the present and upgraded test bench will be reported as well as the first extracted beam analysis.

Electron cyclotron resonance 140 mA D(+) beam extraction optimization for IFMIF EVEDA accelerator.

Based on the experience of the SILHI electron cyclotron resonance (ECR) ion source for the IPHI accelerator, which produces routinely 100-120 mA H(+) beam, the CEA-Saclay is in charge of the design and realization of the 140 mA cw deuteron source for the IFMIF project (International Fusion Materials Irradiation Facility). IFMIF is an accelerator-based neutron irradiation facility consisting of two accelerators of 125 mA D(+) beam at 40 MeV that hit in parallel a lithium target. IFMIF utilizes the deuteron-lithium (d-Li) neutron, producing a reaction to simulate the 14 MeV neutron environment in deuterium-tritium (D-T) fusion reactors. In the framework of the IFMIF EVEDA phase (Engineering Validation and Engineering Design Activities), we are studying a cw ECR ion source with a new extraction system to allow high current extraction while keeping a low divergence as well as a small emittance. Starting from SILHI five-electrode system with H(+) ions, the extracted beam characteristics as well as electric field conditions are compared with the cases of four- and three-electrode extraction systems. Experimental results made on the SILHI source with H(+) ions are briefly discussed. Extensive experimental results on the new source test bench BETSI are expected as soon as the design and fabrication of a dedicated extraction system with a new set of electrodes will be finished.

Numerical experiments on quantum chaotic billiards.

A recently proposed numerical technique for generation of high-quality unstructured meshes is combined with a finite-element method to solve the Helmholtz equation that describes the quantum mechanics of a particle confined in two-dimensional cavities. Different shapes are treated on equal footing, including Sinai, stadium, annular, threefold symmetric, mushroom, cardioid, triangle, and coupled billiards. The results are shown to be in excellent agreement with available measurements in flat microwave resonator counterparts with nonintegrable geometries.

A phase I dose-finding study of combined treatment with an antisense Bcl-2 oligonucleotide (Genasense) and mitoxantrone in patients with metastatic hormone-refractory prostate cancer.

PURPOSE: Bcl-2 is a negative prognostic indicator in prostate cancer, implicated in the development of androgen independence and treatment resistance, and is overexpressed in hormone-refractory prostate cancer (HRPC). Genasense is a phosphorothioate antisense oligonucleotide complementary to the bcl-2 mRNA open reading frame that in preclinical studies has shown significant activity in inhibiting expression of Bcl-2, delaying androgen independence, and improving chemosensitivity in prostate and other cancer models. In this dose escalation study, we evaluated the combination of Genasense and mitoxantrone, a standard chemotherapy for patients with HRPC. DESIGN: Twenty-six patients with HRPC were treated at seven dose levels receiving Genasense at a dose ranging from 0.6 to 5.0 mg/kg/day and mitoxantrone from 4 mg/m(2) to 12 mg/m(2). Genasense was administered as a 14-day i.v. continuous infusion every 28 days with mitoxantrone given as an i.v. bolus on day 8. RESULTS: No dose-limiting toxicities were observed. Hematological toxicities were transient and included neutropenia, thrombocytopenia, and lymphopenia. Nonhematological toxicities included fatigue, fever, nausea, arthralgias, myalgias, and transient elevations in serum creatinine, none of which were severe. Two patients had >50% reductions in prostate-specific antigen. One patient, who received six cycles of Genasense at 1.2 mg/kg/day and a low dose (4 mg/m(2)) of mitoxantrone, also had symptomatic improvement in bone pain. Peripheral blood lymphocyte Bcl-2 protein expression decreased in five of five patients given Genasense at 5mg/kg/day (mean change from baseline, -12.8%; SD, 16.4%) as assessed by flow cytometry. Serum concentrations of Genasense given at doses of 3 mg/kg/day and greater, exceeded 1 microg/ml. CONCLUSIONS: Genasense and mitoxantrone are well tolerated in combination, and mitoxantrone can be delivered at a standard dose with biologically active doses of Genasense without significant additional toxicity. This observation allays concerns about trials that combine Genasense with full doses of other cytotoxic agents seeking greater evidence of activity.

Effects of Bcl-2 modulation with G3139 antisense oligonucleotide on human breast cancer cells are independent of inherent Bcl-2 protein expression.

We have investigated the effects of transient Bcl-2 down-regulation induced by the Bcl-2 antisense oligodeoxynucleotide (ODN) G3139 (Genta Incorporated) in high Bcl-2 protein expressing, estrogen receptor (ER) positive MCF-7 and low Bcl-2 expressing, ER negative MDA435/LCC6 human breast cancer cells. Treatment with Bcl-2 antisense ODN in vitro caused > 80% reduction of Bcl-2 protein levels in a sequence specific manner for both cell lines. Maximum mRNA reduction was achieved within 24 h of the first antisense ODN exposure whereas full protein down-regulation required antisense exposure over 48 h. This Bcl-2 reduction was associated with 80-95% loss of viable cells compared to untreated cells. Similar cytotoxic effects were observed in both cell lines despite a nine-fold intrinsic difference in Bcl-2 protein expression suggesting that the relative degree of down-regulation of Bcl-2 is more important than the absolute reduction. Cell death associated with G3139 exposure exhibited properties indicative of apoptosis such as mitochondrial membrane depolarization and caspase activation. Combined treatment with G3139 and cytotoxic agents resulted in additive cytotoxicity in both cell lines. However, under most conditions studied, the direct cytotoxic activity of G3139 antisense was not synergistic with the cytotoxic agents. These results suggest that while Bcl-2 clearly constitutes an attractive therapeutic target due to its role in regulating apoptosis in breast cancer cells, additional mechanisms are important in the control of apoptosis arising from exposure to anticancer agents in vitro.

Quantitative fluorescence cytometric analysis of Bcl-2 levels in tumor cells exhibiting a wide range of inherent Bcl-2 protein expression: correlation with Western blot analysis.

BACKGROUND: A protocol to measure a wide range of Bcl-2 protein expression using quantitative fluorescence cytometry (QFCM) in different cell types was developed for use with flow cytometry. Bcl-2 measurements obtained by flow cytometry were correlated with Western blot Bcl-2 measurements to confirm specificity of the Bcl-2-FITC staining. This protocol was applied to measure absolute levels of Bcl-2 protein in different tumor cell lines including Bcl-2-transfected breast carcinoma cell lines and in peripheral blood lymphocytes (PBL). METHODS: HL-60, K562, DOHH2, Jurkat, MDA435/LCC6, MCF7 cell lines, and PBL derived from normal donors were fixed, permeabilized, stained with anti-Bcl-2-FITC antibody and evaluated by QFCM. In parallel, the same cells were evaluated for Bcl-2 protein expression by Western blot analysis. Mitochondrial localization of anti-Bcl-2-FITC antibody inside cells was confirmed using fluorescence imaging microscopy. RESULTS: Bcl-2 expression in different cell types could be accurately quantified based on antibody-binding capacity (ABC) ranging from 12.6 x 10(3) antibody-binding sites in HL-60 cells to 1.64 x 10(6) antibody-binding sites in a Bcl-2-transfected MDA435/LCC6 clone. The data from flow cytometry analysis correlated well with Western analysis (R(2) = 0.78). Bcl-2-FITC staining colocalized with dyes specific for mitochondria. CONCLUSIONS: The Bcl-2 staining protocol described here was shown to be specific, sensitive, and it was able to provide higher resolution as well as more reproducible quantitation of Bcl-2 protein content in cells when compared with Western blot methods. Quantitation of Bcl-2 content in cells by QFCM may be useful for monitoring Bcl-2 expression in cells undergoing various treatments in vitro and in vivo.

Molecular and pharmacokinetic properties associated with the therapeutics of bcl-2 antisense oligonucleotide G3139 combined with free and liposomal doxorubicin.

Bcl-2 is a key apoptosis-regulating protein that has been implicated in mechanisms of chemoresistance for a variety of malignancies by blocking programmed cell death. This study investigated the activity of the Bcl-2 antisense oligodeoxynucleotide (AS ODN) G3139 combined with free doxorubicin (F-DOX) or sterically stabilized liposomal doxorubicin (SL-DOX) to determine the role that drug pharmacodistribution properties may have on antitumor activity using a Bcl-2-expressing human breast solid tumor xenograft model. Administration of G3139 was able to delay the growth of MDA435/LCC6 cells compared with control ODN-treated animals; however, in all of the cases, tumors reestablished after AS ODN treatment. Western blot analyses of Bcl-2 levels of solid tumors showed a sequence-specific down-regulation of the Bcl-2 protein after four daily doses of G3139, which correlated with histological evidence of tumor cell death. Interestingly, the expression of Bcl-2 returned to pretreatment levels during the course of subsequent ODN administration, which suggested the development of resistance to continued Bcl-2 ODN treatment. The antitumor activity of ODN given in conjunction with either F-DOX or SL-DOX was also examined. The combination of G3139 and F-DOX was able to suppress the growth of MDA435/LCC6 cells beyond that obtained with either of the treatments given alone, indicative of synergistic action. Examination of the pharmacokinetics of F-DOX with systemic G3139 administration revealed that elevated tumor drug DOX levels were obtained compared with DOX treatment in the absence of G3139. This effect was sequence-specific and plasma DOX levels were unaffected by G3139 treatment, which indicated possible positive ODN-drug interactions at the tumor site. Combining G3139 with SL-DOX further increased the degree of antitumor activity. The improved efficacy of this combination was attributed to increased tumor drug levels that arise from the ability of SL-DOX to passively accumulate in solid tumors. These results suggest that additional benefits of Bcl-2 antisense ODN may be obtained when it is combined with liposomal formulations of anticancer drugs such as DOX.

Selective targeting of immunoliposomal doxorubicin against human multiple myeloma in vitro and ex vivo.

Circulating malignant CD19(+) B cells have been implicated in the pathogenesis and relapse of multiple myeloma (MM). This study investigated the therapeutic applicability of using long-circulating liposome-encapsulated doxorubicin (DXR) targeted against the internalizing CD19 antigens present on human MM cells. In vitro binding studies using the CD19(+) MM cell line ARH77 demonstrated that CD19-directed immunoliposomes (SIL[anti-CD19]) specifically attached to these cells. Formulations of immunoliposomal doxorubicin (DXR-SIL[anti-CD19]) showed a higher association with, and higher cytotoxicity against, ARH77 cells than did non-targeted liposomal doxorubicin (DXR-SL) or isotype-matched controls (DXR-NSIL[IgG2a]). By using the pH-sensitive fluorophore, 1-hydroxypyrene-3,6, 8-trisulfonic acid, binding of SIL[anti-CD19] to CD19 antigens was shown to trigger receptor-mediated internalization of the antibody-antigen complexes into endosomes. Targeting of SIL[anti-CD19] to CD19(+) B cells was also demonstrated in a heterogeneous mixture of peripheral blood mononuclear cells (PBMC) from MM patients. A decrease in cellular DNA (which is an indicator of apoptosis) caused by the cytotoxicity of DXR-SIL[anti-CD19] to myeloma PBMC was determined by using flow cytometry. While PBMC treatment with free DXR resulted in non-specific cytotoxicity to both B and T cells, DXR-SL were only minimally cytotoxic to either. In contrast, DXR-SIL[anti-CD19] were selectively cytotoxic for B cells in PBMC, indicating that this treatment may be effective in eliminating circulating malignant B cells in MM patients.

Effects of pentoxifylline and nabumetone on the serum levels of IL-1beta and TNFalpha in rats with adjuvant arthritis.

OBJECTIVE AND DESIGN: This study examined whether serum levels of cytokines, IL-1beta and TNFalpha were elevated in rats with adjuvant arthritis in relation to disease progression, and if so, to verify the treatment effects of nabumetone (20 mg/kg, p. o.), a COX-2 inhibitor and pentoxifylline (20 mg/kg, p.o.), a type-4 phosphodiesterase (PDE4) inhibitor, alone or in combination. MATERIALS AND METHODS: Female Wistar rats were used. Freund's complete adjuvant (FCA) was used to induce arthritis. The increment in contralateral hind paw volume (the secondary lesion) was determined by plethysmometry and the serum cytokines were measured by ELISA. RESULTS: In control rats, the serum IL-1beta and TNFalpha levels were greatly elevated on the very first day i.e. 5 h after FCA, and thereupon a progressive decrease in IL-1beta but not TNFalpha was observed until day 30. The secondary arthritic lesion began to increase on day 14 (125+/-26 microl), and attained its peak (330+/-79 microl) on day 21 post-adjuvant injection. The peak arthritic lesion was significantly (p<0.001) less in rats that received nabumetone and pentoxifylline, alone or in combination (20+/-8, 41+/-15 and 65+/-10 microL, respectively). When serum cytokine levels were analysed on day 20 postadjuvant injection, rats treated with pentoxifylline or in association with nabumetone, but not nabumetone alone showed significantly lowered levels of serum TNFalpha. Unlike TNFalpha, serum IL-1beta did not vary significantly. CONCLUSIONS: The drugs nabumetone and pentoxifylline although appearing to produce differential effects on serum cytokine levels, seem to be equally efficacious in attentuating the progression of FCA-induced arthritis. Serum cytokine levels may not accurately reflect the treatment efficacy.

In vitro and in vivo targeting of immunoliposomal doxorubicin to human B-cell lymphoma.

The ability to selectively target liposomal anticancer drugs via specific ligands against antigens expressed on malignant cells could improve the therapeutic effectiveness of the liposomal preparations as well as reduce adverse side effects associated with chemotherapy. Long-circulating formulations of liposomes containing lipid derivatives of poly(ethyleneglycol) [sterically stabilized liposomes (SLs)] have been described previously, and new techniques have recently been developed for coupling monoclonal antibodies (Abs) at the poly(ethyleneglycol) terminus of these liposomes. Ab-targeted SLs [immunoliposomes (SILs)] containing entrapped anticancer drugs are predicted to be useful in the treatment of hematological malignancies such as B-cell lymphomas or multiple myeloma, in which the target cells are present in the vasculature. The specific binding, in vitro cytotoxicity, and in vivo antineoplastic activity of doxorubicin (DXR) encapsulated in SILs coupled to monoclonal Ab anti-CD19 (SIL[anti-CD19]) were investigated against malignant B cells expressing CD19 surface antigens. Binding experiments with SIL[anti-CD19] resulted in a 3-fold higher association of the SILs with a human CD19+ B lymphoma cell line (Namalwa) in comparison with nontargeted SLs. Using flow cytometry, fluorescently labeled SIL[anti-CD19] bound to B cells with no recognition of T cells in a mixture of B cells and T cells in culture. Nontargeted SLs demonstrated significantly lower recognition of either B cells or T cells. Targeted DXR-SIL[anti-CD19] displayed a higher cytotoxicity to B cells relative to DXR entrapped in nontargeted SLs. Therapeutic experiments in severe combined immunodeficient mice implanted with Namalwa cells by the i.v. or i.p. routes resulted in significantly increased effectiveness of DXR-SIL[anti-CD19] compared to similar amounts of free DXR, DXR-SL (no Ab), or isotype-matched nonspecific Abs attached to DXR-SL. Single doses (3 mg/kg) of DXR-SIL[anti-CD19] administered i.v. resulted in a significantly improved therapeutic benefit, including some long-term survivors. From our results, we infer that targeted anti-CD19 liposomes containing the anticancer drug DXR may be selectively cytotoxic for B cells and may be useful in the selective elimination of circulating malignant B cells in vivo.