Diagnóstico en el síndrome antifosfolipídico: desde una perspectiva histórica a la aparición de nuevos autoanticuerpos / Diagnosis of antiphospholipid syndrome: From an historical perspective to the emergence of new autoantibodies

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Repeated intraperitoneal injections of liposomes containing phosphatidic acid and cardiolipin reduce amyloid-ß levels in APP/PS1 transgenic mice.

The accumulation of extracellular amyloid-beta (Aß) peptide and intracellular neurofibrillary tangles in the brain are two major neuropathological hallmarks of Alzheimer's disease (AD). It is thought that an equilibrium exists between Aß in the brain and in the peripheral blood and thus, it was hypothesized that shifting this equilibrium towards the blood by enhancing peripheral clearance might reduce Aß levels in the brain: the 'sink effect'. We tested this hypothesis by intraperitoneally injecting APP/PS1 transgenic mice with small unilamellar vesicles containing either phosphatidic acid or cardiolipin over 3weeks. This treatment reduced significantly the amount of Aß in the plasma and the brain levels of Aß were lighter affected. Nevertheless, this dosing regimen did modulate tau phosphorylation and glycogen synthase kinase 3 activities in the brain, suggesting that the targeting of circulating Aß may be therapeutically relevant in AD. FROM THE CLINICAL EDITOR: Intraperitoneal injection of small unilamellar vesicles containing phosphatidic acid or cardiolipin significantly reduced the amount of amyloid-beta (Aß) peptide in the plasma in a rodent model. Brain levels of Aß were also affected - although to a lesser extent - suggesting that targeting of circulating Aß may be therapeutically relevant of Alzheimer's disease.

Increasing levels of cardiolipin differentially influence packing of phospholipids found in the mitochondrial inner membrane.

It is essential to understand the role of cardiolipin (CL) in mitochondrial membrane organization given that changes in CL levels contribute to mitochondrial dysfunction in type II diabetes, ischemia-reperfusion injury, heart failure, breast cancer, and aging. Specifically, there are contradictory data on how CL influences the molecular packing of membrane phospholipids. Therefore, we determined how increasing levels of heart CL impacted molecular packing in large unilamellar vesicles, modeling heterogeneous lipid mixtures found within the mitochondrial inner membrane, using merocyanine (MC540) fluorescence. We broadly categorized lipid vesicles of equal mass as loosely packed, intermediate, and highly packed based on peak MC540 fluorescence intensity. CL had opposite effects on loosely versus highly packed vesicles. Exposure of loosely packed vesicles to increasing levels of CL dose-dependently increased membrane packing. In contrast, increasing amounts of CL in highly packed vesicles decreased the packing in a dose-dependent manner. In vesicles that were categorized as intermediate packing, CL had either no effect or decreased packing at select doses in a dose-independent manner. Altogether, the results aid in resolving some of the discrepant data by demonstrating that CL displays differential effects on membrane packing depending on the composition of the lipid environment. This has implications for mitochondrial protein activity in response to changing CL levels in microdomains of varying composition.

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations.

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.

In-vitro and in-vivo anti-cancer activity of a novel gemcitabine-cardiolipin conjugate.

Our objectives were to study the biological activity of a novel gemcitabine-cardiolipin conjugate (NEO6002) and compare that with gemcitabine. Cytotoxicity in vitro was determined against several gemcitabine-sensitive parental and gemcitabine-resistant cancer cell lines using the sulforhodamine B assay. The in vivo toxicity was examined by changes in body weight and hematologic indices of conventional mice. Immunodeficient SCID mice bearing P388 and BxPC-3 tumor xenografts were used to evaluate the in-vivo therapeutic efficacy. Both NEO6002 and gemcitabine showed pro-apoptotic and cytotoxic effects against all gemcitabine-sensitive cell lines tested. Unlike gemcitabine, the cytotoxicity of NEO6002 was independent of nucleoside transporter (NT) inhibitors, indicating a different internalization route of NEO6002. The conjugate demonstrated a favorable activity not only in ARAC-8C, a NT-deficient gemcitabine-resistant human leukemia cell line, but also in several other gemcitabine-resistant cell lines. At the in-vivo level, a comparative toxicity study showed a significant body weight loss and a decrease in white blood cell counts in gemcitabine-treated mice, whereas the influence of NEO6002 was mild. Treatment of NEO6002 at 27 micromol/kg increased the median survival of CD2F1 mice bearing P388 cells by up to 73%, while at the same doses and schedule of gemcitabine resulted in toxic deaths of all treated mice. At a dose of 18 micromol/kg, NEO6002 inhibited the growth of BxPC-3 xenografts by 52%, while only 32% of tumor inhibition was achieved with gemcitabine. We conclude that NEO6002 may be an effective chemotherapeutic agent with improved tolerability and can potentially circumvent NT-deficient, gemcitabine-resistant tumors.

IL-4 and T cells are required for the generation of IgG1 isotype antibodies against cardiolipin.

Infection with Mycobacterium tuberculosis induces Abs against a vast array of mycobacterial lipids and glycolipids. One of the most prominent lipid Ags recognized is cardiolipin (CL). The kinetics of the generation of anti-CL Abs during infection reveals that IgM titers to CL increase over time. Interestingly, at day 30 postinfection CL-specific IgG1 appears, an isotype usually dependent on T cell help. Using an immunization schedule with CL/anti-CL Ab complexes, which induces antiphospholipid syndrome in mice, we show that the generation of IgG1 to CL requires IL-4 and that optimal production is T cell dependent. IgG1 production to CL was impaired in nude (nu/nu) mice devoid in conventional T cells, but was not affected in mice deficient for either alphabeta TCR(+), gammadelta TCR(+), CD4(+), CD8(+), or NK1.1(+) T cells. We conclude that IgG1 production to CL depends on T cell help and IL-4, which can be provided by different T cell populations. This is the first report that IL-4 is indispensable for the induction of IgG1 Abs to lipid Ags.

Phospholipid-bound beta 2-glycoprotein I induces the production of anti-phospholipid antibodies.

Apoptotic-cell-bound beta2-glycoprotein I (beta2GPI), but not apoptotic cells or beta2GPI alone, can induce the production of anti-phospholipid (anti-PL) antibodies (Ab) in normal mice. Although it is presumed that beta2GPI binds to anionic phospholipid (PL) exposed on the apoptotic cell membrane, the precise nature of this complex and its immunogenicity is unclear. To address these issues, we investigated the structure and immunogenicity of human beta2GPI in the presence of different PL that may be expressed on the surface of apoptotic cells. BALB/c mice were immunized intravenously (iv) with beta2GPI in the presence of cardiolipin (CL), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylcholine (PC), or PS/PC (25%/75%) vesicles. Cardiolipin+beta2GPI induced the highest levels of anti-beta2GPI and anti-CL IgG Ab and lupus anticoagulant (LA) activity, while beta2GPI with PC or PS/PC vesicles produced no significant anti-PL Ab. PS+beta2GPI was somewhat immunogenic, but less so than PG+beta2GPI. beta2GPI was immunogenic in the presence of native (CL(N)), but not hydrogenated (CL(H)), CL. Circular dichroism analysis demonstrated that the structure of beta2GPI was altered specifically by interaction with CL(N), but not other anionic PL, including CL(H). Similarly, the structure of CL(N)was affected by interaction with beta2GPI, as detected by(31)P nuclear magnetic resonance. These findings demonstrate that beta2GPI complexed with CL(N)is structurally altered, highly immunogenic, and induces the production of IgG anti-PL Ab. Furthermore, the structural modification and the generation of immunogenic epitopes on beta2GPI upon interaction with CL(N)require the presence of unsaturated fatty acid chains, suggesting a role for oxidation in this process.

Replacement for 30-milliliter flat-bottomed, glass-stoppered, round bottles used in VDRL antigen preparation.

When the flat-bottomed, glass-stoppered, round bottle traditionally used to make VDRL antigen was discontinued, an appropriate substitute was needed. Although many laboratories have switched to one of the other nontreponemal tests for syphilis serology screening, the VDRL test remains the only approved procedure for testing spinal fluids of patients with possible neurosyphilis. We tested 25-ml glass-stoppered, convex-bottomed Erlenmeyer flasks to determine if these could be used as appropriate substitutes. We tested 52 reactive sera and 54 nonreactive sera by using one reference antigen prepared in the traditional flat-bottomed bottles and five antigens prepared in the Erlenmeyer flasks. Results with all serum samples were comparable. We also tested two lots of a commercial antigen plus an additional lot of reference antigen. Again there was no difference in the reactivity of the antigens. Therefore, we conclude that 25-ml glass-stoppered Erlenmeyer flasks can be used as an appropriate substitute for glass-stoppered, flat-bottomed, round glass bottles in the making of VDRL antigen.

Augmentation and suppression of release of tumor necrosis factor from macrophages by negatively charged phospholipids.

We recently reported that some lipid species of cell membranes and lipoproteins induced the growth of peripheral macrophages. In this study, the effects of phospholipids on tumor necrosis factor (TNF)-releasing activity of macrophages were examined. Ten to 20 micrograms/ml of cardiolipin, which is a suboptimal concentration for macrophage growth-stimulation, augmented macrophage TNF release triggered by lipopolysaccharide (LPS) in vitro. This priming effect appeared with 1 day of preincubation and was still potent on day 3, whereas the priming effect of interferon-gamma (IFN-gamma) peaked at 3 h and then gradually decreased. In contrast, a high concentration of cardiolipin (40 micrograms/ml) which is optimal for the induction of macrophage growth, completely suppressed LPS-triggered TNF release from not only untreated macrophages but also IFN-gamma-primed macrophages. The suppressive effect was potent even with 3 h preincubation, was still potent on day 3, and was not abolished by indomethacin. Cardiolipin had scarcely any effect on the triggering activity of LPS. Similar augmentative and suppressive activities were observed in peroxidized phosphatidylserine, which is also highly active in inducing macrophage growth, but was not found in native phosphatidylserine, which is less active in inducing macrophage growth, but was not found in native phosphatidylserine, which is less active, nor in phosphatidylcholine, which is an inactive species toward macrophage growth. These results suggest that lipids may be important endogenous factors in regulating both activation and growth states of peripheral macrophages.

Immunotoxicity of multiple dosing regimens of free doxorubicin and doxorubicin entrapped in cardiolipin liposomes.

We have shown that doxorubicin entrapped in cardiolipin liposomes retain antitumour efficacy in mice but had diminished cardiac uptake and cardiotoxicity. Such liposomes are preferentially taken up by spleen. In a previous study we showed that a single dose of liposomal doxorubicin is not more toxic than free doxorubicin with regard to immunologic parameters including generation of cytotoxicity for histocompatibility alloantigens and mitogenic responsiveness. In the present study, we have explored clinically relevant multiple dosing at weekly intervals, 2, 3, or 4 times. Again, despite splenic localization of liposomal doxorubicin, the depressive effect on these immunological parameters is not greater than the effect of free drug, and, in addition, the damage is repaired earlier.