Antiphospholipid autoantibodies as blood biomarkers for detection of early stage Alzheimer's disease.

A robust blood biomarker is urgently needed to facilitate early prognosis for those at risk for Alzheimer's disease (AD). Redox reactive autoantibodies (R-RAAs) represent a novel family of antibodies detectable only after exposure of cerebrospinal fluid (CSF), serum, plasma or immunoglobulin fractions to oxidizing agents. We have previously reported that R-RAA antiphospholipid antibodies (aPLs) are significantly decreased in the CSF and serum of AD patients compared to healthy controls (HCs). These studies were extended to measure R-RAA aPL in serum samples obtained from Alzheimer's Disease Neuroimaging Initiative (ADNI). Serum samples from the ADNI-1 diagnostic groups from participants with mild cognitive impairment (MCI), AD and HCs were blinded for diagnosis and analyzed for R-RAA aPL by ELISA. Demographics, cognitive data at baseline and yearly follow-up were subsequently provided by ADNI after posting assay data. As observed in CSF, R-RAA aPL in sera from the AD diagnostic group were significantly reduced compared to HC. However, the sera from the MCI population contained significantly elevated R-RAA aPL activity relative to AD patient and/or HC sera. The data presented in this study indicate that R-RAA aPL show promise as a blood biomarker for detection of early AD, and warrant replication in a larger sample. Longitudinal testing of an individual for increases in R-RAA aPL over a previously established baseline may serve as a useful early sero-epidemiologic blood biomarker for individuals at risk for developing dementia of the Alzheimer's type.

Phosphatidylethanolamine at the luminal endothelial surface--implications for hemostasis and thrombotic autoimmunity.

OBJECTIVE: Accumulating evidence suggests that phosphatidylethanolamine (PE) is physically present at the luminal endothelial surface, where it tentatively functions as a critical anticoagulant. The goal of the current investigation was 3-fold: to characterize the distribution profile of PE at the luminal endothelial surface; to examine the immunoreactivity to the vascular endothelium by anti-PE (aPE) sera from patients presenting with thrombosis; and to discuss the potential mechanism of PE upregulation by endothelial cells. METHODS: The rat aortic arch was selected as major conduit vessel under significant hemodynamic burden. The presence of PE and the antigenic profile of aPE sera at the luminal endothelial surface were examined using duramycin as a PEbinding probe and immunohistochemistry. Phosphatidylethanolamine upregulation at endothelial cell surface was investigated using cultured monolayer subject to laminar shear stress or thrombin treatment. RESULTS: High levels of PE were detected at the luminal endothelial surface of aortic flow dividers, the ascending aorta, and the outer curvature of the aortic arch. The antigenic profiles of aPE sera, which are highly associated with elevated thrombotic risks in patients, are consistent with PE distribution along the endothelial surface. Finally, PE is upregulated at the surface of cultured endothelial cells in response to luminal shear stress but not thrombin. CONCLUSIONS: The current data describe the physical distribution of vascular PE at the blood-endothelium interface. The luminal PE presents a vulnerability to anti-PE autoimmunity and is consistent with the association between aPE and elevated risk for idiopathic thrombosis.

Autoantibody potential of cancer therapeutic monoclonal antibodies.

We and others have reported that multiple autoantibodies are unmasked in human polyclonal antibody preparations after exposure to physiological oxidizing agents (hemin) or electromotive force. We now have asked if oxidation unmasks autoantibody reactivities in monoclonal antibodies (mAb). To do this, we have studied 9 FDA approved mAb used therapeutically, including 4 chimeric, 4 humanized and 1 chemically modified chimeric Fab that were exposed to the physiological oxidizing agent hemin at 36 degrees C for 20 hr. These mAb were studied for autoantibody activity to phospholipids and DNA before and after oxidation with hemin and found to develop autoantibody activities after oxidation, while retaining their original specificity as measured by mAb anti-glycophorin A binding of erythrocytes, CD 19 binding to B lymphocytes and anti-HLA-A29 binding to A29-positive lymphocytes. The finding that certain mAb have the potential to unmask autoantibody activities as a consequence of exposure to physiological redox reactions in vitro gives pause to our present understanding of the immunological basis of tolerance and concern for potential autoimmune side effects in patients receiving mAb for diagnosis or treatment.

Redox-reactive antiphospholipid antibody differences between serum from Alzheimer's patients and age-matched controls.

There is no universally acceptable inclusive laboratory biomarker for the diagnosis and staging of neurodegenerative diseases, for example, Alzheimer's. There is an abnormal increase of oxidative stress in the central nervous system (CNS) of Alzheimer's patients that causes oxidation of proteins, lipids and DNA. We have published that the antiphospholipid (aPL) autoantibodies that are members of the redox-reactive autoantibody (R-RAA) family, are significantly decreased or absent in the cerebrospinal fluids of autopsy-confirmed Alzheimer's disease (AD) patients. Because of the known elevation of oxidation-induced damage in the CNS and the abnormal enrichment of redox reactive metals in postmortem AD brains, we questioned if the R-RAA in the blood of AD patients might also show a departure from the normal aPL levels. We compared 16 AD serum samples to 17 serum samples, from age-matched volunteer blood donors. Each serum was tested before and after oxidation for four aPL specificities by using an in-house ELISA. Comparisons between the AD and normal populations revealed highly significant differences. In-sample Fisher's linear discriminate analysis found a sensitivity of 88% and a specificity of 94%. In-sample Classification and Regression Tree analysis (CART) found a sensitivity of 84% and a specificity of 100%. This study is the first to indicate that blood tests for R-RAA may be used as a laboratory criterion for an Alzheimer's diagnosis.

Redox-reactive autoantibodies: biochemistry, characterization, and specificities.

Oxidation-reduction (redox) reactions can "unmask" autoantibody activity in blood and other body fluids from normal, healthy individuals. These "unmasked" autoantibodies are similar if not identical to autoantibodies associated with autoimmune diseases. The agents responsible for this unmasking are physiological oxidants such as hemin and likely other naturally occurring molecules in the body that contain transitional metals available for participation in redox reactions. Laboratory comparisons between oxidized and non oxidized IgG fail to show differences to account for the oxidation-induced alteration of antibody specifics. The autoantibodies unmasked by redox reactivities represent a growing list of specificities, many that are responsible for modulating and/or regulating intracellular functions. In contrast, alloantibodies, such as anti-HLA antibodies, do not exhibit susceptibility to oxidation-induced autoantibody alterations, suggesting differences in the amino acids responsible for forming the complementarity determining regions of these respective antibody molecules. We have proposed that such reversible oxidative conversions of antibody reactivities represent a heretofore undiscovered, but an evolutionary-conserved, resource of innate humoral immunity destined to maintain an immunological homeostasis.

Anti-phospholipid antibodies in cerebrospinal fluid but not serum from a boy with psychosis.

A 12-year-old African American boy with mental retardation and Asperger's disorder presented with acute psychosis. Antiphospholipid antibody testing with enzyme-linked immunosorbent assay showed increased levels of immunoglobulin G anticardiolipin antibodies in the cerebrospinal fluid, but not in the serum. Although antiphospholipid antibodies have been reported in the serum of patients with thrombotic and neurologic disorders, there are only a few reports of these antibodies in cerebrospinal fluid. This finding is consistent with a recent report of antiphospholipid antibodies found in the cerebrospinal fluid of adults with acute psychosis.

Antiphospholipid antibodies in blood and cerebrospinal fluids of patients with psychosis.

Antiphospholipid antibodies (aPL) have been reported in the cerebrospinal fluids (CSF) of neurology patients but no CSF studies with psychiatric patients exist. We tested serum from 100 hospitalized psychotic patients having hallucinations and/or delusions for aPL. Patients with positive serum aPL findings were asked to submit CSF for aPL testing. Five CSF samples had aPL specificities not found in the patient's serum suggesting the possibility of intrathecal synthesis. Specificity and isotype discordance between CSF and blood aPL in these psychiatric patients implicates a central nervous system independent autoimmune process that may have an underlying association with the pathophysiology of their diseases.

Redox-reactive autoantibodies in cerebrospinal fluids.

The oxidative stress associated with increased transitional metal concentrations in neurodegenerative diseases served as the impetus for our testing the status of redox-reactive autoantibodies in the cerebrospinal fluids from autopsy-confirmed Alzheimer's patients. Here we describe a novel family of autoantibodies capable of recognizing autoantigens subsequent to in vitro oxidation-reduction (redox) reactions in the blood and spinal fluids of all normal individuals tested. Redox autoantibodies are not detected in conventional immunoassays, thereby differentiating them from natural and hidden autoantibodies described by others. Whereas blood-borne redox autoantibodies can be IgG, IgM, and/or IgA, in spinal fluid the antibody isotype is limited to IgG. Autoantibodies in certain patients are reversible and disappear when exposed to oxidizing agents in vitro. One mechanism proposed to modulate the autoantibody unmasking-masking reactions relies upon redox-driven nitrosylation of an amino acid-containing aromatic ring, which is found within the complementarity-determining regions (CDR) of the antibodies' antigen-binding sites. The evolutionary persistence of this novel autoantibody family indicates that they are important for immunological homeostasis and suggests that they perform necessary physiological functions. The dramatic difference in the presence of such antibodies in normal versus Alzheimer's disease (AD) suggests an important immune system dysfunction in AD.

Redox-reactive autoantibodies in Alzheimer's patients' cerebrospinal fluids: preliminary studies.

Oxidation of cerebrospinal fluid (CSF) causes differential unmasking of autoantibodies in control CSF vs. that obtained from postmortem CSF samples from autopsy confirmed Alzheimer's disease (AD) cases. This study demonstrates that normal CSF from both living patients and from non-demented autopsy cases contains redox-reactive autoantibodies with specificities that include antiphospholipid antibodies (aPL). In contrast, CSF from autopsy confirmed AD subjects contained little or no redox-reactive aPL autoantibodies. Tests using an in vitro rat synaptosome model showed that the oxidized CSF autoantibodies from a normal individual can cause ERK1/2 phosphorylation at a level consistent with reports of pathogenic changes found in brain tissues from AD patients. The decrease or absence of redox-reactive antibodies in CSF from Alzheimer's patients suggests that these antibodies may have been previously unmasked by the oxidative conditions that exist in the CNS in AD patients. These unmasked autoantibodies could then bind to neuronal tissues and possibly participate in the initial cascade leading to the dementia in Alzheimer's. To our knowledge, this is the first description of resident autoantibodies with the potential to cause brain cell damage documented in CSF without a breech in the blood-brain barrier. The untimely and inappropriate physiological unmasking of these redox-reactive autoantibodies in AD patients CSF may represent a valuable biomarker for diagnosis and progression of this and perhaps other neurodegenerative diseases which also have oxidative stress components. These novel autoantibody observations may stimulate thoughts about additional therapeutic approaches and warrant similar studies for other neurodegenerative diseases.

Fatal hemorrhagic diathesis associated with mild factor IX deficiency in pl/J mice.

Surgical implantation of devices into the abdomen of PL/J mice was associated with fatal hemorrhage at 9 to 11 d after surgery. Coagulation profiles were evaluated to determine the underlying cause of this effect. The mean activated partial thromboplastin time (aPTT) of untreated PL/J mice was significantly higher than that of BALB/cByJ and C57BL/6J strains. The addition of human plasmas deficient in factors VIII, XI, or XII, prekallikrein, or high molecular-weight kininogen corrected the elevated aPTT of PL/J mice, but correction did not occur when factor IX-deficient human plasma was added. When compared to an assigned factor IX activity of 100% for pooled plasma from BALB/cByJ mice, C57BL/6J and PL/J mice revealed percent activities of 67% and 16%, respectively. PL/J mice could represent a new model for the study of pathogenesis and therapy of mild factor IX deficiency that is expressed and becomes clinically apparent secondary to major surgery.

IgG-antiphospholipid antibodies in follicular fluid of IVF-ET patients are related to low fertilization rate of their oocytes.

PROBLEM: Patients undergoing in vitro fertilization and embryo transfer (IVF-ET) failures show an increased incidence of antiphospholipid antibodies (aPL) in their blood. The physiological manifestations of aPL in this patient group are nonetheless controversial. Pathological effects of aPL on embryos in vitro have been documented. We questioned whether aPL if found in follicular fluids (FFs) could result in embryonic damage. METHOD OF STUDY: Blood from 44 patients with three or more IVF-ET failures were tested by enzyme-linked immunosorbent assays (ELISA) for the presence of immunoglobulin (Ig)G, IgM and IgA aPL. Both the 29 aPL-positive and 15 aPL-negative patients gave permission for FF collection during their next IVF-ET attempt for additional aPL determinations. RESULTS: Patients with no aPL in their blood, had no aPL in their FFs. Patients with IgG and/or IgM aPL in their blood had IgG but not IgM in their respective FFs. CONCLUSIONS: The presence of IgG aPL in FFs and increased infertility length were significantly related to lower fertilization rates, independently. Follicular fluid IgG aPL appears as a risk factor in association with successful IVF-ET outcomes.

Redox-reactive autoantibodies: detection and physiological relevance.

We recently described a hitherto unrecognized family of autoantibodies that become unmasked (detectable) subsequent to oxidation-reduction (redox) reactions. These masked redox-reactive autoantibodies are not detectable by using conventional immunoassays. Additional experimentation has demonstrated that autoantibodies in the blood of patients with autoimmune diseases can be masked (become undetectable) by exposure to oxidizing agents. Simultaneous masking and unmasking of different autoantibodies in a given patient's serum or plasma is evidence that immune complexes are not the source of redox-reactive autoantibodies. We propose that a mechanism responsible for unmasking-masking antibody specificities requires nitrosylation of tyrosine residues in the hypervariable or complementarity determining regions of the antibodies' antigen-binding sites. Other laboratories, selected by us for their respective expertise, have studied our redox-reacted and control serum and/or antibody preparations and have found an expanding array of autoantibody specificities. The gathering data suggest that certain autoimmune diseases may involve redox disorders rather than a failure to deplete, suppress, tolerate or divert self-directed B cell activity. The persistence and fluctuation of redox-reactive autoantibodies suggest that they are manifestations of an as yet undefined natural selective pressure on the evolution of the immunological system. We propose that they are the "contrivances" suggested by Paul Ehrlich more than a hundred years ago, and that these antibodies perform important physiological and pathophysiological functions.

Autoantibodies unmasked by redox reactions.

Blood from healthy donors was found to contain a variety of autoantibodies after being cultured overnight in commercial blood culture bottles. Paradoxically some autoantibodies in the blood of patients with autoimmune diseases were no longer detectable when similarly cultured. By a process of elimination it was revealed that hemin was responsible for the conversion of antibody-negative blood to antibody-positive blood, as well as for the conversion of antibody-positive blood to antibody-negative blood. By using a purified component system of hemin and immunoglobulin, and an iron-free congener of hemin, we have shown that the appearance and/or disappearance of antibodies occur uniquely in the presence of coordinated iron and in the absence of antioxidants such as vitamin C. The oxidation-reduction (redox) reactions used to demonstrate the appearance and disappearance of autoantibodies have been performed in vitro. Whether the reactions we have observed have a parallel in vivo remains to be determined. It is clear from our findings that normal individuals have immunoglobulin molecules which can exhibit autoantibody binding capacity, and that at least some autoantibodies in autoimmune individuals can have their binding capacity masked by exercise of a natural redox system. These preliminary findings need further investigation but they already hint that some of our apparently well based views on autoimmunity might be expanded to include a role for masked and unmasked autoantibodies.

The appearance and disappearance of antiphospholipid autoantibodies subsequent to oxidation--reduction reactions.

The mechanisms that cause the appearance of autoantibodies are not understood. Compared to normal antibody production, factors responsible for autoantibody synthesis are more complex; they are thought to disrupt the normal mechanisms proposed to eliminate or down-regulate self-antibodies or to interfere with anti-self-receptor editing. Data presented show that autoantibodies exist in the blood of all normal individuals. The autoantibodies appear after simple oxidation-reduction (redox) reactions and react by ELISA, immunofluorescence, flow cytometry, Western blots, and in lupus anticoagulant (LA) assays. Antiphospholipid antibody (aPL) specificities detected after redox are cardiolipin (aCL), antiphosphatidylserine (aPS), antiphosphatidylethanolamine (aPE), antiphosphatidylcholine (aPC), and LA. These antibody activities were confirmed in several outside laboratories. The aPL isotypes detected in ELISA are plasma protein-dependent and include IgG, IgA, and IgM. Oxidizing agents tested to date include hemin, KMnO4, and NaIO4. Furthermore, aPL appear after exposure to direct current (DC)-mediated electromotive force. Alternating current (AC) is ineffective. Commercial IvIg preparations, also a source of IgG autoantibodies, provide a less complex milieu than plasma or serum for studying the biology of aPL redox-mediated mechanisms. Inhibition of hemin-mediated IvIg aPL conversion can be achieved by the addition of antioxidants, e.g., ascorbic acid, hemopexin, apotransferrin, and by addition of normal plasma or serum. Remarkably, the aPL specificities in the blood of autoimmunity patients disappear subsequent to application of redox reactions. These data document the hitherto unknown existence of redox-reactive autoantibodies in all normal individuals. The evolutionary persistence of these redox-sensitive antibodies raises interesting possibilities about their potentially beneficial role in immunological homeostasis.

Antiphospholipid antibodies: discovery, definitions, detection and disease.

Antiphospholipid antibodies (aPL) are immunoglobulins of IgG, IgM and IgA isotypes that target phospholipid (PL) and/or PL-binding plasma proteins. Detection of aPL in the laboratory is done currently by both immunoassays and functional coagulation tests. Convention defines aPL specificity in immunoassays according to the particular PL substrate present, for example aPS represents antiphosphatidylserine antibodies. This may be technically incorrect inasmuch as a particular PL may be responsible for binding and highly concentrating a specific plasma protein, the latter then becomes the target for the aPL. The binding of beta(2)GP-I (apolipoprotein H) to the negatively charged PL, cardiolipin (CL) provides a good example of this circumstance. In contrast, aPL which specifically prolong coagulation times in in vitro are called lupus anticoagulants (LA). The precise PL target(s) of the aPL responsible for LA activities are unknown and often debated. The persistent finding of aPL in patients in association with abnormal blood clotting and a myriad of neurological, obstetrical and rheumatic disorders often compounded by autoimmune diseases has led to an established clinical diagnosis termed antiphospholipid syndrome (APS). The common denominator for these APS patients is the presence of circulating aPL on two or more occasions and the observation of events attributable to abnormal or accelerated blood clotting somewhere in vivo. The purpose of this review is to collect, collate, and consolidate information concerning aPL.

Frequency and specificities of antiphospholipid antibodies (aPL) in volunteer blood donors.

In the State of Indiana, blood donors are screened by a written questionnaire prior to donation to identify potential exposures to infectious diseases and to assess medications. The potential donor is not asked about aPL-related events. Animal experiments have shown, however, that passive transfer of aPL can produce aPL-associated pathology in the recipient. We determined the incidence of antiphospholipid antibodies (aPL) in 775 volunteer blood donors in Indiana. Tubing segments containing 2 ml of anticoagulated blood were obtained from donor units. The average donor age was 43 years (range 17-82); 45% were female. Our in-house aPL ELISA tested for IgG, IgA and IgM to cardiolipin (aCL), phosphatidylserine (aPS), phosphatidylethanolamine (aPE) and phosphatidylcholine (aPC). The plasmas were tested with and without supplemental phospholipid (PL)-binding plasma proteins from adult bovine plasma (ABP). A total of 24 tests were performed for each donor. Normal ranges were determined for 98% of the donors by calculating multiples of the means (MoM) of OD values for each antigen-isotype combination. No decimals were used, all MoMs were rounded up to the next whole number. An additional two MoMs were added to the calculated cutoff values to eliminate borderline positive values. Results revealed that 63 (8.1 %) donors had positive findings for one or more aPL. The relatively high number of aPL-positive individuals reflects the observation that different donors were often in the highest 2% of each antigen-isotype combination tested. The aPL specificities, isotypes, and PL-binding protein dependence are summarized in this report.