'Non-criteria antiphospholipid antibodies': bridging the gap between seropositive and seronegative antiphospholipid syndrome.

OBJECTIVE: We aimed to analyse the prevalence of non-criteria anti-phospholipid (aPL) antibodies and their role in the diagnosis, treatment and prognosis in a cohort of patients with clinical features consistent with a diagnosis of antiphospholipid syndrome (APS), but persistently negative for criteria aPL - anti-cardiolipin antibodies (aCL), anti-ß2-glycoprotein I antibodies (aß2-GPI) and lupus anticoagulant (LA) - named seronegative APS (SN-APS). METHODS: Sera from SN-APS patients were tested for aCL by TLC-immunostaining, anti-vimentin/cardiolipin (aVim/CL) and anti-phosphatidylserine/prothrombin (anti-PS/PT) by ELISA. Control groups of our study were APS patients and healthy controls. RESULTS: We enrolled 114 consecutive SN-APS patients, 69 (60.5%) resulted positive for at least one non-criteria test in two occasions 12 weeks apart. Among the persistently positive patients to these tests, 97% resulted positive for aCL by TLC-immunostaining, 52.3% for aVim/CL and 17.4% for aPS/PT. SN-APS patients with double positivity (aCL by TLC-immunostaining and aVim/CL) showed a likelihood positive ratio of 8 to present mixed thrombotic and obstetrical features. Among SN-APS patients tested positive, after the therapeutic changes, three cases of recurrent thrombosis were observed [median follow-up 41 months (IQR 39.5)]. Twenty pregnancies were recorded in 17 SN-APS patients after the detection of unconventional aPL and 12 of them (60%) experienced a good outcome under conventional treatment for APS. CONCLUSIONS: This is the largest monocentric study demonstrating that aCL tested by TLC-immunostaining and aVim/CL can detect aPL positivity in SN-APS. It may encourage clinicians to monitor and provide adequate targeted therapy, which improve SN-APS prognosis.

A woman with systemic lupus erythematosus, multiple pregnancy complications, and cerebral infarction who was only positive for phosphatidylserine-dependent antiprothrombin antibodies.

A woman with systemic lupus erythematosus (SLE) had a history of two abortions before the 10th week, two foetal deaths with normal morphology, and one premature before the 34th week with early-onset hypertensive disorder of pregnancy (HDP) and placental dysfunction. Although she did not have any conventional antiphospholipid antibodies (aPLs), antiphospholipid syndrome (APS) was strongly suspected based on her obstetric history and renal biopsy findings consistent with aPL-associated nephropathy (APLN). Eventually, she was found to be positive for phosphatidylserine-dependent antiprothrombin antibodies (aPS/PTs). A healthy baby was born with anticoagulation and intravenous immunoglobulin (IVIG) therapy during pregnancy. aPS/PT titres gradually increased after delivery. Cerebral infarction occurred at 9 years after birth. If APS is clinically suspected but the antibodies included in the classification criteria for APS are all negative, we should consider an association with unconventional aPLs and manage according to APS.

Immunization With Mycobacterium tuberculosis Antigens Encapsulated in Phosphatidylserine Liposomes Improves Protection Afforded by BCG.

Liposomes have been long considered as a vaccine delivery system but this technology remains to be fully utilized. Here, we describe a novel liposome-based subunit vaccine formulation for tuberculosis (TB) based on phosphatidylserine encapsulating two prominent TB antigens, Ag85B, and ESAT-6. We show that the resulting liposomes (Lipo-AE) are stable upon storage and can be readily taken up by antigen presenting cells and that their antigenic cargo is delivered and processed within endosomal cell compartments. The Lipo-AE vaccine formulation combined with the PolyIC adjuvant induced a mixed Th1/Th17-Th2 immune response to Ag85B but only a weak response to ESAT-6. An immunization regimen based on systemic delivery followed by mucosal boost with Lipo-AE resulted in the accumulation of resident memory T cells in the lungs. Most importantly though, when Lipo-AE vaccine candidate was administered to BCG-immunized mice subsequently challenged with low dose aerosol Mycobacterium tuberculosis, we observed a significant reduction of the bacterial load in the lungs and spleen compared to BCG alone. We therefore conclude that the immunization with mycobacterial antigens delivered by phosphatidylserine based liposomes in combination with Poly:IC adjuvant may represent a novel BCG boosting vaccination strategy.

Antibodies to phosphatidylserine/prothrombin (aPS/PT) enhanced the diagnostic performance in Chinese patients with antiphospholipid syndrome.

BACKGROUND: Increasing evidence has highlighted the role of non-criteria antiphospholipid antibodies (aPLs) as important supplements to the current criteria aPLs for the diagnosis of antiphospholipid syndrome (APS). In this retrospective study, we evaluated the clinical relevance of antibodies to phosphatidylserine/prothrombin (aPS/PT) in Chinese patients with APS. METHODS: A total of 441 subjects were tested, including 101 patients with primary APS (PAPS), 140 patients with secondary APS (SAPS), 161 disease controls (DCs) and 39 healthy controls (HCs). Serum IgG/IgM aPS/PT was determined by ELISA. RESULTS: The levels of IgG/IgM aPS/PT were significantly increased in patients with APS compared with DCs and HCs. IgG and IgM aPS/PT were present in 29.7% and 54.5% of PAPS, and 42.1% and 53.6% of SAPS, respectively. For diagnosis of APS, IgG aCL exhibited the highest positive likelihood ratio (LR+) of 21.60, followed by LA (13.84), IgG aß2GP1 (9.19) and IgG aPS/PT (8.49). aPS/PT was detected in 13.3% of seronegative PAPS patients and 31.3% of seronegative SAPS patients. LA exhibited the highest OR of 3.64 in identifying patients with thrombosis, followed by IgG aCL (OR, 2.63), IgG aPS/PT (OR, 2.55) and IgG aß2GP1 (OR, 2.33). LA and IgG aCL were correlated with both arterial and venous thrombosis, whereas IgG aPS/PT and IgG aß2GP1 correlated with venous or arterial thrombosis, respectively. CONCLUSIONS: Our findings suggest that the inclusion of IgG/IgM aPS/PT may enhance the diagnostic performance for APS, especially in those in whom APS is highly suspected, but conventional aPLs are repeatedly negative. In addition, IgG aPS/PT may contribute to identify patients at risk of thrombosis.

Takayasu Arteritis With Antiphosphatidylserine/Prothrombin Antibody-Positive Antiphospholipid Syndrome: Case Report and Literature Review.

A relationship between Takayasu arteritis (TA) and positive antiphospholipid antibody states has been pointed out, but patients with TA complicated with antiphospholipid antibody syndrome (APS) are rare. Here we report the case of a 17-year-old Japanese man diagnosed with TA based on pulselessness of the left brachial artery, discrepancy of blood pressure between the upper extremities, and arterial wall thickening and narrowing of artery in contrast computed tomography. He was also diagnosed with provisional APS based on a pulmonary infarction without narrowing of the pulmonary artery and positive antiphosphatidylserine/prothrombin antibody. The patient also had concurrent Crohn's disease (CD) based on histopathological findings, which may have been associated with TA. We started high-dose corticosteroid therapy and anticoagulation therapy, and his symptoms including fever, dizziness, chest pain, and lower-right uncomfortable abdomen improved.We reviewed 9 cases of TA with APS including our patient by conducting a PubMed search. Based on past reports, we considered the relationship among TA, APS, and CD.Clinicians should bear in mind that many etiologies can exist in 1 patient, and differential diagnoses are essential.

Pandemic influenza immunization in primary antiphospholipid syndrome (PAPS): a trigger to thrombosis and autoantibody production?

OBJECTIVE: The objective of this report is to conduct short- and long-term evaluation of a large panel of antiphospholipid (aPL) autoantibodies following pandemic influenza A/H1N1 non-adjuvant vaccine in primary antiphospholipid syndrome (PAPS) patients and healthy controls. METHODS: Forty-five PAPS and 33 healthy controls were immunized with H1N1 vaccine. They were prospectively assessed at pre-vaccination, and three weeks and six months after vaccination. aPL autoantibodies were determined by an enzyme-linked immunosorbent assay (ELISA) and included IgG/IgM: anticardiolipin (aCL), anti-beta2glycoprotein I (anti-ß2GPI); anti-annexin V, anti-phosphatidyl serine and anti-prothrombin antibodies. Anti-Sm was determined by ELISA and anti-double-stranded DNA (anti-dsDNA) by indirect immunofluorescence. Arterial and venous thrombosis were also clinically assessed. RESULTS: Pre-vaccination frequency of at least one aPL antibody was significantly higher in PAPS patients versus controls (58% vs. 24%, p = 0.0052). The overall frequencies of aPL antibody at pre-vaccination, and three weeks and six months after immunization remained unchanged in patients (p = 0.89) and controls (p = 0.83). The frequency of each antibody specificity for patients and controls remained stable in the three evaluated periods (p > 0.05). At three weeks, two PAPS patients developed a new but transient aPL antibody (aCL IgG and IgM), whereas at six months new aPL antibodies were observed in six PAPS patients and none had high titer. Anti-Sm and anti-dsDNA autoantibodies were uniformly negative and no new arterial or venous thrombosis were observed throughout the study. CONCLUSIONS: This is the first study to demonstrate that pandemic influenza vaccine in PAPS patients does not trigger short- and long-term thrombosis or a significant production of aPL-related antibodies (ClinicalTrials.gov, #NCT01151644).

Exposure to factor VIII protein in the presence of phosphatidylserine induces hypo-responsiveness toward factor VIII challenge in hemophilia A mice.

Administration of recombinant factor VIII (FVIII), an important co-factor in blood clotting cascade, elicits unwanted anti-FVIII antibodies in hemophilia A (HA) patients. Previously, FVIII associated with phosphatidylserine (PS) showed significant reduction in the anti-FVIII antibody response in HA mice. The reduction in the immune response to FVIII-PS could be due either to a failure of the immune system to recognize the antigen (i.e. immunological ignorance) or to an active induction of an antigen-specific nonresponsiveness (i.e. immunological tolerance). If it were a result of tolerance, one would predict that pre-exposure to FVIII-PS would render the mice hypo-responsive to a subsequent FVIII challenge. Here, we have demonstrated that naive HA mice that were pretreated with FVIII-PS showed a significantly reduced FVIII immune response to further challenge with native FVIII and that this decreased responsiveness could be adoptively transferred to other mice. An increase in number of FoxP3-expressing CD4(+) regulatory T-cells (Treg) was observed for the FVIII-PS-immunized group as compared with animals that received FVIII alone, suggesting the involvement of Treg in PS-mediated hypo-responsiveness. The PS-mediated reduction in antibody response was reversed by the co-administration of function-blocking anti-TGF-ß antibody with FVIII-PS. The decreased response to FVIII induced by FVIII-PS was determined to be antigen-specific because the immune response to another non-cross-reactive antigen (ovalbumin) was not altered. These results are consistent with the notion that FVIII-PS is tolerogenic and suggest that immunization with this tolerogenic form of the protein could be a useful treatment option to minimize immunogenicity of FVIII and other protein-based therapeutics.

Development of bavituximab, a vascular targeting agent with immune-modulating properties, for lung cancer treatment.

Bavituximab is a chimeric monoclonal antibody directed against the membrane phospholipid phosphatidylserine. Phosphatidylserine exposure is increased on endothelial cells and apoptotic cancer cells in solid tumors, allowing tumor-specific targeting of bavituximab. Bavituximab binding results in tumor vessel occlusion and enhanced antitumor immunity. Preclinical investigations have demonstrated efficacy as monotherapy and in combination with other modalities against multiple cancer types. Phase I clinical trials of bavituximab monotherapy and in combination with chemotherapy in adults with refractory solid tumors have been completed. Phase II trials of bavituximab in combination with chemotherapy for the first- and second-line treatment of advanced non-small-cell lung cancer are currently ongoing. This article summarizes the preclinical development and clinical experience with bavituximab in non-small-cell lung cancer.

Reassessment of autoreactivity of the broadly neutralizing HIV antibodies 4E10 and 2F5 and retrospective analysis of clinical safety data.

BACKGROUND: The broadly neutralizing recombinant human HIV-1 antibodies 4E10, 2F5 and Igh1b12 are reported to have autoreactive potential, which is significant for HIV-1 vaccine development and passive immunotherapy using these antibodies. OBJECTIVE: To investigate the clinical relevance of these findings in subjects receiving passive immunotherapy with these antibodies. METHODS: Four types of investigations were performed: (1) Investigation of clotting parameters in an ongoing clinical study with 4E10, 2F5 and 2G12. (2) Mixing experiments of pooled plasma with the same antibodies. (3) Retrospective analysis of serum from patients who received passive immunotherapy with 4E10, 2F5 and 2G12 either alone or in combination. (4) Assessment of clinical safety data obtained after 418 infusions with these antibodies. RESULTS: Standard clinical assays confirmed that 4E10 showed low-level cross-reactivity with cardiolipin, while previously reported cardiolipin cross-reactivity for 2F5 could not be confirmed. High serum titers of 4E10 induced mild prolongation of the activated partial thromboplastin time, which resolved with the wash out of 4E10. Neither 2F5 nor 2G12 affected coagulation. Repeated high-dose infusions of the monoclonal antibody combination were well tolerated with no incidence for thrombotic complications after 418 infusions in 39 subjects. CONCLUSIONS: Monoclonal antibody 4E10 but not 2F5 or 2G12 showed autoreactive binding specificities. Infusion of 4E10 resulted in transient low anticardiolipin titers. Although an increased thromboembolic risk cannot definitely be excluded, this risk appears to be low and likely depend on underlying disorders.

Plasma protein beta-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells.

A promising target on tumor vasculature is phosphatidylserine (PS), an anionic phospholipid that resides exclusively on the inner leaflet of the plasma membrane of resting mammalian cells. We have shown previously that PS becomes exposed on the surface of endothelial cells (EC) in solid tumors. To target PS on tumor vasculature, the murine monoclonal antibody 3G4 was developed. 3G4 localizes to tumor vasculature, inhibits tumor growth, and enhances anti-tumor chemotherapies without toxicity in mice. A chimeric version of 3G4 is in clinical trials. In this study, we investigated the basis for the interaction between 3G4 and EC with surface-exposed PS. We demonstrate that antibody binding to PS is dependent on plasma protein beta-2-glycoprotein 1 (beta2GP1). beta2GP1 is a 50-kDa glycoprotein that binds weakly to anionic phospholipids under physiological conditions. We show that 3G4 enhances binding of beta2GP1 to EC induced to expose PS. We also show that divalent 3G4-beta2GP1 complexes are required for enhanced binding, since 3G4 Fab' fragments do not bind EC with exposed PS. Finally, we demonstrate that an artificial dimeric beta2GP1 construct binds to EC with exposed PS in the absence of 3G4, confirming that antibody binding is mediated by dimerization of beta2GP1. Together, these data indicate that 3G4 targets tumor EC by increasing the avidity of beta2GP1 for anionic phospholipids through formation of multivalent 3G4-beta2GP1 complexes.

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.

Comparative assessment of phospholipid-binding antibodies indicates limited overlapping.

The implication of autoantibodies with anticoagulant and/or so-called antiphospholipid activities, under clinical circumstances with vascular obliteration, has led to the development of various types of tests allowing their detection. The most used tests involve investigation of the presence of an anticoagulant effect and of anticardiolipin IgG. It has also been proposed that the reactivity of patient samples toward other phospholipids or proteins be tested, but it remains difficult to appreciate which tests are redundant or complementary. Here we investigated whether the dissociation or association of anticoagulant and anticardiolipin correlated with specific ELISA reactivity to five other phospholipids: phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylcholine, and phosphatidylethanolamine. The study was performed with 70 samples, evenly partitioned as positive for either anticardiolipin antibodies or anticoagulant effect, or both. Our data clearly confirm that cardiolipin reactivity is an individual entity, likely to be complementary to other assays. Neither anticardiolipin nor anticoagulant levels correlated with assays investigating antibody levels toward the five other phospholipids, although higher mean levels were noted when both lupus anticoagulant and anticardiolipin antibodies are present. Individual patterns were evidenced in all groups. These data support the interest of current and further studies exploring the clinical relevance of individual reactivities to phospholipids.

Inhibition by cholesterylphosphorylserine of T-cell-mediated immune responses in mice.

The synthetic analogue of phosphatidylserine, cholesterylphosphorylserine (CPHS) inhibits T-cell-mediated immune responses in mice. Tested in cultured mouse spleen cells, CPHS inhibits concanavalin A-induced activation of DNA synthesis (IC50, 3.5 microM). Injected i.p. during the efferent phase, CPHS (25-100 mg/kg) inhibits the manifestations of delayed-type of hypersensitivity. The compound (25 mg/kg i.p., daily) reduces the acute graft-versus-host reaction when given for 5 days to donor mice before the isolation of spleen cells used for the inoculum. These data suggest that the addition of a phosphorylserine group to a steroid ring may produce immunoregulatory compounds.

The role of myelin P2 protein in the production of experimental allergic neuritis.

Myelin P2 protein has been proposed as the primary antigen in whole myelin-induced experimental allergic neuritis (EAN). We investigated the neuritogenic properties of P2 by sensitizing Lewis rats with complete Freund's adjuvant (CFA) containing P2, P2 plus phosphatidyl serine, or whole myelin containing an equivalent amount of P2. Animals were examined using a battery of clinical, electrophysiological, immunological, and morphological methods. Myelin-immunized rats developed the characteristic features of EAN. P2-sensitized rats developed a similar but much less intense disorder. When rats were sensitized with P2 in the presence of phosphatidyl serine, however, they developed radiculoneuropathy that was indistinguishable from myelin-induced EAN. Inoculation with phosphatidyl serine plus complete Freund's adjuvant or complete Freund's adjuvant alone had no detectable effect on peripheral nerves. These studies demonstrate that sensitization of rats with a single myelin antigen, P2 protein, is sufficient to induce the clinical, electrophysiological, and neuropathological features of EAN.

Induction of allergic neuritis in rhesus monkeys.

Experimental allergic neuritis (EAN) was induced in rhesus monkey (Macaca mulatta) following sensitization with rabbit nerve (PNS) myelin in complete Freund's adjuvant (CFA) or with bovine P2 protein complexed with phosphatidyl serine (P2-lipid) in CFA. The response of monkeys receiving PNS myelin in CFA differed from the previous studies where monkeys developed clinical signs of fatal EAN within 15-20 days following sensitization. The monkeys in this study (6) showed a much longer delay (40-114 days) before the appearance of severe clinical signs, and 4 of the 6 animals survived without further attack (1 year). Monkeys (4) injected with P2-lipid (2:1 ratio; w/w) developed severe clinical signs of EAN which was fetal in 3 cases. Peripheral lymphocytes from monkeys sensitized to the P2-lipid showed a much stronger mitogeneic response to P2 protein than those from the PNS myelin-sensitized monkeys. on quantitation of the circulating anti-P2 antibodies, the P2-sensitized monkeys generally had much titers than those sensitized with PNS myelin.