Testing for antiphospholipid antibody (aPL) specificities in retrospective "normal" cerebral spinal fluid (CSF).

Antiphospholipid antibodies (aPL) have been found in the blood of patients with systemic and neurological disease. The rare reports of aPL in cerebral spinal fluid (CSF) have been limited mostly to IgG and IgM anticardiolipin (aCL). Our published finding of IgA aPE in the CSF of a young stroke victim prompted us to establish "normal" CSF aPL values for a panel of aPL, which included aCL, antiphosphatidylserine (aPS), antiphosphatidylethanolamine (aPE) and antiphosphatidylcholine (aPC). CSF samples were tested by ELISA for IgG, IgM and IgA aPL. In addition, the CSF samples were tested for activity in the presence and absence of phospholipid (PL) binding plasma-proteins. A total of 24 data points were obtained for each CSF sample. We tested 59 CSF samples obtained from 59 patients who were undergoing evaluation for systemic or neurologic diseases. All CSF samples had normal protein, glucose and cell counts. Ten of the 59 CSF samples (17%) had elevated aPL optical density (OD) values an order of magnitude higher than the other 49 CSF samples for one or more aPL specificity and/or isotype. One CSF sample had both PL-binding protein dependent and independent IgG aPE activity. Another CSF sample showed both IgG aPE and aPC reactivity. The remaining eight CSF samples showed single aPL findings; IgG aPE (5), IgG aPC (1), IgG aCL (1) and IgM aPC (1). Seven of 10 patients with elevated CSF values were females. As expected, most "normal" aPL OD values were substantially lower in CSF than those we have reported in blood samples from volunteer blood donors.

Antiphospholipid antibodies and renal transplantation: a risk assessment.

Data from 110 transplanted patients show that the presence of antiphospholipid antibodies (aPL) at the time of transplantation is an important risk factor for early renal allograft failure. Sera were tested for IgG, IgM and IgA to CL, PS, PE and PC. Haemodialysis patients had a significantly higher incidence of aPL compared to patients who did not receive haemodialysis (P = 0.0171). aPL-positive ESRD patients on peritoneal dialysis (CAPD) or who had never received haemodialysis were at maximal risk; 100% failure (P = 0.0022). aPL-positive patients receiving haemodialysis were not at such risk. Biopsy findings from the failed kidneys show abundant fibrin deposition in the microvasculature. Serial blood samples from transplanted patients showed aPL titres to decrease immediately after transplant and increase after removal of the failed graft, indicating that aPL specifically target the allografts. To confirm this, we were able to isolate aPL from a failed graft after transplant nephrectomy. Ninety-seven per cent of the aPL-positive patients' historic pre-transplant serum samples demonstrated the presence of the same aPL specificity detected in the final crossmatch sera. The exposure to heparin during haemodialysis suggested to us that heparin reduces the risk of clotting in aPL positive transplant candidates. To lessen the risk of graft loss in aPL positive kidney transplant patients (including CAPD), subcutaneous heparin was administered peri- and post-operatively. To date, none of the heparin-treated aPL-positive transplanted patients suffered an early graft loss. Further, they experienced fewer rejection episodes requiring biopsy and thus are prescribed less steroid therapy than patients not treated with heparin.

Immune responses to native beta(2)-glycoprotein I in patients with systemic lupus erythematosus and the antiphospholipid syndrome.

OBJECTIVE: To identify HLA class II associations with anti beta(2)-glycoprotein I (beta2GPI) antibodies in a cohort of Caucasian patients with systemic lupus erythematosus (SLE) and to determine whether these HLA genotypes act as restriction elements for lymphocyte proliferation to native human beta2GPI in vitro. METHODS: Anti-beta2GPI antibodies were detected in patient sera using enzyme-linked immunosorbent assays (ELISAs). HLA class II alleles (DRB1, DQB1) were determined by polymerase chain reaction-based DNA genotyping. In vitro peripheral blood mononuclear cell (PBMC) responses to native human beta2GPI were measured in a 7-day proliferation assay. RESULTS: We identified three groups of Caucasian SLE patients using these ELISAs. In group 1, 16 out of 18 SLE patients (89%) with anti-beta2GPI antibodies were positive for HLA-DRB1*0401/4/8, DR11 or DRB1*1302 (P=0.001 vs controls) compared with 23 out of 53 patients (43%) in group 2 with anti-cardiolipin antibodies only, 57 out of 151 patients (38%) in group 3 (SLE patients without anticardiolipin antibodies) and 109 out of 225 controls (48%). Fourteen patients with anti-beta2GPI antibodies had greater median stimulation indices to beta2GPI in vitro compared with the 15 controls studied (P=0.04). CONCLUSION: The HLA class II and PBMC proliferation data suggest that beta2GPI may be both a T- and B-cell autoantigen in SLE.

Left ventricular assist system recipients exposed to bovine thrombin preparations have a higher frequency of antiphospholipid antibodies than nonexposed recipients.

After left ventricular assist system (LVAS) placement, recipients often develop antiphospholipid antibodies (aPL) that are associated with thrombosis. Fibrin glue containing a bovine thrombin preparation is used routinely in LVAS placement surgery. We investigated whether exposure to the thrombin preparation is responsible for stimulating aPL development in LVAS recipients. Pre-LVAS and weekly post-LVAS sera from six fibrin glue-exposed LVAS recipients and five nonexposed recipients were tested by enzyme-linked immunosorbent assay for IgG, IgA, and IgM anti-phosphatidylserine (aPS), anticardiolipin (aCL), anti-phosphatidylethanolamine (aPE), and anti-phosphatidylcholine (aPC). Fibrin glue exposed recipients developed a significantly greater number of aPL than the nonexposed recipients (24 vs. 8; p = 0.0069). In particular, a higher frequency of IgG aCL (6/6 vs. 1/5; p = 0.015) and IgG aPE (4/6 vs. 0/5; p = 0.045) were noted. Exposure to the bovine thrombin component of fibrin glue seems to stimulate aPL development in LVAS recipients.

Antiphospholipid antibodies. Risk assessments for solid organ, bone marrow, and tissue transplantation.

The literature pertaining to transplantation of solid organs, bone marrow, and other tissues in aPL-positive patients has been reviewed. The effects that aPL have relative to BMT are altogether different than those ascribed to solid organs and tissues. By definition, the transplantation of allogeneic bone marrow serves to reconstitute the recipient with a completely new and genetically different repertoire of antibody-producing cells. Previously aPL-positive bone marrow recipients become aPL-negative subsequent to transplantation assuming that the marrow donor is aPL-negative. These observations are the basis for contemporary experimental approaches to curing certain autoimmune diseases with BMT. Similarly, it would follow that an aPL-negative patient provided cells from an aPL-positive donor could become aPL-positive and suffer increased risk for thrombosis. From the data provided in most of the non-bone marrow publications, the presence of aPL should be considered a grave risk factor for any potential solid organ or tissue transplant candidate. Peritoneal dialysis patients seem to be at maximal risk. Given the serious emotional and economic impact of irreversible thrombotic loss suffered by organ transplant recipients, these factors alone should justify the modest expense of pretransplant aPL screening. In the United States, the average cost of losing a kidney transplant to aPL-associated thrombosis was estimated from 1996 data to be $82,000. The cost of losing a heart or liver is measured not only in dollars but often in the patient's life. The encouraging news, however, is that once aPL are identified before transplantation, prophylactic anticoagulation seems to be capable of forestalling untoward aPL-associated allograft events. Clearly, much remains to be discovered in exploring the pathobiologic characteristics of aPL in the laboratory as well as in neutralizing their procoagulant effects at the bedside.

Report of a new DRB1*13 allele: DRB1*1336.

This brief communication describes the characterization of a new allele, DRB1*1336.

Decreased suppression of antibody-dependent cellular cytotoxicity by seminal plasma in unexplained infertility.

OBJECTIVE: To determine whether seminal plasma (SP) from unexplained infertile males has different suppressive activity on antibody-dependent cellular cytotoxicity (ADCC) than SP from fertile males or SP from males of couples with known infertility factor. DESIGN: Comparative clinical/experimental study. SETTING: In vitro fertilization program in a university hospital and a hospital research laboratory. PATIENT(S): A total of 245 SP samples from 174 infertile and 16 fertile couples were compared. INTERVENTION(S): SP suppression of ADCC was measured by using human 51chromium-labeled red blood cells (RBC), sensitized with IgG-rabbit-anti-human-RBC as targets and peripheral blood lymphocytes as effector cells. MAIN OUTCOME MEASURE(S): Suppressive activity of each sample was determined by calculating 51Cr-release in the presence and absence of SP. RESULT(S): When analyzed with respect to sperm number, motility, and morphology, suppressive activities of samples with normal semen analyses (n = 142) were significantly higher (x = 37% +/- 14%) than suppressive activities of abnormal samples (n = 103; x = 32% +/- 13%). There was no strong correlation of suppressive activity to other semen parameters. Within the andrologically normal males, SP from the unexplained infertile couples (n = 15) showed significantly lower suppressive activity (x = 24% +/- 11%) compared with the SP from fertile males (n = 16; x = 35% +/- 13%) and from couples with female infertility factor (n = 65; x = 39% +/- 14%). CONCLUSION(S): Loss of suppressive activity is associated with unexplained infertility, even in male patients who previously were considered normal by traditional methods of semen analysis.

Spectrum of antiphospholipid antibodies (aPL) in patients with cerebrovascular disease.

BACKGROUND: The association of stroke and antiphospholipid antibodies (aPL) other than anticardiolipin antibodies (aCL) is not well documented. OBJECTIVE: To report the distribution of aCL, antiphosphatidylethanolamine (aPE), and antiphosphatidylserine (aPS) aPL among patients with symptomatic cerebrovascular disease evaluated by our Stroke Service at Indiana University Hospital from January 1997 to November 1999. METHODS: We retrospectively reviewed medical records from 1997 to 1999 at Indiana University Hospital for all patients with symptomatic cerebrovascular disease using the International Statistical Classification of Diseases, 9th Revision, (ICD-9) codes. We identified patients with elevated titers of aPL. Sera from these patients were obtained within the first 30 days of the index event. We included only those patients for whom the serum samples were tested in a single laboratory by an in-house enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) immunoglobulin A (IgA) and immunoglobulin M (IgM) aCL, aPE, and aPS. We examined the clinical presentation, stroke risk factors, associated rheumatologic disorders, and distribution of aPL specificity and isotype. RESULTS: Thirty-four of 185 patients, 26 women (76%), with a mean age of 46 years, and 8 men (24%) with a mean age of 46 years, had aPL. Nine patients had transient ischemic attacks (TIA), 25 suffered strokes, 23 had ischemic infarcts, and 2 had hemorrhagic infarcts (1 had a superior sagittal sinus thrombosis with bilateral hemispheric hemorrhagic infarcts, and one had bilateral hemorrhagic infarcts associated with systemic lupus erythematosus [SLE]). Six patients had SLE. The most common stroke risk factors were cigarette smoking (38%) and arterial hypertension (26%). Approximately two thirds (60%) of patients had a single positive aPL finding: aPE in 35%, aCL in 18%, and aPS in 6%. Multiple specificities were seen in 40%. IgA was the only aPL antibody isotype detected in 26% of the patients, IgG was the lone isotype in 24%, and IgM alone in 12%. Multiple aPL isotypes were detected in 38% of patients. Five patients (15%) presented with aPE IgA as the exclusive aPL. CONCLUSION: In our series, aPE was the most frequent finding in stroke patients who were suspected to have an associated aPL syndrome. These specific types of aPL may be present relatively often in stroke patients and are often not assessed. Further studies are needed to determine how specific these aPL are in stroke versus other acute illnesses and versus healthy controls, and how these aPL are associated with stroke risk.

Risk of early renal allograft failure is increased for patients with antiphospholipid antibodies.

Renal allograft thrombosis can cause transplant failure. Because antiphospholipid antibodies (aPA) are associated with thrombosis, we investigated pretransplant sera from patients with early renal allograft failure to determine if aPA were present. Fifty-six final cross-match (FxM) sera from patients whose transplant failed within 16 days were compared to FxM sera from the next sequential transplant patients. The sera were tested for IgG, IgM, and IgA antibodies to cardiolipin, phosphatidylserine, and phosphatidylethanolamine. aPA were identified in 57% of FxM sera from patients with early non-function versus 35% of FxM sera from patients with functioning grafts (P = 0.02). Historical sera from 11 aPA-positive patients contained aPA up to 18 months prior to transplantation. Since aPA were present in historical sera, testing for aPA can identify certain patients at risk for early allograft failure. The involvement of aPA in early allograft loss is supported by studies demonstrating aPA recovery from an explanted failed transplant.

Henoch-Schönlein purpura and stroke: antiphosphatidylethanolamine antibody in CSF and serum.

A 15-year-old girl with features of Henoch-Schönlein purpura and brain infarct had a transient IgA antiphosphatidylethanolamine antibody (aPE) in her serum and CSF that disappeared 5 months after presentation. Serum aPE is known to be associated with thrombotic events. The authors found no aPE in the CSF of two control individuals or in the serum of two patients with active Henoch-Schönlein purpura without neurologic involvement. The patient may represent a variant of antiphospholipid antibody syndrome.

Anti-phosphatidylethanolamine (aPE) antibodies: a survey.

Reports of anti-phosphatidylethanolamine antibodies (aPE) with similar or identical pathogenic associations as those described for anticardiolipin (aCL) and anti-phosphatidylserine (aPS) are found in the literature. In some instances, aPE is the sole antiphospholipid antibody (aPL) observed. Lupus anticoagulant antibodies (LA) appear to represent a subset of aPE as hexagonal phase PE can specifically inhibit the LA ability to prolong clotting times. As documented for aPL to the negatively charged phospholipids (PL), plasma proteins have been implicated for a positive aPE signal in the ELISA. Other aPE appear independent of known PE-binding plasma proteins. Among the described PE-binding proteins are high and low molecular weight kininogens (HMWK and LMWK) and the HMWK-binding proteins, factor XI and prekallikrein. Recently prothrombin has been added to this list. The reports of aPE published since 1986 are reviewed and discussed in this presentation.

Antiphospholipid antibodies are a risk factor for early renal allograft failure.

BACKGROUND: Biopsy specimens of transplanted kidneys that fail to function reveal cellular infiltrates, infarcts, and thrombi. Because antibodies to phospholipids (aPA) and/or phospholipid-binding proteins have been associated with thrombosis, we asked whether aPA are a risk factor for early allograft failure. METHODS: Final crossmatch sera from 56 patients with primary nonfunctioning renal allografts were tested for aPA. Serum from the next consecutive patient to undergo transplantation served as transplantation controls. Both groups were compared with aPA values obtained from testing 252 control individuals. The ELISA was designed to detect IgG, IgM, and IgA antibodies to phosphatidylserine, cardiolipin, and phosphatidylethanolamine. RESULTS: Patients were evaluated based upon the aPA ELISA findings. aPA were present in 57% of the patients with early nonfunction renal allografts and 35% of the patients with functioning grafts (P=0.0234). aPA in previously hemodialyzed patients did not predict allograft failure or success (P=0.3766). In contrast, all nonhemodialysis patients who had aPA at the time of transplantation experienced early allograft failure (P=0.0022). CONCLUSIONS: These data show that aPA are an important risk factor for early renal allograft failure. Furthermore, aPA-positive patients who have no history of hemodialysis are at the greatest risk. Pretransplantation aPA screening of renal transplant candidates forewarns of early graft failure and indicates which patients may benefit from anticoagulant therapy.

Increased incidence of antiphospholipid antibodies in left ventricular assist system recipients.

BACKGROUND: Antiphospholipid antibodies are associated with thrombosis. Because thromboembolic complications are often observed in recipients of a left ventricular assist system, we questioned if antiphospholipid antibodies were present in these patients. We report results from 10 patients who received a Novacor left ventricular assist system. METHODS: Serum samples were collected before left ventricular assist system placement and weekly thereafter until discharge after cardiac transplantation. Samples were tested for IgG, IgA, and IgM antiphosphatidylserine, anticardiolipin, and antiphosphatidylethanolamine using an enzyme-linked immunosorbent assay. RESULTS: Development of phospholipid-binding plasma protein-dependent antiphospholipid antibodies was observed in 9 of the 10 patients. Before placement of the assist system, 3 patients had IgG antiphospholipid antibodies, and 9 were positive after placement. None had IgA antiphospholipid antibodies before placement, whereas 5 seroconverted for IgA after placement. One patient had IgM antiphospholipid antibodies before placement, and 1 additional patient became positive after placement. In patients with a preexisting antibody, increased titers and additional specificities developed subsequent to placement. CONCLUSIONS: All but 1 patient showed development of phospholipid-binding plasma protein-dependent antiphospholipid antibodies after left ventricular assist system placement.

DQB1*0202 and the new DQB1*0203 allele: a fourth pair of DQB1 alleles differing only at codon 57.

Amino acid 57 of DQ beta chains is of functional importance as it influences peptide binding, is part of B and T cell epitopes, and is associated with susceptibility and resistance to insulin-dependent diabetes mellitus and humoral immunodeficiencies. Polymorphism of codon 57 is conserved in primates and in HLA class II B genes implying that balancing selection operates on this residue. Previously, three DQB1 allele pairs have been described, that only differ at residue 57. In an African-American Black individual with the HLA phenotype A23.30;B58,63;Cw6;DR18,12;DR52;DQ5,2, we found a fourth example of this dimorphism: the new DQB1*0203 allele, that was identical to DQB1*0202 except for codon 57, which encodes aspartic acid and alanine respectively in the two alleles. The class II haplotype carrying the new allele was deduced to be DRB1*0302,DRB3*0101,DQA1*05011,DQB1*0203.

Identification of structural mutations in the fifth domain of apolipoprotein H (beta 2-glycoprotein I) which affect phospholipid binding.

Apolipoprotein H (apoH), also known as beta 2-glycoprotein-I, is considered to be a cofactor for the binding of certain antiphospholipid autoantibodies to negatively charged phospholipids. Genetically determined structural abnormalities in the lipid binding domain(s) of apoH can affect its ability to bind lipid and consequently the production of the autoantibodies. In this study we have identified two common structural mutations at codons 316 and 306 in the fifth domain of apoH which rendered apoH unable to bind to negatively charged phosphatidylserine (PS). The missense mutation at codon 316 (TGG --> TCG) replaces Trp316 with Ser316 and disrupts the integrity of four highly conserved hydrophobic amino acids sequence at positions 313-316, which is a potential protein-lipid hydrophobic interaction site. The missense mutation at codon 306 (TGC --> GGC) involves the substitution of Cys306 by Gly306 which causes the disruption of a disulfide bond between Cys281 and Cys306 and affects the normal configuration of the fifth domain of apoH that appears to be critical for clustering positively charged amino acids along with four hydrophobic amino acids sequence. ApoH from the two homozygotes (Ser316/Ser316) and all seven compound heterozygotes (Ser316/Gly306) failed to bind to PS; all heterozygotes at one or the other sites and wild type showed normal PS binding. These data indicate that the fifth domain of apoH harbors the lipid binding region. An estimated 2 million Caucasians in the United States, who are compound heterozygotes for the two mutations, may be precluded from producing apoH-dependent antiphospholipid autoantibodies.

Analyses of HLA-DQ alleles in recurrent spontaneous abortion (RSA) couples.

PROBLEM: The influence of HLA sharing on pregnancy outcome is controversial. In renal transplantation, HLA-DQB1 donor-recipient mismatches have been shown beneficial for long-term transplant success. Since pregnancy is defined as Nature's allograft, we investigated the relevance of HLA-DQ mismatching in normal reproducing couples compared to couples experiencing RSA. METHOD: Unexplained RSA couples referred to our laboratory for immunological testing were classified by immunological findings and obstetrical history. Primary RSA couples shared > or = 2 HLA-A, B, or DR antigens, had no cytotoxic anti-paternal antibodies, and no gestation beyond 20 weeks. Secondary RSA couples had cytotoxic anti-paternal antibodies and RSA after a live birth. HLA-DQA1 and DQB1 alleles were identified by PCR-SSP. RESULTS: No differences in DQA1 and DQB1 mismatch were observed among RSA patients and controls. DQA1-DQB1 haplotype mismatches were not different among the three groups of couples. CONCLUSIONS: In contrast to renal transplant, HLA-DQ incompatibility did not differ among RSA couples compared with successful reproducing couples.