16(th) IHIW: global distribution of extended HLA haplotypes.

This report describes the project to identify the global distribution of extended HLA haplotypes, a component of 16th International HLA and Immunogenetics Workshop (IHIW), and summarizes the initial analyses of data collected. The project aims to investigate extended HLA haplotypes, compare their distribution among different populations, assess their frequency in hematopoietic stem cell unrelated donor registries and initiate an international family studies database and DNA repository to be made publicly available. HLA haplotypes compiled in immunogenetics laboratories during the evaluation of transplant candidates and related potential donors were analysed. Haplotypes were determined using the pedigree analysis tool publicly available from the National Marrow Donor Program (NMDP) website. Nineteen laboratories from 10 countries (11 laboratories from North America, five from Asia, two from Latin America and one from Australia) contributed data on a total of 1719 families comprised of 7474 individuals. We identified 10393 HLA haplotypes, of which 1682 haplotypes included high-resolution typing at HLA-A, B, C, DRB1 and DQB1 loci. We also present haplotypes containing MICA and other HLA loci and haplotypes containing rare alleles seen in these families. The project will be extended through the 17th IHIW, and investigators interested in joining the project may communicate with the first author.

Anisotropy of resistivity in hexagonal late transition metals and their alloys.

Transport properties of hexagonal transition metals Co, Ru, and Os at finite temperatures are studied by means of ab initio electronic structure techniques and the Kubo linear response theory. An alloy analogy model for a quantitative treatment of the electrical conductivities due to temperature-induced lattice vibrations (phonons) and spin fluctuations is applied with focus on anisotropy induced by the hexagonal structure. The resistivity anisotropy in Co is found opposite to that in Ru and Os, in agreement with existing experimental data. This result is ascribed to the strong itinerant ferromagnetism of Co which leads to profound differences in the electronic structure and conductivities in the majority and minority spin channels. A similar sensitivity to spin polarization is predicted for the anisotropy of residual resistivity in random hexagonal Co-rich Co-Ni and Co-Ni-Fe alloys.

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.

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.

Changes in beta 2-glycoprotein I antigenicity induced by phospholipid binding.

beta 2-glycoprotein I (beta 2GPI) or apolipoprotein H has been described as a necessary cofactor for antiphospholipid antibody (aPA) binding in ELISA. Some investigators disagree with the beta 2-GPI requirement whereas data from other laboratories indicate that beta 2GPI, not phospholipid (PL), is the antigen for aPA. To investigate the cofactor we have produced three IgG1 monoclonal antibodies (mAb) to human beta 2GPI; 3G9, 1B4 and 3D11. Western blot analyses showed the mAb to bind human beta 2GPI (40 kDa), but no reactivity was observed with adult or fetal bovine sera. In contrast, rabbit anti-beta 2GPI reacted with both human and bovine sera. None of the mAb reacted with phosphatidylserine (PS) or cardiolipin (CL) by ELISA. There were no significant differences in ELISA binding to purified beta 2GPI when the mAb were adjusted to the same concentration. mAb 3G9 and 1B4 gave stronger signals in ELISA after beta 2GPI bound to PS; the increase for 3G9 was significantly greater than for 1B4 (p < 0.002). mAb 3D11 was unique inasmuch as it failed to recognize beta 2PGI bound to PS. In comparison, the rabbit anti-beta 2GPI was unaffected by PS-beta 2GPI binding. These observations indicate that the mAb recognize three distinct epitopes on beta 2GPI. The data suggest that beta 2GPI undergoes conformational changes subsequent to binding PL. Our findings are consistent with the hypothesis that aPA recognize a beta 2GPI neotope formed subsequent to binding PL.

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.

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.

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.

Antiphospholipid antibodies in heart transplant recipients.

Antiphospholipid antibodies (aPA) are found in patients with systemic lupus erythematosus, aPA syndrome, myocardial infarction, and stroke. The presence of aPA may predict recurrent events in certain victims of heart attack and stroke. Blood samples from 105 cardiac transplant recipients (81 men, 24 women) were tested by enzyme-linked immunosorbent assay (ELISA) for the presence of IgG, IgM, and IgA aPA to phosphatidylserine (PS), cardiolipin (CL), and phosphatidylethanolamine (PE). Patients' ages ranged from 17 to 70 years (mean 51 years). Collection times ranged from Day 1 to 9 years post transplant (mean 36 months). All patients received triple immunosuppressive therapy. We report our aPA ELISA results in multiples of the normal median (MoM) of the OD405 values calculated for 252 healthy individuals. A positive MoM is greater than the MoM that encompasses 95% of the controls; for example, above 3 MoM is considered positive for IgG anti-CL, IgA anti-PS, and CL. Above 4 MoM is positive for IgG anti-PS and PE and IgM anti-PS and CL. Thirty-nine patients had IgG anti-PS (range 4.1-14.8 MoM), 63 had IgG anti-CL (3.1-9.4 MoM), 7 had IgM anti-PS (4.1-12.1 MoM), 1 had IgM anti-CL (14 MoM), 47 had IgA anti-PS (3.1-13.1 MoM), and 58 had IgA anti-CL (3.1-11.5 MoM). In our patient population, the incidence of IgG and/or IgA aPA was significantly higher (p < 0.001) than IgM. Few patients showed specificity for either PS, CL, or PE, and many were positive with more than one antibody isotype. Because aPA were evaluated in these patients, we investigated pretransplant serum samples which are available from 79 of the 105 recipients, and found aPA in 52 of 79 (66%) patients before transplantation. Longitudinal studies were done in three patients: two had increasing IgA aPA, beginning on Days 13 and 26 post transplant, whereas the third patient showed an increased aPA on Day 8 but a decrease on Day 23. Studies are in progress to determine whether a correlation exists between the presence of aPA, immunocytochemical (biopsy) findings, and clinical outcome.

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.

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.

Formulation of a stable parenteral product; Clonidine Hydrochloride Injection.

Clonidine Hydrochloride Injection (Duraclon) is a clear, colorless, preservative-free, pyrogen free, aqueous solution of clonidine hydrochloride. The indication for this product is for use as an adjunct in pain management, administered epidurally, when opiates are insufficient. The drug formulation was evaluated under both normal and stress conditions in the preformulation/formulation studies. The list of studies conducted includes a light sensitivity study, an oxygen sensitivity study, a pH/stability study, a stopper compatibility evaluation, a freeze-thaw study, and a stability study. Samples from the light, oxygen, pH/stability, and stability studies were evaluated for color, visual clarity, pH, potency, and chromatographic purity. Samples from the freeze-thaw study were evaluated for all of the above except chromatographic purity. The results for these studies demonstrate the stability of the product as formulated. The pH of this unbuffered product was consistently within the acceptance criteria. The product remained clear and colorless for the duration of each study. The values obtained for the potency and chromatographic purity assays showed no evidence of degradation. The reasons for the lack of degradation can be found in the molecular structure of the drug substance and the formulation of the drug product. Since the molecular structure is that of a Schiff base, it is theoretically possible, although difficult, to cleave the molecule. A catalyst would be required, and none of the possible catalysts are present in the formulation. The molecule could also be cleaved upon exposure to light, and the evidence indicates that the molecule does interact with light. This interaction is not to the degree, however, that product stability is affected. The formulation contains only the active drug substance and sodium chloride in water for injection with a pH of approximately 6. Although the product is unbuffered, the influence of the stoppers and glass vials upon the formulation pH was minimal. In addition, the stopper compatibility of the product is enhanced by the absence of chelating agents, preservatives, acids, and bases. Since the dilute concentrations of both the active and excipient are well below their solubility limits, no solubility related issues would be expected upon freezing and subsequent thawing. Clonidine Hydrochloride Injection, as formulated, does not require protection from light, oxygen, or freezing. The product shows acceptable stability within the pH range, and the rubber closure is compatible with the product. Real time stability data combined with statistical projections support a 36-month expiration date.

SNP identification, linkage and radiation hybrid mapping of the porcine lamin A/C (LMNA) gene to chromosome 4q.

The lamins are components of nuclear lamina and they have a profound influence on nuclear structure and functions. They are encoded by three genes, LMNA, LMNB1 and LMNB2. A genomic fragment of the porcine LMNA gene (822 bp; from exons 7 to 9) was amplified by polymerase chain reaction and comparatively sequenced. Four single nucleotide polymorphisms (SNPs) were identified in intronic sequences: G162A, G208A, T367G and C618T. The SNPs are within the restriction sites for enzymes Bsh1236I, HpaII, AluI and Bsh1236I respectively. Allele frequencies at SNPs G208A, T367G and C618T were determined by using eight pig breeds. Linkage analysis in the Hohenheim Meishan x Piétrain family placed the LMNA gene in the chromosome 4q linkage group, between MEF2D and GBA (MEF2D - 3.0 cM - LMNA - 0.2 cM - GBA). In radiation hybrid mapping LMNA was most significantly linked to SW270 on chromosome 4 (39 cR; LOD = 7.86). The LMNA gene is located in the quantitative trait loci region for some carcass traits on chromosome 4q.