L. Gaur

Glycoprotein Ia (alpha2 integrin) is a subunit of the heterodimeric membrane complex (GPIa/IIa) that mediates platelet adhesion to collagen. Several nucleotide sequence variations of GPIa have been described. A nucleotide 1648 G/A dimorphism that leads to a Glu/Lys substitution at amino acid 505 is responsible for the human platelet antigen system, HPA-5. Recently, two other linked GPIa nucleotide dimorphisms involving codons Phe224 and Thr246 were identified: a C/T substitution at nucleotide 807 and a G/A substitution at nucleotide 873(1). Using restriction enzyme digestion of amplified GPIa genomic DNA fragments (PCR-RFLP) to distinguish genotypes, we have determined the allele frequencies of the GPIa 807C/T and Glu/Lys505 dimorphisms in three North American populations, and a panel of non-human primates. Our results indicate a genetic relationship between the 807C/T and Glu/Lys505 dimorphisms that leads to an evolutionary model of GPIa isoforms.

The Consortium for Blood Group Genes is a worldwide organization whose goal is to have a vehicle to interact, establish guidelines, operate a proficiency program, and provide education for laboratories involved in DNA and RNA testing for the prediction of blood group, platelet, and neutrophil antigens. Immunohematology 2009;25:75–78.

SummaryGlycoprotein Ia (α2 integrin) is a subunit of the heterodimeric membrane complex (GPIa/IIa) that mediates platelet adhesion to collagen. Several nucleotide sequence variations of GPIa have been described. A nucleotide 1648 G/A dimorphism that leads to a Glu/Lys substitution at amino acid 505 is responsible for the human platelet antigen system, HPA-5. Recently, two other linked GPIa nucleotide dimorphisms involving codons Phe224 and Thr246 were identified: a C/T substitution at nucleotide 807 and a G/A substitution at nucleotide 873 (1). Using restriction enzyme digestion of amplified GPIa genomic DNA fragments (PCR-RFLP) to distinguish genotypes, we have determined the allele frequencies of the GPIa 807C/T and Glu/Lys505 dimorphisms in three North American populations, and a panel of non-human primates. Our results indicate a genetic relationship between the 807C/T and Glu/Lys505 dimorphisms that leads to an evolutionary model of GPIa isoforms.

Three leucoreduction filters were evaluated - when used alone or combined with centrifuge leucoreduction (C-LR) - to prevent alloimmune platelet refractoriness in a dog platelet transfusion model. Donor platelet-rich plasma (PRP) or buffy coat (BC) platelets were either filter leucoreduced (F-LR) or F-LR/C-LR, (51) Cr radiolabelled and transfused. Weekly transfusions were given for up to 8 weeks or until platelet refractoriness. Recipients who accepted treated transfusions were then given non-leucoreduced (non-LR) platelets to determine whether donor-specific tolerance had been induced. Acceptance of F-LR PRP transfusions ranged from 29% to 66%. F-LR/C-LR transfusions prepared from PRP were accepted by 92%, from BC by 63% and from pooled PRP by 75% of recipients (p=NS); overall acceptance rate of F-LR/C-LR transfusions was 83%. Tolerance to subsequent non-LR transfusions occurred in 45% of the F-LR-/C-LR-accepting recipients unrelated to DR-B compatibility between donors and recipients (P = 0·18). In a dog platelet transfusion model, acceptance of donor platelets required combining F-LR with C-LR as apparently each process removes different immunizing WBCs.

The monoclonal antibody 1F5 recognizes human B-cell surface protein CD20 and can activate resting B cells; with this antibody we found CD20 to be a 35/37-kDa non-disulfide-linked protein. The protein has a pI of 7.5-8.0 and is phosphorylated in B-cell lines, tonsillar B cells, and peripheral blood B cells. Both CD20 surface expression and phosphorylation are increased on buoyant tonsillar B cells activated in vivo. Because phorbol 12-myristate 13-acetate (PMA) supports the activation signal initiated by monoclonal antibody 1F5, we studied the effect of PMA on CD20 expression. After brief incubation with mitogenic levels of PMA, the number of dense tonsillar B cells positive for CD20 protein transiently decreased. Paradoxically, the cells remaining positive had more surface CD20 than did control cells, and these remaining surface CD20 molecules were hyperphosphorylated. Furthermore, PMA not only induced phosphorylation of CD20 protein on Raji cells but also increased the internalizat...

In utero hematopoietic stem cell transplantation could potentially be used to treat many genetic diseases but rarely has been successful except in severe immunodeficiency syndromes. We explored two ways to potentially increase chimerism in a nonhuman primate model: (a) fetal immune suppression at the time of transplantation and (b) postnatal donor stem cell infusion. Fetal Macaca nemestrina treated with a combination of the corticosteroid betamethasone (0.9 mg/kg) and rabbit thymoglobulin (ATG; 50 mg/kg) were given haploidentical, marrow-derived, CD34+ -enriched donor cells. Animals treated postnatally received either donor-derived T cell-depleted or CD34+ -enriched marrow cells. Chimerism was determined by traditional and real-time polymerase chain reaction from marrow, marrow progenitors, peripheral blood, and mature peripheral blood progeny. After birth, the level of chimerism in the progenitor population was higher in the immune-suppressed animals relative to controls (11.3% +/- 2.7% and 5.1% +/- 1.5%, respectively; p = .057). Chimerism remained significantly elevated in both marrow (p = .02) and fluorescence-activated cell sorted and purified CD34+ cells (p = .01) relative to control animals at > or = 14 months of age. Peripheral blood chimerism, both at birth and long term, was similar in immune-suppressed and control animals. In the animals receiving postnatal donor cell infusions, there was an initial increase in progenitor chimerism; however, at 6-month follow-up, the level of chimerism was unchanged from the preinfusion values. Although fetal immune suppression was associated with an increase in the level of progenitor and marrow chimerism, the total contribution to marrow and the levels of mature donor progeny in the peripheral blood remained low. The level of long-term chimerism also was not improved with postnatal donor cell infusion.

In utero hematopoietic stem cell transplantation is a therapeutic procedure that could potentially cure many developmental diseases affecting the immune and hematopoietic systems. In most clinical and experimental settings of fetal hematopoietic transplantation the level of donor cell engraftment has been low, suggesting that even in the fetus there are significant barriers to donor cell engraftment. In postnatal hematopoietic transplantation donor cells obtained from mobilized peripheral blood engraft more rapidly than cells derived from marrow. We tested the hypothesis that use of donor hematopoietic/stem cells obtained from mobilized peripheral blood would improve engraftment and the level of chimerism after in utero transplantation in non-human primates. Despite the potential competitive advantage from the use of CD 34(+) from mobilized peripheral blood, the level of chimerism was not appreciably different from a group of animals receiving marrow-derived CD 34(+) donor cells. Based on these results, it is unlikely that this single change in cell source will influence the clinical outcome of fetal hematopoietic transplantation.

Membranous nephropathy is the most common cause of idiopathic nephrotic syndrome in adults. Some cases are associated with autoimmune disorders (for example, systemic lupus erythematosus), infections (for example, hepatitis B), drugs (for example, gold or penicillamine), heavy metals (for example, mercury), organic solvents, or cancer, but most cases are idiopathic [1, 2]. We describe four patients who developed membranous nephropathy after exposure to toxic concentrations of formaldehyde commonly found in homes with certain types of insulation or cabinetry. Formaldehyde may induce membranous nephropathy in genetically susceptible persons. Methods Four patients with membranous nephropathy were referred to us with possible exposure to toxic concentrations of formaldehyde. Air-borne formaldehyde concentrations were measured as recommended by the National Institute of Occupational Safety and Health [3]. Standard microcytotoxicity assays were used to serologically type histocompatibility leukocyte (HLA) antigens. We identified HLA-DR sequences from enzymatically amplified DNA by a modified reverse blot assay [4] that used sequence-specific oligonucleotide probes. Results All four patients presented with the nephrotic syndrome. Biopsy specimens showed typical membranous nephropathy (Table 1). All had worked with materials that contained formaldehyde or had moved into new homes that had substantial amounts of formaldehyde in the insulation or cabinetry. In three of four cases, the patient or a family member had a history of eye or upper respiratory problems related to the formaldehyde fumes. No patient had evidence for any known cause of membranous nephropathy. However, atypical features included mild eosinophilia (two patients), low-positive antinuclear antibody levels with a negative anti-double-stranded DNA antibody test result (two patients), and borderline-low C4 (one patient) or CH50 (one patient). Histocompatibility leukocyte antigen typing showed the A1 B8 DR3 haplotype in two patients and the B18 DR2 haplotype in one patient. These haplotypes have been associated with membranous nephropathy in Europe and Japan, respectively [1]. However, sequence-specific oligotyping showed that all four patients inherited an HLA-DR allele at either the DR 1 or DR 5 locus, which encodes an identical amino acid sequence (amino acids 35-39) in the peptide-binding region. Two of the patients are described below. Table 1. Characteristics of Four Patients with Membranous Nephropathy and Formaldehyde Exposure* Patient 1 A 39-year-old male contractor built a cottage using wood paneling that was bonded with phenol formaldehyde resin. The floor consisted of urea formaldehyde-bonded particle board. While working, the patient noted a strong odor that was associated with burning eyes and headaches. Eight months later, hypertension and the nephrotic syndrome developed (serum creatinine level, 1.4 mg/dL; urine protein concentration, 9 g/d). A renal biopsy specimen showed membranous nephropathy. Patient 2 The cottage built by patient 1 was intermittently occupied by a family of four in the year after construction. A strong odor was again noted, and several family members and visitors reported burning eyes, nausea, vomiting, diarrhea, or upper respiratory tract symptoms when in the house. After a year of intermittent exposure, the 13-year-old son developed the nephrotic syndrome (serum creatinine level, 0.6 mg/dL; urine protein concentration, 6 g/d). A renal biopsy specimen showed membranous nephropathy. Discussion Formaldehyde is commonly used to bond particle board, fiber board, and plywood and was also used in the 1960s and 1970s for building insulation [5-7]. Whereas concentrations of formaldehyde in urban air are usually less than 0.01 parts per million (ppm), concentrations of formaldehyde in mobile homes or homes constructed with particle board are often increased (0.1 to 2 ppm or greater) [5-7]. Concentrations of 0.05 ppm may be associated with a strong odor and upper respiratory or eye irritation in exposed persons [5, 6]. Formaldehyde exposure may also be associated with dermatitis, urticaria, and possibly sinonasal cancer [5, 6]. Our patients developed membranous nephropathy after documented exposure to formaldehyde at a concentration of at least 0.1 ppm. A similar case has also been reported, although that patient was exposed to organic solvents as well [2]. Although specific epidemiologic studies are necessary to confirm this association, two recent studies showed a twofold increase in chronic nephritis as a cause of death in persons involved in the use [7] or manufacture [8] of formaldehyde. Membranous nephropathy is probably an autoimmune disease induced by an antibody directed against an antigen on the glomerular epithelial cell [1]. Formaldehyde exposure may induce autoimmune reactions, including antinuclear autoantibody production and autoimmune hemolytic anemia [5, 9]. Because many people exposed to formaldehyde do…

A common concern with many autoimmune diseases of unknown etiology is the extent to which tissue T-lymphocyte infiltrates, versus a nonspecific infiltrate, reflect a response to the causative agent. Lyme arthritis can histologically resemble rheumatoid synovitis, particularly the prominent infiltration by T lymphocytes. This has raised speculation about whether Lyme synovitis represents an ongoing response to the causative spirochete, Borrelia burgdorferi, or rather a self-perpetuating autoimmune reaction. In an effort to answer this question, the present study examined the repertoire of infiltrating T cells in synovial fluid from nine Lyme arthritis patients, before and after stimulation with B. burgdorferi. Using a highly sensitive and consistent quantitative PCR technique, a comparison of the T-cell antigen receptor (TCR) beta-chain variable (Vbeta) repertoires of the peripheral blood and synovial fluid showed a statistically significant increase in expression of Vbeta2 and Vbeta...