Anti-Galα1–3Gal antibodies (antiαGal Ab) are a major barrier to clinical xenotransplantation as they are believed to initiate both hyperacute and acute humoral rejection. Extracorporeal immunoadsorption (EIA) with αGal oligosaccharide columns temporarily depletes antiαGal Ab, but their return is ultimately associated with graft destruction. We therefore assessed the ability of two immunotoxins (IT) and two monoclonal antibodies (mAb) to deplete B and/or plasma cells both in vitro and in vivo in baboons, and to observe the rate of return of antiαGal Ab following EIA.
Differential expression of CD20 surface antigen in B-cell neoplasms at different sites is largely unknown. The number of CD20 antibodies bound per cell (CD20 ABC) in bone marrow (BM), peripheral blood (PB), and lymph node aspirate (LNA) samples from patients with B-cell chronic lymphocytic leukemia (B-CLL) or other B-cell disease was studied using quantitative flow cytometry. CD20 ABC differed significantly with the specimen type in B-CLL, being highest in PB (mean, 9,051) and lower in BM (mean, 4,067) and LNA (mean, 3,951). No difference in CD20 ABC between BM and PB samples was found in splenic lymphoma, mantle cell lymphoma, or follicular lymphoma. Also, we found a significant difference of CD20 ABC by type of disease: lowest in B-CLL; higher in splenic, follicular, and mantle cell lymphoma; and highest in hairy cell leukemia. The lower CD20 surface antigen levels in BM and LNA than in PB in B-CLL may have clinical relevance with regard to the efficacy of rituximab therapy.
Osteoarthritis (OA) is a leading cause of disability in Western society with multiple risk factors, including a complex genetic pattern. Identifying loci involved in the heredity of OA might lead to insights into the molecular pathogenesis of this common disorder. A previous genome scan mapped a primary hip OA susceptibility locus to chromosome 2q with a maximum multipoint logarithm of odds score of 1.6 in 378 affected sibling pair families. Here, microsatellite targeting of eight candidate genes in this region from 2q23-2q32 demonstrated significant associations with the tumor necrosis factor alpha-induced protein 6 gene in all probands and the integrin alpha 6 and frizzled motif associated with bone development (FRZB) genes in female probands. However, genotyping showed lack of association for a nonsynonymous single-nucleotide polymorphism in tumor necrosis factor alpha-induced protein 6, whereas a single-nucleotide polymorphism in FRZB resulting in an Arg324Gly substitution at the carboxyl terminus was associated with hip OA in the female probands (P = 0.04). This association was confirmed in an independent cohort of female hip cases (n = 338; P = 0.04). In addition, a haplotype coding for substitutions of two highly conserved arginine residues (Arg200Trp and Arg324Gly) in FRZB was a strong risk factor for primary hip OA, with an odds ratio of 4.1 (P = 0.004). FRZB encodes secreted frizzled-related protein 3, which is a soluble antagonist of wingless (wnt) signaling. Variant secreted frizzled-related protein 3 with the Arg324Gly substitution had diminished ability to antagonize wnt signaling in vitro. Hence, functional polymorphisms within FRZB confer susceptibility for hip OA in females and implicate the wnt signaling pathway in the pathogenesis of this disease.
B-cell depletive therapies have beneficial effects in patients suffering from rheumatoid arthritis. Nevertheless, the role of B cells in the pathogenesis of the disease is not clear. In particular, it is not known how the regeneration of the B-cell repertoire takes place. Two patients with active rheumatoid arthritis were treated with rituximab, and the rearranged immunoglobulin heavy-chain genes (Ig-VH) were analysed to follow the B-cell regeneration. Patient A was treated with two courses of rituximab, and B-cell regeneration was followed over 27 months by analysing more than 680 Ig-VH sequences. Peripheral B-cell depletion lasted 7 months and 10 months, respectively, and each time was accompanied by a clinical improvement. Patient B received one treatment course. B-cell depletion lasted 5 months and was accompanied by a good clinical response. B cells regenerated well in both patients, and the repopulated B-cell repertoire was characterised by a polyclonal and diverse use of Ig-VH genes, as expected in adult individuals. During the early phase of B-cell regeneration we observed the expansion and recirculation of a highly mutated B-cell population. These cells expressed very different Ig-VH genes. They were class-switched and could be detected for a short period only. Patient A was followed long term, whereby some characteristic changes in the VH2 family as well as in specific mini-genes like VH3–23, VH 4–34 or VH 1–69 were observed. In addition, rituximab therapy resulted in the loss of clonal B cells for the whole period.
Our data show that therapeutic transient B-cell depletion by anti-CD20 antibodies results in the regeneration of a diverse and polyclonal heavy-chain repertoire. During the early phase of B-cell regeneration, highly mutated B cells recirculate for a short time period in both the patients analysed. The longitudinal observation of a single patient up to 27 months shows subtle intraindividual changes, which may indicate repertoire modulation.
A variety of cell surface markers are being used to identify B cell subpopulations in peripheral blood. Currently at least eight subpopulations have been identified. Analyses of healthy individuals indicate that in general the various B cell subpopulations exist in relatively similar ratios in unrelated individuals. It has been demonstrated that B lymphocyte homeostasis is disturbed during infection and autoimmune disease. In this review we compare the distribution of B cell subpopulations in the peripheral blood of patients with systemic lupus erythematosus, rheumatoid arthritis and primary Sjogren's syndrome with each other, and with healthy individuals. The different autoimmune diseases have distinct changes in the B cell subpopulations. Understanding the nature of these B subpopulation signatures will potentially impact understanding the mechanisms of disease, diagnosis and therapy.
To confirm the association of 2 variants of the Frizzled-related protein gene (FRZB) with osteoarthritis (OA) of the hip, and to investigate whether these variants also associate with other heritable generalized OA phenotypes.
An association analysis of 2 variants (R200W and R324G) of FRZB was performed in a random sample of 1,369 subjects (ages 55-70 years) from a population-based cohort (the Rotterdam Study) scored for radiographic characteristics of OA in the hip, hand, spine, and knee and in a patient population of Caucasian probands (ages 40-70 years) and their siblings selected for the presence of primary symptomatic OA at multiple sites.
The allele frequency of the 2 variants was not significantly different between subjects with hip radiographic OA (ROA) and controls. The frequency of the G allele of the R324G variant was significantly increased in subjects with generalized ROA from the Rotterdam Study (0.10) and in subjects from the Genetics, osteoARthritis and Progression study (0.11) compared with that in controls from the Rotterdam Study (0.08). Carriers of this G allele had increased susceptibility for generalized ROA (odds ratio [OR] 1.4, 95% confidence interval [95% CI] 0.9-1.9, P = 0.10) or familial symptomatic OA at multiple sites (OR 1.6, 95% CI 1.1-2.3, P = 0.02).
Our results confirm that the R324G variant of the FRZB gene is involved in OA and indicate a role of this variant in several generalized OA phenotypes. A more extended OA phenotype may indeed be expected from genetic variation in an essential pathway of skeletal development such as Wnt signaling.
To examine the association of the Arg200Trp and Arg324Gly variants of FRZB with the risk and phenotype of radiographic osteoarthritis (OA) of the hip and serum levels of Frizzled-related protein (FRP) in a prospective cohort of elderly Caucasian women.
Radiographic hip OA status of patients was defined by the presence of severe joint space narrowing (JSN) (feature grade>or=3), a summary grade>or=3, or definite osteophytes (grade>or=2) and JSN (grade>or=2) in the same hip. Genotypes were obtained in 569 patients with radiographic OA of the hip and in 1,317 and 4,136 controls for the Arg200Trp and Arg324Gly variants, respectively. Serum FRP levels were measured by enzyme-linked immunosorbent assay. Multivariate logistic regression was performed.
The minor allele frequency for the Arg200Trp polymorphism was 0.12 in the control group compared with 0.14 in the group with radiographic OA of the hip (P=0.12), and the minor allele frequency for the Arg324Gly variant was 0.083 in the control group compared with 0.088 in the group with radiographic OA of the hip (P=0.63). The multilocus genotypes available in 1,886 subjects suggested that inheritance of both minor alleles was a risk factor for developing OA characterized by JSN (P<0.01). Patients with radiographic OA of the hip who were homozygous for the Arg200Trp minor allele had higher serum FRP levels than controls who were homozygous for the major allele.
Our data confirm findings of another study, that a rare haplotype with both Arg200Trp and Arg324Gly FRZB variants contributes to the genetic susceptibility to hip OA among Caucasian women, and that these polymorphisms may contribute to increased serum levels of proteins as biomarkers of OA.
Recon-stitution of peripheral blood B cells after depletion with rituximab in patients with rheumatoid arthritis
Leandro Mj G Cambridge
Leandro MJ, Cambridge G, Ehrenstein MR, Edwards JC. Recon-stitution of peripheral blood B cells after depletion with rituximab in patients with rheumatoid arthritis. Arthritis Rheum 2006;54: 613–20.