Hunting for the elusive target antigen in gestational alloimmune liver disease (GALD).

The prevailing concept is that gestational alloimmune liver disease (GALD) is caused by maternal antibodies targeting a currently unknown antigen on the liver of the fetus. This leads to deposition of complement on the fetal hepatocytes and death of the fetal hepatocytes and extensive liver injury. In many cases, the newborn dies. In subsequent pregnancies early treatment of the woman with intravenous immunoglobulin can be instituted, and the prognosis for the fetus will be excellent. Without treatment the prognosis can be severe. Crucial improvements of diagnosis require identification of the target antigen. For this identification, this work was based on two hypotheses: 1. The GALD antigen is exclusively expressed in the fetal liver during normal fetal life in all pregnancies; 2. The GALD antigen is an alloantigen expressed in the fetal liver with the woman being homozygous for the minor allele and the father being, most frequently, homozygous for the major allele. We used three different experimental approaches to identify the liver target antigen of maternal antibodies from women who had given birth to a baby with the clinical GALD diagnosis: 1. Immunoprecipitation of antigens from either a human liver cell line or human fetal livers by immunoprecipitation with maternal antibodies followed by mass spectrometry analysis of captured antigens; 2. Construction of a cDNA expression library from human fetal liver mRNA and screening about 1.3 million recombinants in Escherichia coli using antibodies from mothers of babies diagnosed with GALD; 3. Exome/genome sequencing of DNA from 26 presumably unrelated women who had previously given birth to a child with GALD with husband controls and supplementary HLA typing. In conclusion, using the three experimental approaches we did not identify the GALD target antigen and the exome/genome sequencing results did not support the hypothesis that the GALD antigen is an alloantigen, but the results do not yield basis for excluding that the antigen is exclusively expressed during fetal life., which is the hypothesis we favor.

Blood cell gene expression profiling in rheumatoid arthritis. Discriminative genes and effect of rheumatoid factor.

UNLABELLED: To study the pathogenic importance of the rheumatoid factor (RF) in rheumatoid arthritis (RA) and to identify genes differentially expressed in patients and healthy individuals, total RNA was isolated from peripheral blood mononuclear cells (PBMC) from eight RF-positive and six RF-negative RA patients, and seven healthy controls. Gene expression of about 10,000 genes were examined using oligonucleotide-based DNA chip microarrays. The analyses showed no significant differences in PBMC expression patterns from RF-positive and RF-negative patients. However, comparisons of gene expression patterns from all fourteen RA patients and healthy controls identified a subset of discriminative genes. These results were validated by real time reverse transcription polymerase chain reaction (RT-PCR) on another group of RA patients and healthy controls. This confirmed that the following genes had a significantly higher expression in RA patients than in healthy controls: CD14 antigen, defensin alpha-1 and alpha-3 (DEFA), fatty-acid-Coenzyme A ligase, long-chain 2 (FACL), ribonuclease 2 (RNASE2), S100 calcium-binding protein A8 and A12 (S100A8 and S100A12). In contrast, the expression of MHC class II, DQ beta1 (HLA-DQB1) was significantly reduced in RA patients compared to healthy controls. CONCLUSIONS: With the analytical procedure employed, we did not find any indication that RF-positive and RF-negative RA are two fundamentally different diseases. Most of the genes discriminative between RA patients and healthy individuals are known to be involved in immunoinflammatory responses, especially those related to altered phagocytic functions.

Vaccination for birch pollen allergy. Induction of affinity-matured or blocking IgG antibodies does not account for the reduced binding of IgE to Bet v 1.

Specific allergy vaccination (SAV) is associated with increased levels of allergen specific IgG in serum. It is not clear, however, to what extent qualitative changes in allergen binding to IgG may be induced as well. We therefore analyzed the binding of the major allergen in pollen of birch (Betula verrucosa) (Bet v 1), the major allergen in birch pollen, to serum IgG and IgE, separately and in competition. Sera from six birch pollen-allergic patients were obtained before and after 5 years of SAV, and binding was assessed with 125I-Bet v 1. Before SAV, IgG bound more than eight times the amount of Bet v 1 compared with IgE, and together they accounted for more than 85% of the serum binding capacity. While SAV induced minimal changes in IgE binding, the IgG binding capacities increased 6-32 times. In contrast, the binding avidities (K(d) 28-40pM) changed less than 20%, pre- and post-SAV IgG provided similar inhibition of Bet v 1 binding to IgE at equimolar levels, and cross inhibition studies between IgG and IgE showed low inter-individual differences. Following SAV, all sera reduced Bet v 1 binding to CD23(+) cells, correlating with reduced binding of Bet v 1 to IgE (P<0.001). These results show that high avidity IgG of low inter-individual difference in Bet v 1 binding quality is the dominant binding factor of Bet v 1 in sera of birch pollen-allergic patients, and that SAV-induced inhibition of binding of Bet v 1 to IgE can be explained mainly or solely by increased amounts of IgG.