Bjørn Skogen

Transformation of Amyloid Precursor SAA to Protein AA and Incorporation in Amyloid Fibrils in Vivo

Experimental amyloidosis was induced in mice by in t raped toneal injections of endotoxin (lipopolysaccharidc (LPS)). In addition to LPS. a group of mice received high‐density lipoprotein (HDL)‐SAA complexes isolated from human acute‐phase serum, whereas a group of control mice received saline in addition to LPS. Isolated amyloid fibrils from the mice given HDL‐SAA contained human AA protein, as shown by immunodiffusion, immunoblot. and enzyme‐linked Immunosorbent assay techniques, in addition to mouse AA. In contrast, amyloid from the control mice contained exclusively AA of mouse origin. Thus, the experiments provided solid evidence that SAA is the precursor for amyloid fibril proiein AA.

A prospective study of maternal anti-HPA 1a antibody level as a potential predictor of alloimmune thrombocytopenia in the newborn

Background Neonatal alloimmune thrombocytopenia is most commonly due to transplacental passage of maternal anti-HPA 1a antibodies. A prospective study was carried out to evaluate the pattern and quantity of maternal anti-HPA 1a antibodies in order to predict the level of thrombocytopenia in the neonates. Design and Methods A monoclonal antibody immobilization of platelet antigen assay was used to detect antibodies in maternal samples from 1,990 HPA 1bb women. HLA DRB3*0101 typing was performed in all immunized women by sequencing the HLA DRB3 gene when present. Results Primary immunization more often took place in connection with delivery than during the first pregnancy. There was a strong correlation between maternal antibody levels and the platelet counts in the newborn (R2=0.49, p<0.001). A maternal antibody level above 3.0 IU/mL measured in gestational week 22 or 34 had a diagnostic sensitivity and specificity of 93% and 63%, respectively, for predicting the grade of neonatal thrombocytopenia. The women who were negative for HLA DRB3*0101 had significantly lower anti-HPA 1a antibody levels than those who were HLA DRB3*0101 positive (p<0.007). In contrast to primigravida, in whom anti-HPA 1a antibody levels increased during pregnancy, the antibody level decreased in 92 of 147 women who had been pregnant previously (P92 or more of 147 = 0.003). The anti-HPA 1a antibody level regularly increased after delivery. Conclusions Maternal anti-HPA 1a antibody levels in weeks 22 and 34 of pregnancy are good predictors of the degree of thrombocytopenia in the newborn both in the first and subsequent pregnancies. Most mothers became immunized at the time of delivery.

A prominent lack of IgG1 Fc-fucosylation of platelet-alloantibodies in pregnancy

Immunoglobulin G (IgG) formed during pregnancy against human platelet antigens (HPAs) of the fetus mediates fetal or neonatal alloimmune thrombocytopenia (FNAIT). Because antibody titer or isotype does not strictly correlate with disease severity, we investigated by mass spectrometry variations in the glycosylation at Asn297 in the IgG Fc because the composition of this glycan can be highly variable, affecting binding to phagocyte IgG-Fc receptors (FcγR). We found markedly decreased levels of core fucosylation of anti-HPA-1a-specific IgG1 from FNAIT patients (n = 48), but not in total serum IgG1. Antibodies with a low amount of fucose displayed higher binding affinity to FcγRIIIa and FcγRIIIb, but not to FcγRIIa, compared with antibodies with a high amount of Fc fucose. Consequently, these antibodies with a low amount of Fc fucose showed enhanced phagocytosis of platelets using FcγRIIIb(+) polymorphonuclear cells or FcγRIIIa(+) monocytes as effector cells, but not with FcγRIIIa(-) monocytes. In addition, the degree of anti-HPA-1a fucosylation correlated positively with the neonatal platelet counts in FNAIT, and negatively to the clinical disease severity. In contrast to the FNAIT patients, no changes in core fucosylation were observed for anti-HLA antibodies in refractory thrombocytopenia (post platelet transfusion), indicating that the level of fucosylation may be antigen dependent and/or related to the immune milieu defined by pregnancy.

Neonatal alloimmune thrombocytopenia due to anti‐HPA 1a antibodies; the level of maternal antibodies predicts the severity of thrombocytopenia in the newborn

Eleven thousand one hundred pregnant women were genotyped for human platelet antigen HPA 1, and 198 HPA 1bb women were followed in the pregnancy with quantitative assay for anti‐HPA 1a antibodies. Antibodies were detected in 24 women, and nine children were born with severe thrombocytopenia (< 50×109/L). All mothers with high levels of antibodies were delivered of children with severe thrombocytopenia. None of the newborn infants had clinical signs of intra‐cranial haemorrhage. The level of maternal anti‐HPA 1a antibodies is predictive for fetal thrombocytopenia and may be used in decisions related to time and mode of delivery.

Amyloid and amyloidosis 1990

Amyloid and amyloidosis 1990 , Amyloid and amyloidosis 1990 , کتابخانه دیجیتالی دانشگاه علوم پزشکی و خدمات درمانی شهید بهشتی

Selecting and deselecting imatinib-resistant clones: observations made by longitudinal, quantitative monitoring of mutated BCR-ABL

Resistance to imatinib during the treatment of chronic myeloid leukaemia (CML) is frequently associated with point mutations in the ABL gene encoding the ATP binding region likely to cause disease relapse. Early diagnosis and monitoring of these mutations may be important in order to prevent rapid expansion of resistant clones. We describe a quantitative mutation-specific PCR assay based on the readily available Taqman platform. Selectivity for the mutated target is conferred by mutation-specific primers destabilised by additional mismatches. The assay can be carried out in parallel to standard BCR-ABL quantification and is therefore more quickly compared to standard sequencing procedures. The sensitivity of the assay reaches 0.1%. It also allows for quantitative assessment of mutated clones. By analysing sequential samples of resistant subjects, we show how mutated clones were selected, maintained or deselected depending on the individual treatment setting. The high sensitivity and practical merits of this method makes it a good candidate for prospective molecular surveillance of patients at high risk for imatinib resistance.

Characterization of amyloid proteins AA and SAA as apolipoproteins of high density lipoprotein (HDL). Displacement of SAA from the HDL-SAA complex by apo AI and apo AII.

An AA‐like protein with a molecular weight of 8600 complexed to high‐density lipoprotein (HDL) was demonstrated in several acute‐phase sera with high levels of SAA. The protein ‘apo AA’(to distinguish it from tissue AA) was isolated by elution from sodium dodecyl sulphate (SDS)‐polyacrylamide gel, and showed antigenic identity with purified tissue protein AA in double immunodiffusion. Normal HDL was shown to bind purified tissue AA in vitro. When the in vitro‐associated HDL‐AA complexes were given intravenously to mice during induction of amyloidosis, human AA was incorporated in the amyloid fibrils. Both apo AI and apo AII were shown to displace SAA from acute phase HDL when added to HDL‐SAA complexes in vitro. This might be of importanee in amyloidogenesis, as the liver and the small intestine, which are the main sites for AI and AII synthesis, are also sites of early amyloid deposition.

Maternal anti-platelet β3 integrins impair angiogenesis and cause intracranial hemorrhage

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disease in which intracranial hemorrhage (ICH) is the major risk. Although thrombocytopenia, which is caused by maternal antibodies against β3 integrin and occasionally by maternal antibodies against other platelet antigens, such as glycoprotein GPIbα, has long been assumed to be the cause of bleeding, the mechanism of ICH has not been adequately explored. Utilizing murine models of FNAIT and a high-frequency ultrasound imaging system, we found that ICH only occurred in fetuses and neonates with anti-β3 integrin-mediated, but not anti-GPIbα-mediated, FNAIT, despite similar thrombocytopenia in both groups. Only anti-β3 integrin-mediated FNAIT reduced brain and retina vessel density, impaired angiogenic signaling, and increased endothelial cell apoptosis, all of which were abrogated by maternal administration of intravenous immunoglobulin (IVIG). ICH and impairment of retinal angiogenesis were further reproduced in neonates by injection of anti-β3 integrin, but not anti-GPIbα antisera. Utilizing cultured human endothelial cells, we found that cell proliferation, network formation, and AKT phosphorylation were inhibited only by murine anti-β3 integrin antisera and human anti-HPA-1a IgG purified from mothers with FNAIT children. Our data suggest that fetal hemostasis is distinct and that impairment of angiogenesis rather than thrombocytopenia likely causes FNAIT-associated ICH. Additionally, our results indicate that maternal IVIG therapy can effectively prevent this devastating disorder.

T-cell responses associated with neonatal alloimmune thrombocytopenia: isolation of HPA-1a-specific, HLA-DRB3*0101-restricted CD4+ T cells.

T-cell responses have been implicated in the development of HPA-1a-induced neonatal alloimmune thrombocytopenia (NAIT). However, HPA-1a-specific T cells have neither been isolated nor characterized. Here, we aimed to determine whether HPA-1a-specific T cells could be isolated from HPA-1a-immunized women. In the present study, peripheral blood mononuclear cells (PBMCs) from an HPA-1a-alloimmunized woman were cultured for weeks in the presence of HPA-1a peptide, labeled with CFSE, and assayed for antigen-specific proliferation. Individual proliferating cells were isolated by fluorescence-activated cell sorting and expanded in culture. Antigen specificity and HLA restriction were determined by cytokine secretion (enzyme-linked immunospot [ELISPOT]) and proliferation assays. Several CD3(+)CD4(+) T-cell clones were isolated that proliferated and secreted cytokines in response to HPA-1a peptide. Two of these clones have been established in long-term culture in our laboratory. Both of these recognize synthetic as well as naturally processed HPA-1a antigen, and the recognition is restricted by the MHC molecule HLA-DRB3*0101 that is strongly associated with NAIT. These HPA-1a-specific T-cell clones represent unambiguous evidence for the association of T-cell responses with NAIT, and they will serve as unique tools to elucidate the cellular immune response that may result in NAIT.

Maternal human platelet antigen-1a antibody level correlates with the platelet count in the newborns: a retrospective study

BACKGROUND: Maternal plasma and/or serum levels of anti‐HPA‐1a at delivery were compared to neonatal platelet (PLT) counts.