Anti-PAD4 autoantibodies in rheumatoid arthritis: levels in serum over time and impact on PAD4 activity as measured with a small synthetic substrate

Isoform 4 of the human peptidylarginine deiminase (hPAD4) enzyme may be responsible for the citrullination of antigens in rheumatoid arthritis (RA) and has been shown to be itself the target of disease-specific autoantibodies. Here, we have tested whether the level of serum anti-hPAD4 antibodies in RA patients is stable over a period of 10 years and whether the antibodies influence hPAD4-mediated deimination of the small substrate N-α-Benzoyl-l-arginine ethyl ester. RA sera (n = 128) obtained at baseline and after 10 years were assessed for anti-hPAD4 antibodies by a specific immunoassay. For 118 RA patients, serum anti-hPAD4 IgG levels were stable over 10 years. Seven patients who were negative for anti-PAD4 IgG at baseline had become positive after 10 years. Further, total IgG from selected RA patients and controls were purified, and a fraction was depleted for anti-hPAD4 antibodies. Kinetic deimination assays were performed with total IgG and depleted fractions. The k _cat and K _m values of hPAD4-mediated deimination of N-α-Benzoyl-l-arginine ethyl ester were not affected by the depletion of the anti-hPAD4 antibodies from the total IgG pool. In conclusion, RA patients remain positive for anti-hPAD4 antibodies over time and some patients who are initially anti-hPAD4 negative become positive later in the disease course. The anti-hPAD4 antibodies did not affect the enzymatic activity of hPAD4 when the small substrate N-α-Benzoyl-l-arginine ethyl ester was used. However, this finding may not exclude an effect of these autoantibodies on citrullination of protein substrates in RA.     

POSITIVE SELECTION OF Tac- (CD25) POSITIVE CELLS FOLLOWING T-CELL ACTIVATION: USE OF IMMUNOMAGNETIC SEPARATION AND IMPLICATIONS FOR T-CELL CLONING

We have investigated if positive selection for cells expressing activation antigens, which appear on the cell surface during T-lymphocyte activation, could be used for cloning purposes. For this purpose, we used paramagnetic, monodisperse Dynabeads coated with anti-Tac monclonal antibody, which recognizes CD25 (interleukin-2 receptor light chain). After the first 6–12 h of a primary response, depletion of Tac⁺ cells could largely abrogate the specific response. This indicated that the specifically responding cells were found among the Tac⁺ population. T-cell cloning was thus performed on Tac⁺ blasts positively selected after 18 h of a primary response, at day 6 of a primary response or during secondary stimulation, and gave a high percentage of specific clones. This method is thus a good alternative to established techniques.     

Unanticipated Cesarean Birth from the Father's Perspective

ABSTRACT: Despite the paucity of research on men's experiences of cesarean birth, fathers' attendance at cesareans has become well-established in some areas of the U.S. In this study, interviews were conducted with 46 fathers whose wives had an uncomplicated pregnancy culminating in an unanticipated cesarean birth with a healthy neonate and no major complications for mother and child. Interviews were tape recorded, transcribed and analyzed. Of these 46 fathers, 52 per cent attended the cesarean, and 48 per cent did not, primarily because hospital policy prohibited it. Fathers' predominant emotional reaction to the decision for cesarean was relief (52%); 27 per cent described their reactions as acceptance, 10 per cent expressed moderate disappointment, and 11 per cent were strongly disappointed or angry. Most negative reactions centered not on the cesarean itself, but on policies which excluded fathers from attendance arbitrarily, and on staff behaviors which reflected disregard for the fathers'need to feel included in the birth, whether they were permitted to attend the delivery or not. Seventy per cent of these fathers expressed some displeasure at physician or nurse behaviors, expressing disappointment and resentment about being excluded from discussions leading to the decision for the cesarean, having little previous contact with the obstetrician, the nursing staff failing to provide the father with adequate information and support during and immediately after the cesarean, and being denied access to the wife and infant after the cesarean for apparently arbitrary reasons.     

Oxygenation within the first 120 h following coronary artery bypass grafting. Influence of systemic hypothermia (32 degrees C) or normothermia (36 degrees C) during the cardiopulmonary bypass: a randomized clinical trial

BACKGROUND: Lung function is often impaired after cardiac surgery performed under cardiopulmonary bypass (CPB). Normothermic CPB has become more common, but it remains unknown whether it reduces post-operative lung function compared with hypothermic CPB. The aim of this study was to investigate oxygenation within the first 120 h after systemic hypothermia and normothermia under CPB. METHODS: Thirty patients undergoing coronary artery bypass grafting (CABG) were randomized to either hypothermic (32 degrees C) or normothermic (36 degrees C) CPB. Oxygenation was studied by a simple method for the estimation of intrapulmonary shunt and ventilation-perfusion (V/Q) mismatch pre-operatively and 4, 48 and 120 h post-operatively by changing Fio2 in four to six steps. V/Q mismatch was described with DeltaPo2 (normal values, 0-2.38 kPa). RESULTS: Shunt and V/Q mismatch (DeltaPo2) increased post-operatively in both groups (P<0.01), with no differences between the groups, and with the nadir values 48 h after surgery, i.e. shunt of 15% (5.8-25%) and DeltaPo2 of 3.0 kPa (0.8-14 kPa) [values given as median (range)]. CONCLUSIONS: Impaired oxygenation is prevalent and prolonged following CABG, with equal intensity after hypothermic and normothermic CPB.     

Head injury mortality in the Nordic countries

Traumatic brain injury (TBI) is a major cause of morbidity and mortality in Western countries. Effective management planning for these patients requires knowledge of TBI epidemiology. The purpose of this study was to describe and analyze the development of TBI mortality in the Nordic countries during the period 1987-2001. Data on TBI deaths were retrieved from the national official statistical agencies according to specified diagnostic codes. We also collected data on the number of operations for acute TBI in the year 2000 from all Nordic hospitals admitting trauma patients. Finland had about twice as high a TBI mortality rate as the other countries. Similarly, the Finnish incidence of acute TBI operations was nearly twice that of the other countries. The median TBI death rate for Finland was 21.2 per 100,000 per year, and for Denmark, Norway, and Sweden 11.5, 10.4, and 9.5, respectively. There were more male than female deaths in all countries. The mortality rate from extracranial injuries was relatively equal between the countries. We observed a sizeable reduction in TBI mortality rates for all countries, except in Finland. Younger age groups had the most pronounced decrease in TBI mortality rates. The oldest age group had the least favorable development of TBI mortality rates, and the mean age of TBI casualties increased substantially during the study period. This study demonstrates considerable differences in and between the Nordic countries regarding TBI mortality. Preventive measures and implementation of regional guidelines are needed to assure a positive development in the future.     

Recombinant antibodies for delivery of antigen: a single loop between beta-strands in the constant region can accommodate long, complex and tandem T cell epitopes

Recombinant antibodies are increasingly used for efficient delivery of T cell epitopes to antigen-presenting cells (APCs), both for vaccination purposes and for immune modulation. We have previously shown that recombinant antibodies can accommodate single T cell epitopes inserted into loops between beta-strands in constant (C) domains. Such recombinant antibodies have in addition been equipped with variable regions that target APCs for increased delivery of C region T cell epitopes. We here show that loop 6 (loop FG) in C(H)1 of human gamma 3 can be exchanged with (i) long T cell epitopes up to 37 amino acids, (ii) epitopes with complex secondary structure such as gluten epitopes with a type II polyproline helical confirmation and (iii) two tandemly linked T cell epitopes. T cell responses increased with T cell epitope elongation, presumably due to a positive influence of flanking residues. Recombinant antibodies targeted to either CD14 on monocytes or HLA-DP on monocytes and dendritic cells gave similar results and were 2-4 logs more efficient at stimulating human T cells than were non-targeted controls. Thus, single loops in C regions of recombinant antibodies seem versatile and may be used for delivery of lengthy, complex and multiple T cell epitopes to human APCs.     

The intestinal T cell response to alpha-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase

The great majority of patients that are intolerant of wheat gluten protein due to celiac disease (CD) are human histocompatibility leukocyte antigen (HLA)-DQ2(+), and the remaining few normally express HLA-DQ8. These two class II molecules are chiefly responsible for the presentation of gluten peptides to the gluten-specific T cells that are found only in the gut of CD patients but not of controls. Interestingly, tissue transglutaminase (tTG)-mediated deamidation of gliadin plays an important role in recognition of this food antigen by intestinal T cells. Here we have used recombinant antigens to demonstrate that the intestinal T cell response to alpha-gliadin in adult CD is focused on two immunodominant, DQ2-restricted peptides that overlap by a seven-residue fragment of gliadin. We show that tTG converts a glutamine residue within this fragment into glutamic acid and that this process is critical for T cell recognition. Gluten-specific T cell lines from 16 different adult patients all responded to one or both of these deamidated peptides, indicating that these epitopes are highly relevant to disease pathology. Binding studies showed that the deamidated peptides displayed an increased affinity for DQ2, a molecule known to preferentially bind peptides containing negatively charged residues. Interestingly, the modified glutamine is accommodated in different pockets of DQ2 for the different epitopes. These results suggest modifications of anchor residues that lead to an improved affinity for major histocompatibility complex (MHC), and altered conformation of the peptide-MHC complex may be a critical factor leading to T cell responses to gliadin and the oral intolerance of gluten found in CD.     

Binding of peptides to HLA-DQ molecules: peptide binding properties of the disease-associated HLA-DQ({alpha}1*0501, {beta}1*0201) molecule

Peptide binding to DQ molecules has not previously been described.Here we report a biochemical peptlde-blndlng assay specificfor the BQ2 [I.e. DQ(1^*0501, ß1^*0201)] molecule. Thismolecule was chosen since It shows a strong association to diseasessuch as celiac disease and insulin-dependent diabetes mellitus.Initially we radlolabelled some selected peptides and testedthem for binding to affinity-purified DQ2 molecules. One ofthe peptides, a Mycobacterium bovis (MB) 65 kDa 243–255Ypeptide, displayed a good slgnal-to-noise ratio and was thuschosen as an indicator peptide in the DQ2 binding assay. TheMB 65 kDa 243–255Y peptide bound to DQ2 In a strictlypH-dependent fashion, with optimal binding around pH 5 and onlyweak binding at pH 7.4. The association of the MB 65 kDa 243–255Ypeptide to DQ2 was slow, but once formed, the peptide-HLA complexeswere very stable. The binding of peptides to DQ2 was specific,as shown in Inhibition experiments with a panel of 47 peptides,differing in length, sequence, and origin. The binding of peptidesto DR3 was tested in a similar assay with a Mycobacterium tuberculosis65 kDa 3–13 peptide as the binding indicator. DQ2 andDR3 molecules bound to different sets of peptides. However,the peptide binding to DQ2 and DR3 showed, In general, similarcharacteristics with respect to pH dependence and kinetic parameters,Indicating that the overall rules for peptide binding to DQmolecules are the same as those previously shown for human DRand murlne I-A and I-E molecules.     

Patients with multiple sclerosis carry DQB1 genes which encode shared polymorphic amino acid sequences

Of 61 Norwegian multiple sclerosis patients tested, 59, i.e., 97%, were positive for at least one of the HLA specificities DR2, DR4, or DRw6. Typing with sequence-specific oligonucleotide probes revealed that the same 59 patients carried DR2-, DR4-, or DRw6-associated HLA-DQB1 genes which encode shared polymorphic amino acid sequences in the membrane-distal part of their HLA-DQ beta chains. This shared DQ beta polymorphism may explain previously reported DR associations and could thus be the primary HLA association in MS.