Antibodies to citrullinated proteins: molecular interactions and arthritogenicity

Summary: The discovery of antibodies specific for citrullinated protein epitopes [anti-citrullinated protein antibodies (ACPAs)] is a hallmark for the diagnosis and prognosis of rheumatoid arthritis (RA) and will also be a useful tool for understanding the fundamental pathologic processes. There are several essential questions pertaining to ACPA that remain to be explored, such as understanding the early specificity of the underlying T-cell recognition, whether the production of ACPA is a primary or secondary process, and in the event of such antibodies being arthritogenic, whether they could possibly regulate the disease development. To answer these questions, animal models are needed, but unfortunately ACPA is not a prominent feature of any of the classical animal models of RA. However, we showed recently that ACPA can be isolated from animals susceptible to collagen-induced arthritis that are specific for citrullinated type II collagen (CII). The citrulline specificity could be visualized, and the specificity is determined primarily by a direct interaction with citrulline. We also demonstrated that these antibodies are specific for the citrullinated epitopes and are pathogenic in vivo. A new hypothesis to explain how inflammation in RA can be directed to cartilaginous joints and be self-perpetuating is suggested, which involves recognition of post-translational modifications (glycosylation and citrullination) on CII by T and B cells that can have both arthritogenic and regulatory consequences.     

Bb2Bb3 regulation of murine Lyme arthritis is distinct from Ncf1 and independent of the phagocyte nicotinamide adenine dinucleotide phosphate oxidase

Several quantitative trait loci regulating murine Lyme arthritis severity have been mapped, including a highly significant linkage found on chromosome 5, termed Bb2Bb3. Within this region, the Ncf1 gene of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has recently been identified as a major regulator of arthritis severity in rodent models of rheumatoid arthritis, an effect attributed to protective properties of reactive oxygen species. To assess the role of Ncf1 in Lyme arthritis, we introgressed Bb2Bb3 from severely arthritic C3H/He mice onto mildly arthritic C57BL/6 mice. This increased Lyme arthritis severity, whereas the reciprocal transfer conferred protection from disease. A single nucleotide polymorphism was identified in the Ncf1 gene that did not influence the protein sequence or expression of Ncf1. Although polymorphonuclear leukocytes from C57BL/6 mice generated a greater oxidative burst than polymorphonuclear leukocytes from C3H/He mice, studies with the Bb2Bb3 congenic mice demonstrated this difference was not linked to Ncf1 alleles. Furthermore, Lyme arthritis severity was not altered in mice lacking either the Ncf1 or Gp91phox subunits of the NADPH oxidase complex. Together, these results argue that Ncf1 is not a candidate gene for regulation of Lyme arthritis and reveal Lyme arthritis to be independent of NADPH oxidase activity, distinguishing it from other models of rheumatoid arthritis.     

Complement activation by both classical and alternative pathways is critical for the effector phase of arthritis

To analyze the role of the classical and alternative pathways of complement activation in the effector phase of arthritis, we have induced arthritis in C3- and factor B (FB)-deficient (C3(-/-) and FB(-/-)) DBA/1J mice using well-defined monoclonal IgG2b and IgG2a antibodies to type II collagen. In control DBA/1J mice, severe swelling of the joints, destruction of cartilage and erosion of bone developed very rapidly with a 100% incidence and a peak on days 7-10. Although 75% of C3(-/-) mice developed arthritis, the clinical severity was very mild and the onset was delayed. Severity of arthritis in FB(-/-) mice ranked intermediate in comparison with C3(-/-) and control mice with an incidence of 100%. Immunohistochemical analysis of the inflamed joints demonstrated substantial reduction in macrophage and neutrophilic leukocyte infiltration in both C3(-/-) and FB(-/-) mice, thereby confirming the clinical findings. We conclude that both the classical and the alternative pathways of complement activation are involved in the effector phase of arthritis.     

Erythrocyte filterability in normal and high-risk pregnancy

Erythrocyte filterability was studied longitudinally in normal pregnancy and in certain categories of high-risk pregnancy. Study subjects included ten normal controls, 12 insulin-dependent diabetics, eight gestational diabetics, and five essential hypertensives. Our results indicate that erythrocyte filterability remains relatively stable over the course of normal gestation. We noted no differences between controls and essential hypertensives or gestational diabetics, although a favorable effect of insulin therapy was suggested in gestational diabetics. Erythrocyte filterability and mean arterial blood pressure were not related. Insulin-dependent diabetics demonstrated a significantly elevated and widely varying erythrocyte filterability, and individual patient trends correlated well with outcome. Fibrinogen levels in diabetics rose precipitously and were significantly higher than normal throughout gestation. Fibrinogen levels paralleled changes in erythrocyte filterability, with the two parameters positively correlated. Mean glucose control had no influence on filterability. We conclude that in the diabetic pregnancy, varying erythrocyte filterability is related to altered fibrinogen metabolism and may contribute to perinatal morbidity.     

The influence of direct and antithrombin-dependent thrombin inhibitors on the procoagulant and anticoagulant effects of thrombin

INTRODUCTION: Clinical trials evaluating direct thrombin inhibitors in unstable coronary artery disease (CAD) have been disappointing. The hypothesis tested in the present study was that these agents may inhibit the anticoagulant effect of thrombin to a further extent than the procoagulant effect of thrombin. MATERIALS AND METHODS: We studied both reversible and irreversible thrombin inhibitors and compared the effects of each inhibitor on activated protein C (APC) generation vs. the effect on fibrinopeptide A (FPA) generation. A mixture of protein C, thrombin inhibitor, fibrinogen, fibrin polymerisation blocker and thrombin was incubated with thrombomodulin (TM)-expressing human saphenous vein endothelial cells (HSVECs). The inhibitors investigated were melagatran, inogatran, hirudin, hirugen, D-Phe-D-Pro-D-arginyl chloromethyl ketone (PPACK), and antithrombin (AT) alone or in combination with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). RESULTS: All agents, except hirugen, inhibited APC and FPA generation in a dose-dependent manner. FPA inhibition/APC inhibition ratios, based on IC50 for inogatran, melagatran, hirudin, PPACK, AT, AT-UFH and AT-LMWH were 1.73, 0.85, 0.55, 2.1, 0.5, 0.65 and 3.1 respectively. CONCLUSIONS: All agents, except hirugen, inhibited APC and FPA generation approximately to a similar extent. Thus, it can be inferred that the poor efficacy of thrombin inhibitors in recent clinical trials in patients with unstable CAD is unlikely to be a consequence of their effects on the protein C system.     

Effects of tiagabine, a gamma-aminobutyric acid re-uptake inhibitor, on normal rat bladder function

PURPOSE: Previous reports have demonstrated the inhibitory effect of exogenous gamma-aminobutyric acid (GABA) on micturition. In the current study we tested whether tiagabine (Sanofi Synthelab., Newcastle-upon Tyne, United Kingdom), a GABA re-uptake inhibitor increasing endogenous GABA concentrations, would affect micturition in awake rats or influence rat detrusor contraction in vitro. MATERIALS AND METHODS: Nonanesthetized female Sprague-Dawley rats underwent cystometric investigation in a metabolic cage. Micturition was stimulated by infusing saline intravesically. Micturition parameters were recorded and compared before and after drug administration. In vitro the effects of tiagabine on electrical and carbachol induced contractions in bladder strips were investigated. Furthermore, it was studied whether tiagabine interfered with electrically induced release of acetylcholine. RESULTS: Intravenous administration of 5 and 20 mg. kg.-1 tiagabine in 7 and 9 rats decreased micturition pressure a mean plus or minus standard error of mean of 21% +/- 11% and 42% +/- 9%, and decreased voided volume a mean of 31% +/- 9% and 33% +/- 9%, respectively. At 20 mg. kg.-1 tiagabine intravenously increased post-void residual volume a mean of 300% +/- 120% and decreased bladder capacity a mean of 14% +/- 3%. Tiagabine (100 microg.) intrathecally in 7 rats reduced micturition pressure a mean of 34% +/- 10% and increased bladder capacity a mean of 30% +/- 9% and post-void residual volume a mean of 250% +/- 75%. However, voided volume was not changed. In vitro studies demonstrated that tiagabine attenuated bladder contractions induced by electrical field stimulation to a mean of 69% +/- 6% of controls at 100 microM. but did not affect contractions induced by carbachol. Release studies revealed that tiagabine inhibited electrical induced acetylcholine release to a mean of 82% +/- 5% of controls at 100 microM. CONCLUSIONS: The current results show that tiagabine has an inhibitory action on rat micturition. The site of action may be central and peripheral.     

Initial experiences with 123-Iodine-IBZM neuroreceptor scintigraphy in extrapyramidal disorders

The receptor scintigraphy of the dopaminergic system of the brain is of interest in the evaluation of movement disorders. The 123I-IBZM is a radiopharmaceutical with affinity predominantly to postsynaptic D2 receptors. The aim of this study was to evaluate the role of IBZM SPECT investigations in the differentiation of disorders with Parkinson's syndrome. Eight patients with idiopathic Parkinson's syndrome and 8 patients Parkinson's syndrome with other etiology were investigated with 123I-IBZM SPECT (6 females, 10 males, mean age +/- SD: 59 +/- 9). The patients according to the clinical signs and symptoms, results of CT/MRI and rCBF SPECT investigation were categorized. The reconstructed SPECT slices were evaluated visually and quantitatively. The visual interpretation of the images were performed by two observer and scored the radiopharmaceutical uptake (from 1-3) of the cortex and the striatum separately. For quantification striatum/frontal cortex activity ratio were calculated with ROI technique. The differences between the patient groups were statistically analyzed by two tailed t-test. The IBZM uptake were different in the two group of patients. The striatal IBZM accumulation was higher in the idiopathic Parkinson's syndrome patients compared to the other parkinsonians. The striatum/frontal lobe activity ratio was 1.69 +/- 0.9 (mean +/- SD) in the right, 1.67 +/- 0.04 (mean +/- SD) in the left hemisphere of the patients with idiopathic Parkinson's syndrome. The corresponding data in the nonidiopathic parkinsonian group were 1.53 +/- 0.06 (mean +/- SD), 1.52 +/- 0.04 (mean +/- SD) respectively (p < 0.01). The quantitative data correlated with the results of the visual evaluation. According to the data presented IBZM-SPECT is an effective tool in the differentiation of disorders with Parkinson syndrome.     

Progestational agents and blood coagulation. IV. Changes induced by progestogen alone

To evaluate the effects of chlormadinone acetate upon the coagulation of blood and fibrinolysin systems, 35 healthy, young women voluntarily using some form of birth control were studied. 10 women who served as controls used intrauterine devices; 25 women took either a progestin-estrogen (1 mg norethindrone acetate and 1 mg mestranol) combination or a synthetic progestational agent (0.5 mg chlormadinone acetate) on a coded, double-blind basis. Platelet counts, thrombelastograms, and plasma assays were performed prior to and after 3 and 6 months of treatment. After 3 months, those taking progestin-estrogen showed a highly significant increase toward hypercoagulability in Quick time, Factors II, VII, and X, and increased levels in the thromboplastin generation time (TGT), Factors V and IX, and plasminogen. At 6 months all levels remained elevated except for TGT. Those on chlormadinone acetate had only a slightly significant change toward hypercoagulability in Quick time and Factor VIII, an increase in Factor IX, and a decrease in Factor X. In the control group only TGT was elevated. The progestin alone induced only minimal changes in comparison to the marked rises accompanied with progestin-estrogen therapy.     

Combination of chemotherapy with dual antagonists and radiotherapy in the treatment of neoplastic disease

The theoretical justification for combination chemotherapy and for the combination of radiation and chemotherapy in the treatment of neoplastic diseases is discussed, literature on the synthesis and biological activity of the ‘dual antagonist’ group of drugs is reviewed. Studies on the radiation-potentiating effect of certain members of this group of drugs are reviewed, and prospects for therapeutic applications are discussed.