Abnormal T cell differentiation persists in patients with rheumatoid arthritis in clinical remission and predicts relapse.

OBJECTIVES: An abnormal CD4+ T cell subset related to inflammation exposure (inflammation-related cells, IRC) has been identified in rheumatoid arthritis (RA). Patients with inflammatory and non-inflammatory diseases were used to examine the relationship between inflammation and this T cell subset in vivo. METHODS: Blood was collected from healthy controls and patients with RA (active disease or in clinical remission), Crohn's disease and osteoarthritis. IRC and chemokine receptors were quantified by flow cytometry. Thymic activity and apoptotic factors were measured by real-time polymerase chain reaction. Circulating cytokines were measured by enzyme-linked immunosorbent assay. CXCR4 and SDF1 in synovial biopsies were measured using immunohistochemistry. RESULTS: IRC were identified in patients with RA (p<0.0001) and Crohn's disease (p = 0.005), but not in those with osteoarthritis. In RA in remission, IRC persisted (p<0.001). In remission, hyperproliferation of IRC was lost, chemokine receptor expression was significantly lowered (p<0.007), Bax expression dropped significantly (p<0.001) and was inversely correlated with IRC (rho = -0.755, p = 0.03). High IRC frequency in remission was associated with relapse within 18 months (OR = 6.4, p<0.001) and a regression model predicted 72% of relapse. CONCLUSIONS: These results suggest a model in which, despite the lack of systemic inflammation, IRC persist in remission, indicating that IRC are an acquired feature of RA. They have, however, lost their hyper-responsiveness, acquired a potential for survival, and no longer express chemokine receptors. IRC persistence in remission confirms their important role in chronic inflammation as circulating precursors of pathogenic cells. This was further demonstrated by much higher incidence of relapse in patients with high IRC frequency in remission.

Early rheumatoid arthritis is associated with a deficit in the CD4+CD25high regulatory T cell population in peripheral blood.

OBJECTIVE: Our aim was to test the hypothesis that there is a deficit in the CD4+CD25high regulatory T-cell population in early rheumatoid arthritis (RA), either in size or functional activity. METHODS: Peripheral blood mononuclear cells were examined from subjects with early active RA who had received no previous disease-modifying therapy (n = 43), from individuals with self-limiting reactive arthritis (n = 14), from subjects with stable, well-controlled RA (n = 82) and from healthy controls (n = 72). The frequencies of CD4+CD25high T-cells were quantified using flow cytometry, and function was assessed by the ability to suppress proliferation of CD4+CD25- T-cells. Paired blood and synovial fluid was analysed from a small number of RA and reactive arthritis patients. RESULTS: There was a smaller proportion of CD4+CD25high T-cells in the peripheral blood of early active RA patients (mean 4.25%) than in patients with reactive arthritis or in controls (mean 5.90 and 5.30%, respectively, P = 0.001 in each case). Frequencies in stable, well-controlled RA (mean 4.63%) were not significantly different from early active RA or controls. There were no differences in suppressor function between groups. Higher frequencies of CD4+CD25high T-cells were found in synovial fluid than blood in both RA and reactive arthritis. CONCLUSIONS: These data demonstrate a smaller CD4+CD25high regulatory T-cell population in peripheral blood of individuals with early active RA prior to disease-modifying treatment. This may be a contributory factor in the susceptibility to RA and suggests novel approaches to therapy.

Analysis of the insertion/deletion related polymorphism within T cell antigen receptor beta variable genes in primary Sjögren's syndrome.

OBJECTIVE: To analyse T cell receptor beta variable (TCRBV) gene polymorphisms (insertion/deletion related polymorphism (IDRP) and BV6S7) in primary Sjögren's syndrome (PSS). METHODS: Genomic DNA was extracted from blood samples from patients fulfilling the modified European criteria for PSS (n = 61). Healthy control blood samples were obtained from the Blood Transfusion Service (n = 121). As a disease control group, samples from patients with systemic lupus erythematosus (n = 42) were analysed. BV6S7 was genotyped using an established PCR/RFLP method. The IDRP was determined by comparison of the intensity of PCR product bands from within BV9S2 and an internal control region (BV9S1), to ascertain whether 0, 1, or 2 copies of the insertion were present. RESULTS: There was a decrease (p = 0.018) in the proportion of PSS patients with the deleted/deleted genotype. There was no association with specific BV6S7 alleles or genotypes with either the PSS group or the hypergammaglobulinaemic subgroup. There were no significant differences in haplotype frequencies after Bonferroni correction. CONCLUSIONS: A reduced proportion of patients with PSS have the deleted/deleted genotype. Eighty nine per cent of PSS patients have at least one extra germline copy of BV13S2*1. This may relate to previous observations of increased BV13 specific T cells and mRNA in the salivary glands.

FcgammaRIIIA-158V and rheumatoid arthritis: a confirmation study.

OBJECTIVES: To develop a robust assay for genotyping the FcgammaRIIIA-158V/F polymorphism and to confirm the putative association between the FcgammaRIIIA-158V allele and rheumatoid arthritis (RA). METHODS: This allelic association study examined the FcgammaRIIIA-158V/F polymorphism for association with RA. A novel single-stranded conformational polymorphism assay was used to genotype 828 RA patients and 581 controls from the UK. RESULTS: The FcgammaRIIIA-158V allele was associated with both RA (P=0.02) and nodules (P=0.04). Individuals homozygous for this higher affinity allele had a significantly increased risk of RA (OR 1.53, 95% CI 1.08-2.18) and the development of nodules (OR 2.20, 95% CI 1.20-4.01). There was no evidence of an interaction with the shared epitope. CONCLUSIONS: We have developed a novel assay to genotype the FcgammaRIIIA-158F/V polymorphism and confirmed that homozygosity for the FcgammaRIIIA-158V allele is associated with UK Caucasian RA, particularly in those individuals with nodules, suggesting FcgammaRIIIA may play a role in determining disease severity or in the development of nodules per se.

Coordinated induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of plasminogen activator gene expression by hypoxia promotes pulmonary vascular fibrin deposition.

Oxygen deprivation, as occurs during tissue ischemia, tips the natural anticoagulant/procoagulant balance of the endovascular wall to favor activation of coagulation. To investigate the effects of low ambient oxygen tension on the fibrinolytic system, mice were placed in a hypoxic environment with pO2 < 40 Torr. Plasma levels of plasminogen activator inhibitor-1 (PAI-1) antigen, detected by ELISA, increased in a time-dependent fashion after hypoxic exposure (increased as early as 4 h, P < 0.05 vs. normoxic controls), and were accompanied by an increase in plasma PAI-1 activity by 4 h (P < 0.05 vs. normoxic controls). Northern analysis of hypoxic murine lung demonstrated an increase in PAI-1 mRNA compared with normoxic controls; in contrast, transcripts for both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) decreased under hypoxic conditions. Immunocolocalization studies identified macrophages as the predominant source of increased PAI-1 within hypoxic lung. Using a transformed murine macrophage line, striking induction of PAI-1 transcripts occurred under hypoxic conditions, due to both increased de novo transcription as well as increased mRNA stability. Consistent with an important role of the fibrinolytic system in hypoxia-induced fibrin accumulation, PAI-1 +/+ mice exposed to hypoxia exhibited increased pulmonary fibrin deposition based upon a fibrin immunoblot, intravascular fibrin identified by immunostaining, and increased accumulation of 125I-fibrinogen/fibrin in hypoxic tissue. In contrast, mice deficient for the PAI-1 gene (PAI-1 -/-) similarly exposed to hypoxic conditions did not display increased fibrin accumulation compared with normoxic PAI-1 +/+ controls. Furthermore, homozygous null uPA (uPA -/-) and tPA (tPA -/-) mice subjected to oxygen deprivation showed increased fibrin deposition compared with wild-type controls. These studies identify enhanced expression of PAI-1 as an important mechanism suppressing fibrinolysis under conditions of low oxygen tension, a response which may be further amplified by decreased expression of plasminogen activators. Taken together, these data provide insight into an important potential role of macrophages and the fibrinolytic system in ischemia-induced thrombosis.

Thermoregulatory vasodilation increases the venous partial pressure of oxygen.

UNLABELLED: Thermoregulatory arteriovenous shunt vasoconstriction may facilitate deep-vein thrombosis by producing relative venous stasis and hypoxia. Accordingly, we evaluated the effect of vasomotion on leg blood flow and venous oxygen tension. We studied five male volunteers, each of whom was warmed enough to trigger vasodilation and then cooled sufficiently to provoke thermoregulatory vasoconstriction. The process was then repeated during N2O/desflurane anesthesia. Venous oxygen tension and saturation (with a fraction of inspired oxygen of 1.0) were evaluated in blood samples taken from a catheter that was inserted into a saphenous vein at the ankle and advanced until the tip was proximal to the knee. Thermoregulatory vasodilation with or without general anesthesia significantly increased arteriovenous shunt flow by approximately 10-fold, and increased total leg flow approximately sixfold. However, vasodilated flows were similar with and without general anesthesia, as were vasoconstricted flows. Before induction of anesthesia, thermoregulatory vasodilation increased venous oxygen tension from 46 +/- 6 to 187 +/- 99 mm Hg and venous saturation from 79% +/- 6% to 99% +/- 2%. After induction of anesthesia, thermoregulatory vasodilation increased venous oxygen tension from 55 +/- 11 to 356 +/- 103 mm Hg and venous saturation from 84% +/- 8% to 100% +/- 0%. Our data thus indicate that thermoregulatory vasodilation markedly increases both leg flow and venous oxygenation; and that both factors may help prevent perioperative venous thrombosis. IMPLICATIONS: Thermoregulatory arteriovenous shunt vasoconstriction may facilitate deep-vein thrombosis by producing related venous stasis and hypoxia. In male volunteers, the authors found that when vasodilation induced by warming was produced, both blood flow and venous oxygenation increased, both of which may help prevent perioperative venous thrombosis.

Monocytes and tissue factor promote thrombosis in a murine model of oxygen deprivation.

Clinical conditions associated with local or systemic hypoxemia can lead to prothrombotic diatheses. This study was undertaken to establish a model of whole-animal hypoxia wherein oxygen deprivation by itself would be sufficient to trigger tissue thrombosis. Furthermore, this model was used to test the hypothesis that hypoxia-induced mononuclear phagocyte (MP) recruitment and tissue factor (TF) expression may trigger the local deposition of fibrin which occurs in response to oxygen deprivation. Using an environmental chamber in which inhaled oxygen tension was lowered to 6%, hypoxic induction of thrombosis was demonstrated in murine pulmonary vasculature by 8 h based upon: (a) immunohistologic evidence of fibrin formation in hypoxic lung tissue using an antifibrin antibody, confirmed by 22.5-nm strand periodicity by electron microscopy; (b) immunoblots revealing fibrin gamma-gamma chain dimers in lungs from hypoxic but not normoxic mice or hypoxic mice treated with hirudin; (c) accelerated deposition of 125I-fibrin/fibrinogen and 111In-labeled platelets in the lung tissue of hypoxic compared with normoxic animals; (d) reduction of tissue 125I-fibrin/fibrinogen accumulation in animals which had either been treated with hirudin or depleted of platelets before hypoxic exposure. Because immunohistochemical analysis of hypoxic pulmonary tissue revealed strong MP staining for TF, confirmed by increased TF RNA in hypoxic lungs, and because 111In-labeled murine MPs accumulated in hypoxic pulmonary tissue, we evaluated whether recruited MPs might be responsible for initiation of hypoxia-induced thrombosis. This hypothesis was supported by several lines of evidence: (a) MP depletion before hypoxia reduced thrombosis, as measured by reduced 125I-fibrin/fibrinogen deposition and reduced accumulation of cross-linked fibrin by immunoblot; (b) isolated murine MPs demonstrated increased TF immunostaining when exposed to hypoxia; and (c) administration of an anti-rabbit TF antibody that cross-reacts with murine TF decreased 125I-fibrin/fibrinogen accumulation and cross-linked fibrin accumulation in response to hypoxia in vivo. In summary, these studies using a novel in vivo model suggest that MP accumulation and TF expression may promote hypoxia-induced thrombosis.

Case manager follow-up to failed appointments and subsequent service utilization.

Case manager responses to failed appointments were monitored for 83 seriously mentally ill persons in a rural community mental health center. Case manager actions taken were grouped into four categories of follow-up from most intensive to least intensive: home visit, phone call, letter, and no follow-up. On the whole, case managers most frequently did not follow-up missed appointments (56.7%), followed up by letters (21.3%), and telephone calls (18.7%), and home visits (3.3%). Analyses revealed that home visits were most intensive and all clients who were visited following failed appointments did not fail the subsequent appointment. Clients who received telephone calls or letters were about equally likely to fail the subsequent appointment, but were much more likely to attend the subsequent appointment than were clients who received no follow-up to the failed appointment. Interestingly, clients who failed appointments and received no follow-up were much more likely to need emergency services rather than a regular appointment as their next contact with the clinic.

Hypoxia-induced exocytosis of endothelial cell Weibel-Palade bodies. A mechanism for rapid neutrophil recruitment after cardiac preservation.

The period of hypoxia is an important priming event for the vascular dysfunction that accompanies reperfusion, with endothelial cells (ECs) and neutrophils (PMNs) playing a central role. We hypothesized that EC Weibel-Palade (WP) body exocytosis during the hypoxic/ischemic period during organ preservation permits brisk PMN recruitment into postischemic tissue, a process further amplified in an oxidant-rich milieu. Exposure of human umbilical vein ECs to a hypoxic environment (pO2 approximately 20 torr) stimulated release of von Willebrand factor (vWF), stored in EC WP bodies, as well as increased expression of the WP body-derived PMN adhesion molecule P-selectin at the EC surface. Increased binding of 111In-labeled PMNs to hypoxic EC monolayers (compared with normoxic controls) was blocked with a blocking antibody to P-selectin, but was not affected by a nonblocking control antibody. Although increased P-selectin expression and vWF release were also noted during reoxygenation, hypoxia alone (even in the presence of antioxidants) was sufficient to increase WP body exocytosis. To determine the relevance of these observations to hypothermic cardiac preservation, during which the pO2 within the cardiac vasculature declines to similarly low levels, experiments were performed in a rodent (rat and mouse) cardiac preservation/transplantation model. Immunodepletion of recipient PMNs or administration of a blocking anti-P-selectin antibody before transplantation resulted in reduced graft neutrophil infiltration and improved graft survival, compared with identically preserved hearts transplanted into control recipients. To establish the important role of endothelial P-selectin expression on the donor vasculature, murine cardiac transplants were performed using homozygous P-selectin deficient and wild-type control donor hearts flushed free of blood/platelets before preservation/transplantation. P-selectin-null hearts transplanted into wild-type recipients demonstrated a marked (13-fold) reduction in graft neutrophil infiltration and increased graft survival compared with wild-type hearts transplanted into wild-type recipients. To determine whether coronary endothelial WP exocytosis may occur during cardiac preservation in humans, the release of vWF into the coronary sinus (CS) was measured in 32 patients during open heart surgery. CS samples obtained at the start and conclusion of the ischemic period demonstrated an increase in CS vWF antigen (by ELISA) consisting of predominantly high molecular weight multimers (by immunoelectrophoresis). These data suggest that EC WP exocytosis occurs during hypothermic cardiac preservation, priming the vasculature to recruit PMNs rapidly during reperfusion.

Selective reduction of PVR by inhalation of a cGMP analogue in a porcine model of pulmonary hypertension.

Selective reduction of pulmonary vascular resistance (PVR) remains a therapeutic goal for the treatment of pulmonary hypertension, but current therapeutic options remain limited. Although the gas nitric oxide (NO) selectively dilates the pulmonary vascular bed, it requires special equipment for administration, has a short biologic half-life, and is potentially toxic. We hypothesized that stimulation of the NO pathway at the level of its second messenger, guanosine 3',5'-cyclic monophosphate (cGMP), by targeted pulmonary delivery of a membrane-permeable nonhydrolyzable cGMP analogue would cause selective pulmonary vasodilation. Pulmonary hypertension was induced in 21 pigs by the intravenous infusion of a thromboxane A2 analogue (9,11-dideoxy-9 alpha,11 alpha-epoxymethanoprostaglandin F2 alpha). Inhaled 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) lowered PVR in a time- and dose-dependent manner, with maximal effect achieved after 20 min. Compared with physiological saline control, 8-BrcGMP inhalation (3.0 micrograms/kg) lowered PVR by 25 +/- 3% (P < 0.01), whereas there was no significant decline in systemic vascular resistance (4 +/- 6%); mean pulmonary arterial pressure declined 13 +/- 3% (P < 0.01), whereas there was little change in mean arterial pressure; cardiac output increased 10 +/- 4% (P < 0.05). PVR did not decrease after inhalation of noncyclic 8-bromoguanosine 5'-monophosphate, indicating that stimulation of the NO-cGMP pathway beyond the level of NO results in pulmonary vasodilation independent of stimulation of purinergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Stereophotogrammetric study of growth and development of the nose.

A total of 269 contour maps of the face were measured in three dimensions to study growth and development of the nose. The maps were derived from a mixed longitudinal study of 26 boys and 26 girls between the ages of 9 and 16 years, and were recorded annually. Various nasal parameters were measured to study growth of linear parameters and external nasal volume. Apart from dorsum of the nose between 9 and 11 years of age, all linear parameters were larger for boys by an amount increasing with age. The early growth in girls and late growth in boys suggested the presence of an adolescent growth spurt in the nose, which was confirmed by volumetric measurements. Developmentally the greatest change occurred in anteroposterior prominence of nasal tip in both sexes and the least change occurred in intercanthal width.

Developmental changes in the facial soft tissues.

Short-base stereophotogrammetry was used to study differential growth and development of the soft tissues of the face. Thirteen facial parameters were measured at ages 9, 11, 13, 15, and 16 years on 170 facial contour maps selected from a mixed longitudinal study of 26 boys and 26 girls. Each parameter was measured three-dimensionally, and its developmental progress at the earlier stages was expressed as a percentage of its value at 16 years of age. Standing height development was assessed in the same way. Three parameters that measured soft tissues surrounding the eyes grew little but were very advanced in their development, following a "neural" pattern. The remaining facial parameters grew more but were less advanced, and standing height was least advanced. There appeared to be three separate patterns of development, "neural," "facial," and "skeletal." Girls were, in general, smaller than boys, but their development was more advanced when measured as a percentage of size at 16 years compared with boys.

The adolescent growth spurt in the soft tissues of the face.

The faces of 8 boys and 8 girls were recorded annually by short base stereophotogrammetry. The output for the 16 children consisted of 158 life-size facial maps allowing the serial three-dimensional measurement of 13 soft tissue facial parameters. Three of these parameters (1, 2 and 3) measured the width of the palpebral fissures and the intercanthal distance and were therefore related to the eyes, and 'neural' in character. The remaining 10 parameters (4-13) measured facial characteristics. Standing height was also recorded. The values for each characteristic were age-corrected. Means for the different soft tissue facial parameters are reported for ages 9-16 years. The original individual readings were then three-point smoothed and related first to the year in which peak growth was achieved in standing height, second to the two years preceding and, third, to the two years following that year. The means for the 10 facial parameter growth velocities extending below the eyes showed an adolescent growth spurt related to that in standing height, although not necessarily coinciding with that year. Of the 10 parameters, 5 peaked in PHV year, 3 in -1 PHV year and 2 in +1 PHV year. The facial parameters at eye level, which are really 'neural' parameters, showed much smaller growth velocities and no spurt.

The growth and development of the soft tissues of the human face.

The complexity of shape, soft nature of the facial tissues and the sensitivity of the eyes make their accurate direct measurement difficult. Short base stereophotogrammetry is three dimensional and non-invasive. This technique was used to measure the faces of 52 like-sexed twins between the ages of 9 and 16 years in a mixed longitudinal study. Certain vertical and horizontal parameters defining the eyes, the nose, the mouth and overall facial shape were remeasured and plotted for this study. Relative to the intercanthal line, the alae of nose and angles of mouth are displaced by growth, on or very near to straight lines which were not parallel, indicating a small change of proportion between nose and mouth. Their steepness is a measure of the dominance of vertical over lateral growth. Knowledge of normal growth changes will help to predict soft tissue changes produced by orthodontic or surgical treatment in the faces of children.

Influence of dietary fats on blood coagulation and prostaglandin production in the common marmoset.

Common marmosets were fed a standard marmoset diet (REF) or diets supplemented with 12% (wt/wt) sunflower seed oil (SSO) or sheep fat (SF) for a period of 90 weeks. The values for coagulation indices, clotting time, and Russel viper venom time were consistent with decreased thrombotic tendency of platelets from animals on the SSO diet relative to the low fat, REF diet animals, while an increased tendency to thrombosis was observed with SF-fed marmosets. The SSO- and SF-supplemented marmosets showed a significantly reduced thromboxane (TXB2) generation from platelets aggregating to collagen (ASC) relative to the REF group, while at 50 micrograms/ml ASC this difference was maintained only by the SSO group. The SF diet-fed marmosets showed a reduced prostacyclin (measured as 6-keto-PGF1 alpha) generation from incubated aorta relative to the REF or SSO-fed groups, which were not different from each other. A reduced proportion of platelet phospholipid arachidonic acid (20:4, n-6) and increased alpha-tocopherol concentration was consistent with the decreased aggregability and thromboxane generation of platelets from SSO-fed marmosets relative to the REF and SF groups. The SF diet-fed marmosets, on the other hand, showed minimal change in arachidonic acid, alpha-tocopherol or platelet reactivity from the REF group. These differing responses to dietary fats are discussed in relation to the potential for the development of thrombosis and atherosclerosis.

The influence of dietary fats on plasma lipids, blood pressure and coagulation indices in the rat.

Male Hooded Wistar rats were fed a commercial rat diet supplemented 12% by weight with sheep fat, sunflower seed oil and fish oil (tuna) over a period of 8 months. The influence of these diets on plasma fatty acids, triglycerides and cholesterol, blood pressure, body weight and coagulation indices was assessed. The sheep fat (SF)-fed rats showed a significant increase in body weight over the reference group (C) of 18%, and systolic blood pressure increased by 9.4%, whereas other dietary groups were not significantly affected. The fish oil (TFO)-fed rats showed a significant lowering of plasma cholesterol (-16.6%) and triglyceride (-47%) relative to the reference group, while the sunflower seed oil (SSO) group showed only a lowered plasma triglyceride (-32%). Plasma fatty acids in general reflected closely the dietary fatty acids, with some exceptions. Coagulation indices provided a consistent picture of an increased tendency to thrombosis in SF-fed rats and a significantly reduced tendency in the TFO-fed rats relative to reference rats. Fish oil rich in 20:5 and 22:6 omega 3 long-chain polyunsaturated fatty acids and low in cholesterol appears to have advantages in terms of reducing those parameters identified as risk factors for coronary heart disease in man. Sheep fat supplements rich in saturated fatty acids produce the opposite trend.