A comparative study of the immune modulating properties of antifibrinolytics in cardiac surgery.

PURPOSE: Antifibrinolytics, used in cardiac surgery to abate postoperative blood loss, share anti-inflammatory properties by suppression of pro-inflammatory D-dimer and plasmin levels. Additional drug specific immune modulating qualities are often mentioned in the discussion on which antifibrinolytic can best be used. To determine the extent and relevance of these effects, we investigated cytokine and growth factor plasma levels in cardiac surgery patients randomized to receive either tranexamic acid, aprotinin, or placebo. Corticosteroid-treated patients served to put the effects in perspective. METHODS: Using a biochip immunoassay, plasma of 36 cardiac surgery patients was quantified for 12 cytokines and growth factors, assessed preoperatively and 6, 12, 24, and 48 h after the start of cardiopulmonary bypass. Eight patients were treated with tranexamic acid, nine with aprotinin, and nine received placebo. Ten placebo-treated patients received corticosteroids. RESULTS: IL-1ß, IL-6, IL-8, IL-10, IFN-γ, TNF-α, VEGF, MCP-1, and EGF plasma concentrations significantly changed over time across all patients. Aprotinin-treated patients showed decreased pro-inflammatory TNF-α and peak MCP-1 plasma levels when compared with placebo. However, corticosteroids attenuated the inflammatory response to a much larger extent, lowering postoperative IL-6, IL-10, IFN-γ, and VEGF concentrations also. CONCLUSIONS: Aprotinin attenuates postoperative pro-inflammatory levels TNF-α and MCP-1 whereas tranexamic acid does not. The majority of plasma proteins studied, however, were not affected by the use of antifibrinolytics when compared with placebo. A clinically relevant common anti-inflammatory effect through inhibition of fibrinolysis seems therefore unlikely.

Mechanism of arsenic-induced neurotoxicity may be explained through cleavage of p35 to p25 by calpain.

In recent studies we have demonstrated that arsenic (As) metabolites change the composition of neuronal cytoskeletal proteins in vivo and in vitro. To further examine the mechanism of arsenic-induced neurotoxicity with various arsenate metabolites (iAsV, MMAV and DMAV) and arsenite metabolites (iAsIII, MMAIII and DMAIII), we investigated the role of the proteolytic enzyme calpain and its involvement in the cleavage of p35 protein to p25, and also mRNA expression levels of calpain, cyclin-dependent kinase 5 (cdk5) and glycogen synthase kinase 3 beta (gsk3ss). A HeLa cell line transfected with a p35 construct (HeLa-p35) was used as a model, since all other proteins such as calpain, CDK5 and GSK3beta are already present in HeLa cells as they are in neuronal cells. HeLa-p35 cells were incubated with various As metabolites and concentrations of 0, 10 and 30 microM for duration of 4 h. Subsequently the cells were either lysed to study their relative quantification levels of these genes or to be examined on their p35-protein expression. P35-RNA expression levels were significantly (p<0.01) increased by arsenite metabolites, while p35 protein was cleaved to p25 (and p10) after incubation with these metabolites. The cleavage of p35 is caused by calcium (Ca2+) induced activation of calpain. Inhibition of calpain activity by calpeptin prevents cleavage of p35 to p25. These results suggest that cleavage of p35 to p25 by calpain, probably As-induced Ca2+-influx, may explain the mechanism by which arsenic induces its neurotoxic effects.

Calcineurin activity and inhibition in skin and (epi)dermal cell cultures.

Calcineurin (Cn) is the target of the immunosuppressive drugs cyclosporine A (CsA), tacrolimus (Trl), and pimecrolimus (Prl). Trl and Prl are often used topically for treatment of various skin diseases. The Cn inhibitors CsA and Trl are mostly used for maintenance therapy of transplant patients. Their long-term use, however, causes a dramatic increase in skin cancer risk. By using a newly developed assay for Cn measurement in blood, we were able to demonstrate Cn activity in total skin homogenates. A significantly higher activity was found in epidermis compared to dermis. In skin cell cultures, fibroblasts showed the highest activity as compared to keratinocytes and melanocytes. Of the Cn inhibitors, Trl showed stronger inhibition than CsA and Prl (57 and 55% in fibroblast and keratinocyte cultures, respectively). Also, the lowest IC(50) (the half maximal inhibitory concentration) values were found for Trl (0.5 and 1.3 nM in two different fibroblast cultures). Cn activity and its inhibition can thus be studied in dermatological samples. The effects of Cn inhibition in fibroblasts and keratinocytes may be of influence on the overall functioning of the skin immune system.

Arsenic metabolites affect expression of the neurofilament and tau genes: an in-vitro study into the mechanism of arsenic neurotoxicity.

Neurological studies indicate that the central (CNS) and peripheral nervous system (PNS) may be affected by arsenic (As). As-exposed patients show significantly lower nerve conduction velocities (NCVs) in their peripheral nerves in comparison to healthy subjects. As may play a role in the disruption of neuroskeletal integrity, but the mechanisms by which it exerts a toxic effect on the peripheral and central nervous system are still unclear. In the present study, we examined the neurotoxic effects of various arsenic metabolites (iAs(III), iAs(V), MMA(V) and DMA(V)) on two different cell lines derived from the peripheral (ST-8814) and central (SK-N-SH) nervous system. The effects of the arsenic metabolites were examined on the relative quantification levels of the cytoskeletal genes, neurofilament-light (NEFL), neurofilament-medium (NEF3), neurofilament-heavy (NEFH) and microtubule-associated protein-tau (MAPT), using real-time PCR. Our results show that iAs(III) and iAs(V) have no significant effects on either cell lines. On the other hand, MMA(V) and DMA(V) cause significant changes in expression levels of NEF3 and NEFL genes, while the expression level of the NEFH gene is significantly increased in both cell lines.

Microparticle-associated tissue factor activity: a link between cancer and thrombosis?

BACKGROUND: Cancer, in particular mucinous adenocarcinoma, is associated with venous thromboembolism (VTE). Tissue factor (TF), initiator of coagulation, plays a central role in the paradigm that clotting and tumor growth form a vicious circle, in which hypercoagulability facilitates the aggressive biology of cancer and vice versa. Expression of TF in tumors is associated with poor differentiation and poor prognosis. PATIENT/METHODS: We investigated the association between clinically manifest VTE and procoagulant properties of circulating microparticles (MP) isolated from blood of unselected pancreatic and breast adenocarcinoma patients' consecutive subjects, who presented with ultrasound or CT-scan confirmed VTE, and healthy subjects. RESULTS: Patients with disseminated breast and pancreatic cancer had significantly increased levels of MP-associated TF activity compared with healthy controls, subjects with idiopathic acute VTE and non-metastatic cancer patients. Patients with both high MP-associated TF-activity and MP-associated epithelial mucin (MUC1) had a lower survival rate at 3-9 months follow-up than those with low TF-activity and no MUC1 expression: the likelihood of survival was 0.42 (95% CI: 0.19- 0.94) for an individual with these two predictor variables present, after adjustment for other factors (age cohort, type of cancer, VTE) in the Cox proportional hazards model. CONCLUSIONS: Our results suggest an important role for MP-associated TF and MUC1 in the pathogenesis of thrombosis in disseminated mucinous adenocarcinoma patients. Future studies should reveal the mechanism underlying the observed associations.

Arsenic-induced toxicity: effect on protein composition in sciatic nerve.

Exposure to arsenic compounds may lead to skin and lung cancer and various disorders such as vascular disease and peripheral neuropathy in humans. Peripheral arsenic neurotoxicity has been demonstrated clinically and in electrophysiological studies. Patients intoxicated with arsenic show neurological symptoms in their feet and hands. These patients show significantly lower nerve conduction velocities (NCVs) in their peripheral nerves in comparison with controls. The mechanism of arsenic peripheral nervous system (PNS) toxicity, however, has never been described before. This is the first study to investigate the toxicity of arsenic on the PNS. Male Wistar rats were exposed to arsenite given as a single dose i.v. After sacrifice, sciatic nerves were excised and the protein composition was analysed. Protein analysis of sciatic nerves showed disappearance of neurofilament and fibroblast proteins in rats treated with arsenite doses of 15 and 20 mg/kg in comparison with the control groups. Some fibroblast protein bands had disappeared in the 20-mg/kg dose group. The analysed neurofilament-M and -L proteins decreased dose dependency over time. arsenic affects the composition of proteins in the rat sciatic nerve, especially the neurofilaments. The reduction of signals in Western blot analysis reveals changes in cytoskeletal composition, which may well lead to neurotoxic effects in vivo.

Pro- and non-coagulant forms of non-cell-bound tissue factor in vivo.

BACKGROUND: Concentrations of non-cell-bound (NCB; soluble) tissue factor (TF) are elevated in blood collecting in the pericardial cavity of patients during cardiopulmonary bypass (CPB). Previously, we reported microparticles supporting thrombin generation in such blood samples. In this study we investigated the extent of microparticle association of the NCB form of TF in pericardial and systemic blood, and whether this microparticle-associated form is active in thrombin generation compared with non-microparticle-bound, (fluid-phase) TF. METHODS: Systemic and pericardial blood samples were collected before and during CPB from six patients undergoing cardiac surgery. Microparticles were isolated by differential centrifugation and their thrombin-generating capacity measured in a chromogenic assay. Microparticle-associated and fluid-phase forms of NCB TF were measured by ELISA. Microparticle-associated TF was visualized by flow cytometry. RESULTS: In pericardial samples, 45-77% of NCB TF was microparticle-associated, and triggered factor VII (FVII)-mediated thrombin generation in vitro. Microparticles from systemic samples triggered thrombin generation independently of FVII, except at the end of bypass (P = 0.003). The fluid-phase form of TF did not initiate thrombin generation. Both forms of NCB TF were, at least in part, antigenically cryptic. CONCLUSIONS: We demonstrate the occurrence of two forms of NCB TF. One form, which is microparticle-associated, supports thrombin generation via FVII. The other form, which is fluid-phase, does not stimulate thrombin formation. We hypothesize that the microparticle-associated form of NCB TF may be actively involved in postoperative thromboembolic processes when pericardial blood is returned into the patients.

Microparticles from patients with multiple organ dysfunction syndrome and sepsis support coagulation through multiple mechanisms.

AIM: We investigated the occurrence and thrombin generating mechanisms of circulating microparticles (MP) in patients with multiple organ dysfunction syndrome (MODS) and sepsis. METHODS: MP, isolated from blood of patients (n = 9) and healthy controls (n = 14), were stained with cell-specific monoclonal antibodies (MoAbs) or anti-tissue factor (anti-TF) MoAb and annexin V, and analyzed by flow cytometry. To assess their thrombin-generating capacity, MP were reconstituted in normal plasma. The coagulation activation status in vivo was quantified by plasma prothrombin fragment F1+2- and thrombin-antithrombin (TAT) measurements. RESULTS: Annexin V-positive MP in the patients originated predominantly from platelets (PMP), and to a lesser extent from erythrocytes, endothelial cells (EMP) and granulocytes (GMP). Compared to healthy controls, the numbers of annexin V-positive PMP and TF-exposing MP were decreased (p = <0.001 for both), EMP were decreased (E-selectin, p = 0.003) or found equal (CD144, p = 0.063), erythrocyte-derived MP were equal (p = 0.726), and GMP were increased (p = 0.008). GMP numbers correlated with plasma concentrations of elastase (r = 0.70, p = 0.036), but not with C-reactive-protein or interleukin-6 concentrations. Patient samples also contained reduced numbers of annexin V-negative PMP, and increased numbers of erythrocyte-derived MP and GMP (p = 0.005, p = 0.021 and p <0.001, respectively). Patient MP triggered thrombin formation, which was reduced compared to the healthy controls (p = 0.008) and strongly inhibited by an anti-factor XII MoAb (two patients), by anti-factor XI MoAb (eight patients) or by anti-TF MoAb (four patients). Concentrations of F1+2 and TAT were elevated (p = 0.005 and p = 0.001, respectively) and correlated inversely with the number of circulating MP (and r = -0.51, p = 0.013, and r = -0.65, p = 0.001, respectively) and their thrombin generation capacity (F1+2: r= -0.62, p = 0.013). CONCLUSIONS: In patients with MODS and sepsis relatively low numbers of MP are present that differ from controls in their cellular origin, numbers and coagulation activation mechanisms.

Dexamethasone concentration in the subretinal fluid after a subconjunctival injection, a peribulbar injection, or an oral dose.

PURPOSE: To determine dexamethasone concentrations in the subretinal fluid of patients after a peribulbar injection, a subconjunctival injection, or an oral dose of dexamethasone and to compare the results with those of previous similar studies of dexamethasone concentrations in the vitreous. DESIGN: Prospective, nonrandomized, comparative trial. PARTICIPANTS: One hundred forty-eight patients with a rhegmatogenous retinal detachment. METHODS: Fifty patients received a peribulbar injection of 5 mg dexamethasone disodium phosphate, 49 received a subconjunctival injection of 2.5 mg dexamethasone disodium phosphate, and 49 received an oral dose of 7. 5 mg dexamethasone at various time intervals before surgery. At the time of surgery, a subretinal fluid sample was taken from each patient. MAIN OUTCOME MEASURES: The dexamethasone concentration in the subretinal fluid measured by radioimmunoassay. RESULTS: The estimated maximum dexamethasone concentrations in the subretinal fluid after the peribulbar injection, the subconjunctival injection, and the oral dose were, respectively, 82.2 ng/ml (standard error, 17. 6), 359 ng/ml (standard error, 80.2), and 12.3 ng/ml (standard error, 1.61). Corrected for dose, the maximum dexamethasone concentrations after subconjunctival injection and peribulbar injection were, respectively, 120 (95% confidence interval, 54/180) and 13 (95% confidence interval, 6.8/20) times greater than after oral administration. CONCLUSIONS: A subconjunctival injection of dexamethasone disodium phosphate is more effective in delivering dexamethasone into the subretinal fluid of patients with a rhegmatogenous retinal detachment compared with peribulbar injection or oral administration. The subretinal dexamethasone concentrations were higher than concentrations measured in the vitreous in previous studies with a similar setup after all three delivery methods.

Activated platelets in patients with severe hypertriglyceridemia: effects of triglyceride-lowering therapy.

Hypertriglyceridemia, a risk factor for cardiovascular disease, has been associated with hypercoagulability, but whether platelet activation is implicated is unknown. This study was designed to compare the in vivo platelet activation status between patients with severe hypertriglyceridemia and age- and sex-matched control subjects, and to evaluate the effects of triglyceride-lowering therapy. Sixteen patients with primary hypertriglyceridemia were included in a double-blind, placebo-controlled cross-over trial with 400 mg bezafibrate once daily. Platelet activation was analysed by double label flow cytometry, using monoclonal antibodies against GP53, P-selectin, and platelet-bound fibrinogen. Surface expression of the lysosomal membrane protein GP53 was significantly higher in the hypertriglyceridemic patients at baseline as compared to the group of age- and sex-matched controls (16.3+/-4.8% vs. 8.9+/-3.4%, respectively, P<0.001). No differences in the expression of P-selectin and fibrinogen binding were observed. In response to bezafibrate therapy, the expression of GP53 in the patient group decreased from 16.3+/-4.8% to 13.1+/-4.1% (P=0.018). The expression of P-selectin and fibrinogen binding was not affected by bezafibrate therapy. In conclusion, patients with hypertriglyceridemia have an increased in vivo platelet activation status, which can be improved by bezafibrate therapy.

High concentration of dexamethasone in aqueous and vitreous after subconjunctival injection.

PURPOSE: To determine the dexamethasone concentration in aqueous, vitreous, and serum of patients after a subconjunctival injection with dexamethasone disodium phosphate and to compare the effectiveness of a subconjunctival injection as a method of delivering dexamethasone into the vitreous with that of two previously tested routes: peribulbar injection and oral administration. METHODS: In a prospective study, 50 phakic patients who underwent a pars plana vitrectomy received a single subconjunctival injection with 2.5 mg of dexamethasone disodium phosphate, aqueous solution (after topical anesthesia and a subconjunctival injection with lidocaine) at varied intervals before surgery. An aqueous and a vitreous sample were taken from each patient, and serum samples were collected at multiple time points from nine of 50 patients. Dexamethasone concentrations were measured by radioimmunoassay. RESULTS: The estimated maximum dexamethasone concentration in the aqueous was 858 ng per ml at 2.5 hours after injection, and in the vitreous, 72.5 ng per ml at 3 hours. In serum, a mean maximum concentration of 32.4 ng per ml was measured at approximately 30 minutes after injection. CONCLUSIONS: Subconjunctival injection of 2.5 mg of dexamethasone disodium phosphate resulted in an estimated vitreous dexamethasone peak concentration three and 12 times higher, respectively, than after a peribulbar injection of 5 mg of dexamethasone disodium phosphate and an oral dose of 7.5 mg of dexamethasone. Thus, a subconjunctival injection is the most effective method of delivering dexamethasone into both the anterior and posterior segments of the eye. Systemic drug absorption is considerable and is of the same order of magnitude as after peribulbar injection.

Dexamethasone concentration in vitreous and serum after oral administration.

PURPOSE: To determine the dexamethasone concentration in vitreous and serum of patients after oral administration of dexamethasone and to compare the results with the concentrations in vitreous and serum found in a previous study with peribulbar injection of 5 mg dexamethasone disodiumphosphate. METHODS: In a prospective study, 54 patients who were scheduled for vitrectomy received 7.5 mg dexamethasone orally at varied time intervals before surgery. A vitreous sample was taken from each patient and serum samples were collected at multiple time points from 32 out of 54 patients. Dexamethasone concentrations were measured by radioimmunoassay. RESULTS: Dexamethasone concentrations in serum ranged from 2.5 to 98.1 ng/ml (median, 61.6 ng/ml) between 1 and 3 hours after oral administration of 7.5 mg dexamethasone. Serum concentrations after peribulbar injection of 5 mg dexamethasone disodiumphosphate (containing 3.75 mg dexamethasone) were lower by a factor of 1.5. Concentrations in vitreous ranged from 1.7 to 23.4 ng/ml (median, 5.2 ng/ml) between 4 and 10 hours after oral administration. After peribulbar injection of 5 mg dexamethasone disodiumphosphate, the intravitreal concentrations were 3.9 times higher. CONCLUSIONS: An oral dose of 7.5 mg dexamethasone resulted in an intravitreal corticosteroid concentration with an anti-inflammatory potency that is clearly above physiological level. This concentration, however, is several times lower than is the intravitreal concentration after a peribulbar injection of 5 mg dexamethasone disodiumphosphate, although the two routes of administration resulted in nearly equal dexamethasone concentrations in serum. The higher intravitreal concentration after peribulbar injection is probably caused by diffusion from the serum and additional transscleral diffusion.

Peribulbar corticosteroid injection: vitreal and serum concentrations after dexamethasone disodium phosphate injection.

PURPOSE: To study the dexamethasone level reached in human vitreous after a peribulbar injection of 5 mg of dexamethasone disodium phosphate and to assess its systemic uptake. METHODS: In a prospective study, 61 eyes of 61 patients scheduled for vitrectomy received a single peribulbar injection of 5 mg of dexamethasone disodium phosphate at varied intervals before surgery. At the start of vitrectomy, an undiluted vitreous sample was taken. In 22 patients, multiple serum samples were collected. Dexamethasone concentrations were measured by radioimmunoassay. The physiologic cortisol concentration was determined in the vitreous of 12 eyes of 12 patients who did not receive dexamethasone. RESULTS: An average dexamethasone peak concentration of approximately 13 ng/ml was reached in vitreous 6 to 7 hours after peribulbar injection. In serum the average peak concentration was approximately 60 ng/ml 20 to 30 minutes after peribulbar injection. The average physiologic cortisol concentration in vitreous was 5.1 ng/ml. CONCLUSIONS: After a peribulbar injection of 5 mg of dexamethasone disodium phosphate, an average intravitreal dexamethasone concentration is reached with a 75 times greater anti-inflammatory potency than physiologically present cortisol. Dexamethasone concentration in serum, however, is several times higher. Peribulbar injection is not just a local treatment but results in serum levels comparable to those achieved by a high oral dose.

Glucocorticoid receptors, fibromyalgia and low back pain.

Recently, fibromyalgia (FMS) was shown to be a disorder associated with an altered functioning of the stress response system. FMS patients display a hyperreactive pituitary adrenocorticotropic hormone (ACTH) release in response to corticotropin-releasing hormone (CRH) and to insulin-induced hypoglycemia. We suggested that negative feedback of cortisol could be deranged. Therefore we investigated the properties and function of the glucocorticoid receptors (GR) in FMS patients and compared the results with those of healthy persons and patients with chronic low back pain (LBP a localized pain condition). Forty primary FMS patients (F:M = 36:4), 28 LBP patients (25:3) and 14 (12:2) healthy, sedentary control persons were recruited for the study. Urinary free cortisol excretion in FMS and LBP patients was lower compared to controls. Only FMS patients displayed lower CBG and basal serum cortisol concentrations when compared to controls. However, plasma free cortisol concentrations were similar in the three groups. There was no difference in the number of GR per cell among the three groups (FMS: 6498 +/- 252, LBP: 6625 +/- 284, controls: 6576 +/- 304), but the dissociation constant (Kd) of the FMS (14.5 +/- 0.9 nmol/l) and LBP (14.7 +/- 1.3 nmol/l) subjects was significantly higher than that of the controls (10.9 +/- 0.8 nmol/l) (p < .05). The maximal stimulation of the lymphocytes, as measured by the maximal thymidine incorporation (in the absence of cortisol) in the FMS group was approximately 1.5 times higher (p < .05) than in the control or LBP group. The ED50 (the cortisol concentration giving 50% inhibition of the thymidine incorporation), however, was identical in all three groups. We conclude that FMS patients have a mild hypocortisolemia, increased cortisol feedback resistance in combination probably with a reduced CRH synthesis or release in the hypothalamus. The role of the GR and mineralocorticoid receptor (MR) in the CRH regulation in the FMS patients remains to be solved.