Autoinflammation in pyoderma gangrenosum and its syndromic form (pyoderma gangrenosum, acne and suppurative hidradenitis).

BACKGROUND: Pyoderma gangrenosum (PG) is a rare skin disease characterized clinically by ulcers with undermined borders, and histologically by neutrophil-rich infiltrates. PG may occur alone, in syndromic forms or associated with systemic diseases, such as inflammatory bowel disease and haematological or rheumatological disorders. OBJECTIVES: To determine a specific genetic background related to autoinflammation for PG. METHODS: We assessed autoinflammation by evaluating the cytokine profile and genes involved in classic autoinflammatory diseases in 13 patients with PG and in seven patients with the syndromic form, known as PASH (pyoderma gangrenosum, acne and suppurative hidradenitis). RESULTS: In skin samples, the expression of interleukin (IL)-1ß and its receptors, IL-17 and its receptor, and tumour necrosis factor-α and its receptors were significantly higher in both PG (P = 0·001) and in PASH (P < 0·001) than in controls. The chemokines IL-8; chemokine (C-X-C motif) ligand 1/2/3; chemokine (C-X-C motif) ligand 16; and RANTES (regulated on activation, normal T-cell-expressed and secreted) were also overexpressed. Cases of PG and PASH showed mutations in the autoinflammatory genes MEFV, NLRP3, NLRP12, NOD2, LPIN2 and PSTPIP1. CONCLUSIONS: Overexpression of cytokines/chemokines, along with genetic changes, supports the hypothesis that PG and its syndromic form, PASH, are a spectrum of polygenic autoinflammatory conditions.

Expression of cytokines, chemokines and other effector molecules in two prototypic autoinflammatory skin diseases, pyoderma gangrenosum and Sweet's syndrome.

Pyoderma gangrenosum (PG) and Sweet's syndrome (SS) are two inflammatory skin diseases presenting with painful ulcers and erythematous plaques, respectively; both disorders have a debilitating clinical behaviour and PG is potentially life-threatening. Recently, PG and SS have been included among the autoinflammatory diseases, which are characterized by recurrent episodes of sterile inflammation, without circulating autoantibodies and autoreactive T cells. However, an autoinflammatory pattern clearly supporting this inclusion has never been demonstrated. We studied 16 patients with PG, six with SS and six controls, evaluating, using a sandwich-based protein antibody array method, the expression profile of inflammatory effector molecules in PG, SS and normal skin. The expressions of interleukin (IL)-1 beta and its receptor I were significantly higher in PG (P = 0·0001 for both) and SS (P = 0·004-0·040) than in controls. In PG, chemokines such as IL-8 (P = 0·0001), chemokine (C-X-C motif) ligand (CXCL) 1/2/3 (P = 0·002), CXCL 16 (P = 0·003) and regulated upon activation normal T cell expressed and secreted (RANTES) (P = 0·005) were over-expressed. In SS, IL-8 (P = 0·018), CXCL 1/2/3 (P = 0·006) and CXCL 16 (P = 0·036) but not RANTES were over-expressed, suggesting that chemokine-mediated signals are lower than in PG. Fas/Fas ligand and CD40/CD40 ligand systems were over-expressed in PG (P = 0·0001 for Fas, P = 0·009 for Fas ligand, P = 0·012 for CD40, P = 0·0001 for CD40 ligand), contributing to tissue damage and inflammation, while their role seems to be less significant in SS. Over-expression of cytokines/chemokines and molecules amplifying the inflammatory network supports the view that PG and SS are autoinflammatory diseases. The differences in expression profile of inflammatory effectors between these two disorders may explain the stronger local aggressiveness in PG than SS.

High-molecular-weight kininogen cleavage correlates with disease states in the bradykinin-mediated angioedema due to hereditary C1-inhibitor deficiency.

BACKGROUND: The inherited deficiency of C1-inhibitor (C1-INH), which can be quantitative (type I) or qualitative (type II), is characterized by recurrent attacks of oedema, and it is known as hereditary angioedema due to C1-INH deficiency (HAE-C1-INH). The frequency of symptoms varies widely among patients and in the same patient during life. OBJECTIVE: To identify laboratory markers of disease severity in HAE-C1-INH patients. METHODS: We studied 162 patients with differently severe HAE-C1-INH during remission, 31 HAE-C1-INH patients during attacks, and 81 normal controls, evaluating complement parameters, spontaneous plasma kallikrein activity, the capacity of plasma to inhibit exogenous kallikrein activity, and cleavage of high-molecular-weight kininogen (HK). Sixty-five HAE-C1-INH patients were screened for mutations in the C1-INH gene. RESULTS: As expected, plasma C1-INH levels and activity and C4 levels were low in the HAE-C1-INH patients. Spontaneous plasma kallikrein activity in patients in remission was higher than in controls (P = 0.001) and increased during acute attacks (P = 0.01), whereas the capacity of inhibiting kallikrein activity was lower in patients in remission than in controls (P = 0.001) and further reduced during attacks (P = 0.001). HAE-C1-INH patients in remission had higher levels of cleaved HK than controls (P = 0.001), and these further increased during acute attacks (P = 0.001). Cleaved HK levels were higher in highly symptomatic HAE-C1-INH patients than in those with less frequent attacks (P = 0.001). Thirty-five different mutations in the C1-INH gene were equally distributed in patients with different attack frequencies. CONCLUSIONS: Measuring plasma levels of cleaved HK may be a sensitive mean of assessing disease severity in HAE-C1-INH patients.

Inflammatory cells, cytokines and matrix metalloproteinases in amicrobial pustulosis of the folds and other neutrophilic dermatoses.

Amicrobial pustulosis of the folds (APF) is a rare cutaneous disease characterized by relapsing sterile pustules frequently associated with autoimmune disorders. Although APF pathophysiology is still undefined, scattered reports suggest involvement of neutrophils. The aim of the present study is to evaluate the role of the skin inflammatory infiltrate, selected multifunctional cytokines and effectors of tissue damage in APF and other neutrophilic dermatoses. We studied, by immunohistochemical methods, inflammatory cell markers (CD3, CD163, myeloperoxidase), cytokines (TNF-alpha, IL-8, IL-17), metalloproteinases (MMP-2, MMP-9) and vascular-endothelial-growth-factor (VEGF) in lesional skin from six patients with APF, 11 with pyoderma gangrenosum (PG), 7 with Sweet's syndrome, and in 20 normal skin samples. Immunoreactivities of CD3, CD163, myeloperoxidase, TNF-alpha, IL-8, IL-17, MMP-2, MMP-9 and VEGF were significantly higher in APF, PG and Sweet's syndrome than in controls (p=0.0001). IL-8 was more expressed in PG than in APF (P=0.002) and Sweet's syndrome (p=0.001). In APF, MMP-9 reactivity was higher than in Sweet's syndrome (p=0.035), but less intense than in PG (p=0.020). Our study supports the role of proinflammatory cytokines/chemokines and MMPs as important effectors for the tissue damage in APF similarly to classic neutrophilic dermatoses.

Markers of autoreactivity, coagulation and angiogenesis in patients with nonallergic asthma.

BACKGROUND: Patients with nonallergic asthma frequently show autoreactivity as do subjects with chronic urticaria (CU). Activation of the coagulation cascade and hyper-expression of vascular endothelial growth factor (VEGF) were recently found in CU, and there is sparse evidence that the same may occur in asthma. OBJECTIVE: To investigate autoreactivity, activation of the coagulation cascade, and expression of VEGF in patients with nonallergic asthma. METHODS: Twenty-one adults with nonallergic asthma underwent autologous plasma skin test (APST) and the measurement of plasma levels of the prothrombin fragment F1+2, D-dimer, VEGF, and the inflammatory marker C-reactive protein (CRP). Twenty-one healthy sex- and age-matched subjects served as normal controls. RESULTS: The APST scored positive in 19 of 21 (90%) patients vs 0 controls. Mean fragment F1+2 plasma levels were significantly higher in patients with asthma (267 ± 243 pM) than in controls (150 ± 51 pM; P = 0.0001). Similarly, plasma levels of both D-dimer and VEGF were significantly higher in patients than in controls (D-dimer: 2364 ± 1467 vs 1301 ± 525 pM; P = 0.0001; VEGF: 1721 ± 2566 vs 76 ± 375 fM; P = 0.0001). A trend toward increased levels of F1+2, D-dimer, VEGF, and CRP was found in patients with a more severe disease according to GINA classification. CONCLUSION: Nonallergic asthma is characterized by autoreactivity as well as increased coagulation and angiogenesis markers, which are known to enhance vascular permeability. The presence of circulating vasoactive factors may be relevant to understand the disease pathophysiology and to detect novel therapeutic strategies in nonallergic asthma.

Activation of coagulation in bullous pemphigoid and other eosinophil-related inflammatory skin diseases.

Bullous pemphigoid (BP) is a skin disease caused by autoantibodies to hemidesmosomal proteins BP180 and BP230, with eosinophils participating in blister formation. Tissue factor (TF), the initiator of coagulation, is embodied within the eosinophil granules and exposed upon activation. We evaluated the coagulation activation in patients with BP (63), chronic urticaria (CU; 20), atopic dermatitis (AD; 14), cutaneous drug reactions (CDRs; six), psoriasis (20), dermatitis herpetiformis (DH; four) and primary cutaneous T cell lymphoma (CTCL; five), and in 40 healthy controls. Prothrombin fragment F1+2 and d-dimer (coagulation markers) were measured by enzyme-linked immunosorbent assay (ELISA) in all plasma samples and BP blister fluid. Skin TF expression was evaluated immunohistochemically in the patients and 20 controls. F1+2 and d-dimer levels were higher in BP plasma than in control plasma (P = 0·0001 for both), and dramatically high in blister fluid; both correlated positively with disease severity, esinophil counts and anti-BP180 antibodies (P = 0·006-0·0001). Plasma F1+2 and d-dimer levels were higher in the CU, AD and CDR patients than in controls (P = 0·0001 for all), but normal in the psoriasis, DH and CTCL patients. Skin TF was expressed in the BP (P = 0·0001), CU (P = 0·0001), AD (P = 0·001) and CDR patients (P = 0·01), but not in the psoriasis, DH or CTCL patients. Co-localization confocal microscopy studies confirmed eosinophils as the source of TF in 10 BP patients. The coagulation cascade is activated in BP and other eosinophil-mediated skin disorders, but not in non-eosinophil driven conditions. This hypercoagulability may contribute to inflammation, tissue damage and, possibly, thrombotic risk.

Role of inflammatory cells, cytokines and matrix metalloproteinases in neutrophil-mediated skin diseases.

Pyoderma gangrenosum (PG) is a rare, immune-mediated inflammatory skin disease presenting with painful ulcers having undermined edges. Less commonly, bullous and vegetative variants exist. Histology consists of a neutrophil-rich dermal infiltrate. We characterized immunohistochemically the infiltrate in different variants of PG and in another neutrophilic dermatosis as Sweet's syndrome. We studied 21 patients with PG, eight with Sweet's syndrome and 20 controls, evaluating skin immunoreactivity for inflammatory cell markers (CD3, CD163 and myeloperoxidase), cytokines [tumour necrosis factor (TNF)-α, interleukin (IL)-8 and IL-17], metalloproteinases (MMP-2 and MMP-9) and vascular endothelial growth factor (VEGF). Immunoreactivities of CD3, CD163, myeloperoxidase, TNF-α, IL-8, IL-17, MMP-2, MMP-9 and VEGF were significantly higher in both PG and Sweet's syndrome than in controls (P=0·0001). Myeloperoxidase (neutrophil marker), IL-8 (cytokine chemotactic for neutrophils) and MMP-9 (proteinase-mediating tissue damage) were expressed more significantly in both ulcerative and bullous PG than in vegetative PG as well as in Sweet's syndrome (P=0·008-P=0·0001). In ulcerative PG, the expression of CD3 (panT cell marker) and CD163 (macrophage marker) were significantly higher in wound edge than wound bed (P=0·0001). In contrast, the neutrophil marker myeloperoxidase was expressed more significantly in wound bed than wound edge (P=0·0001). Our study identifies PG as a paradigm of neutrophil-mediated inflammation, with proinflammatory cytokines/chemokines and MMPs acting as important effectors for the tissue damage, particularly in ulcerative and bullous PG where damage is stronger. In ulcerative PG, the wound bed is the site of neutrophil-recruitment, whereas in the wound edge activated T lymphocytes and macrophages pave the way to ulcer formation.

Anti-tumor necrosis factor alpha therapy normalizes fibrinolysis impairment in patients with active rheumatoid arthritis.

OBJECTIVES: Rheumatoid arthritis (RA) is associated with increased cardiovascular risk and involvement of inflammation, coagulation and fibrinolysis. Treatment with infliximab, a tumour necrosis factor-alpha (TNF-alpha) blocking chimeric monoclonal antibody, induces a long-term reduction of inflammation and coagulation, but its effect on fibrinolysis is still unknown. We carried out an observational study investigating plasma biomarkers of inflammation and fibrinolysis in RA patients before and after 14 weeks of infliximab treatment given according to the therapeutic guidelines for RA. METHODS: We studied 20 selected patients with active RA and without any other atherosclerosis risk factor as well as 40 healthy controls. Patients, treated with a stable dose of methotrexate, received infliximab (3 mg/kg) at week 0, 2, 6 and 14. At week 0 and 14, we assessed clinical, inflammatory and fibrinolyitic parameters. RESULTS: At baseline, plasminogen activator inhibitor (PAI-1) antigen, PAI-1 activity and tissue-type plasminogen activator (t-PA) antigen were significantly higher in RA patients than in controls (p=0.01, p=0.001 and p=0.0001 respectively). After 14 weeks of infliximab treatment, the levels of PAI-1 antigen, PAI-1 activity and t-PA antigen significantly decreased till normalization (p=0.0001). Plasma levels of C reactive protein (CRP) and interleukin-6 (IL-6) were directly correlated with levels of PAI-1 antigen (p=0.011 and p=0.0001), PAI-1 activity (p=0.013 and p=0.027) and t-PA antigen (p=0.017 and p=0.040). CONCLUSIONS: This study provides evidence that TNF-alpha blockade by infliximab not only decreases inflammation, but also reduces the inhibition of fibrinolysis. Such a combined effect may be pivotal in reducing the whole thrombotic risk in these patients.

Time course of endogenous nitric oxide inhibitors in severe sepsis in humans.

AIM: Asymmetric and symmetric dimethylarginines (ADMA and SDMA, respectively) are protein breakdown markers; both compete with arginine for cellular transport and both are excreted in urine. Moreover, ADMA is a non-selective inhibitor of nitric oxide (NO) synthase that is metabolized by a specific hydrolase in which the activity during stress remains controversial. While an increase in ADMA is known to be associated with adverse events, little is known about SDMA. We investigated plasma ADMA and SDMA levels during ICU stay to reveal the time course of endogenous NO inhibition in patients with sepsis. METHODS: A post hoc analysis from a prospective random controlled trial conducted in three ICUs was performed to study the pathophysiological pathways of sepsis. ADMA, SDMA, the ratio of ADMA/SDMA (a marker of ADMA catabolism), arginine, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and C reactive protein (CRP) were measured on days 1, 3, 6, 9, 12 and at discharge in 72 consecutive severely septic patients. RESULTS: Fasting basal glycemia, creatinine, IL-6, TNF-alpha, CRP, ADMA, and SDMA were higher than normal. The ADMA/SDMA ratio was decreased by 50%, and arginine levels were low. ADMA levels were related to the total Sequential Organ Failure Assessment (SOFA) scores and arginine levels, and inversely related to IL-6 and CRP levels. SDMA levels were related to Simplified Acute Physiologic Scores II (SAPS II), SOFA scores, blood urea, creatinine, and arginine levels. The ADMA/SDMA ratio was inversely related to IL-6 levels. In 58 ICU survivors, creatinine, IL-6, and CRP levels decreased over time; ADMA levels increased, SDMA levels remained stable, and the ADMA/SDMA ratio increased. In 14 non-survivors, creatinine, IL-6, TNF-alpha, CRP, and ADMA levels were stable, whereas the SDMA levels increased and the ADMA/SDMA ratio remained low. In both ICU survivors and non-survivors, the levels on the last ICU day confirmed the data trends. SDMA, but not ADMA, was associated with ICU mortality. CONCLUSION: ADMA catabolism appears to be activated by inflammation; its increase during the advanced septic phase in surviving patients may suggest an endogenous inhibition of NO synthesis during the full-blown septic phase. In severe sepsis, SDMA, but not ADMA, appears to be a marker of alterations in vital functions and mortality.

Plasma levels of matrix metalloproteinase-9 in chronic urticaria patients correlate with disease severity and C-reactive protein but not with circulating histamine-releasing factors.

BACKGROUND: Matrix metalloproteinase-9 (MMP-9) is an endopeptidase produced by many inflammatory cells that has been found in increased amounts in plasma from patients with chronic urticaria (CU). OBJECTIVE: To evaluate plasma levels of MMP-9 and its tissue inhibitor of metalloproteinase-1 (TIMP-1) in CU patients in relation with disease severity, C-reactive protein (CRP) and circulating histamine-releasing factors. METHODS: Fifty-two consecutive CU patients were included in the study and disease activity was graded from 0 to 3. Plasma MMP-9, TIMP-1 and CRP levels were measured by enzyme immunoassays. Circulating histamine-releasing factors were assessed using in vivo (autologous serum skin test) and in vitro (basophil histamine release) tests. Seven CU patients were studied both during active disease and during remission. Thirty healthy subjects were used as normal controls. RESULTS: Plasma levels of MMP-9, TIMP-1 and CRP were significantly higher in CU patients than in healthy controls (P=0.0001, 0.003 and 0.005, respectively) and a trend towards a higher MMP-9/TIMP-1 molar ratio was found (P=0.051). A significant correlation was found between plasma MMP-9 levels and urticaria severity score (r=0.48, P<0.0001). CRP levels correlated with MMP-9 levels (r=0.37, P=0.008) and CU severity score (r=0.52, P=0.0001), but not with TIMP-1 (r=0.13) concentrations. MMP-9, TIMP-1 and CRP plasma levels and MMP-9/TIMP-1 molar ratio did not differ in patients either with or without an evidence of circulating histamine-releasing factors. Seven patients evaluated during remission showed a significant reduction of MMP-9 and CRP plasma levels. CONCLUSION: Plasma levels of MMP-9 and its inhibitor TIMP-1 are increased in CU patients. MMP-9 levels are associated with disease severity and CRP levels, but not with skin reactivity to autologous serum and with circulating histamine-releasing factors. These findings suggest that in CU there is an ongoing inflammatory process independent of the presence of circulating histamine-releasing factors.

Prothrombotic state in Churg-Strauss syndrome: a case report.

An increased risk of thrombosis has been described in patients with hypereosinophilic syndromes, including Churg-Strauss syndrome (CSS). We report the case of a 43-year-old man with CSS who presented with asthma, pansinusitis, blood eosinophilia (9650/microL), peripheral neuropathy, cutaneous eosinophilic vasculitis, and a positive result for antineutrophil cytoplasmic antibodies. An analysis of plasma during active disease revealed elevated levels of prothrombin fragment 1+2 (marker of thrombin generation) (832 pM; normal range, 68-229 pM) and D-dimer (marker of fibrin degradation) (2300 ng/mL; normal range, 130-250 ng/mL), which indicate an increased risk of thrombosis. Both parameters returned to normal values during remission after immunosuppressive treatment. Skin histology showed leukocytoclastic vasculitis with numerous eosinophils in the dermal infiltrate. Immunohistochemistry revealed expression of tissue factor by skin-infiltrating eosinophils, as confirmed by colocalization with eosinophil cationic protein, a classic marker of eosinophil granulocytes. In conclusion, we present a patient with active CSS and a prothrombotic state that reverted during remission achieved by immunosuppressive therapy.

Plasma levels and skin-eosinophil-expression of vascular endothelial growth factor in patients with chronic urticaria.

BACKGROUND: Although chronic urticaria (CU) is often regarded as autoimmune in nature, only less than 50% of sera from CU patients contain histamine-releasing autoantibodies. This suggests that other factors may contribute to its pathogenesis. We evaluated the possible involvement of vascular endothelial growth factor (VEGF), one of the major mediators of vascular permeability, in CU. METHODS: Eighty consecutive adult patients with CU and 53 healthy subjects were studied. VEGF and prothrombin fragment F(1+2) were measured by enzyme immunoassays. Autologous plasma skin test (APST) was performed in CU patients and, in six of them, skin biopsy specimens were taken from wheals to evaluate the immunohistochemical expression of VEGF and eosinophil cationic protein (ECP). RESULTS: Plasma VEGF concentrations were higher in CU patients (8.00 +/- 0.90 pmol/l) than in controls (0.54 +/- 0.08 pmol/l) (P = 0.0001) and tended to parallel both the severity of CU and to correlate with F(1+2) levels. APST was positive in 85.1% of patients. VEGF concentration was significantly higher in APST-positive than in APST-negative patients (P = 0.0003). Immunohistochemically, all specimens from patients with CU showed a strong expression of VEGF (P = 0.002) that colocalized with ECP, a classic eosinophil marker. CONCLUSIONS: VEGF plasma levels are elevated in CU and parallel the disease severity. This supports a possible role of this molecule in CU pathophysiology. Eosinophils are the main cellular source of VEGF in CU lesional skin.

Antihistamines do not inhibit the flare induced by the intradermal injection of autologous plasma in chronic urticaria patients.

BACKGROUND: There is some evidence suggesting that factors other than autoantibodies to FceRI or IgE and histamine released from mast cells may play a role in skin autoreactivity that characterizes many patients with chronic urticaria (CU) and, possibly, in the pathogenesis of this disease. OBJECTIVE: The effect of antihistamine treatment on autologous plasma skin test (APST) in patients with CU was assessed. METHODS: 24 patients with CU underwent autologous plasma skin test (APST) as well as SPT with histamine 10 mg/ml while taking antihistamines. In 6 cases the same tests had been carried out also before the start of antihistamine treatment. Plasma levels of D-dimer, prothrombin F 1+2 fragment, and vascular endothelial growth factor (VEGF) were measured in 21 patients. RESULTS: 21/24 (87%) patients showed a large flare on APST while taking antihistamines while the skin reaction to histamine 10 mg/ml was abolished or negligible. Little difference in the autologous plasma-induced flare was seen before and after the start of cetirizine therapy in 6 cases, whereas the drug exerted a marked effect on the histamine SPT as well as on the autologous plasma-induced wheal. The APST-induced flare was not associated with patients' response to antihistamine. Plasma levels of VEGF, prothrombin F 1+2 fragment, and D-dimer were increased in plasmas from 8, 9, and 2 patients, respectively. CONCLUSIONS: Factors other than histamine are probably involved in the flare following APST in CU; such factors might play a pathogenic role particularly in patients not responding to standard antihistamine treatments.

Detection of serum histamine-releasing factors in a patient with idiopathic anaphylaxis and multiple drug allergy syndrome.

We describe the case of a 30-year-old woman who reported several episodes of anaphylaxis with angioedema and relapsing urticaria. Some events were related to nonsteroidal anti-inflammatory drug intake and one episode followed alcohol ingestion, but in most cases no triggers could be identified. Specific immunoglobulin E determination was negative for food and drug allergens, C3 and C4 were in the normal range, C1 inhibitor was slightly reduced and serum tryptase was undetectable. In vivo autologous serum skin test and in vitro basophil histamine release assay were positive indicating the presence of circulating histamine-releasing factors. When oral tolerance tests were performed, only doxycycline was tolerated whereas levofloxacin, clarithromycin, nimesulide and tramadol caused mild urticaria. Premedication with cetirizine allowed the patient to tolerate levofloxacin, clarithromycin and nimesulide. The demonstration of circulating histamine-releasing factors in a patient with idiopathic anaphylaxis and multiple drug allergy syndrome provides a new mechanistic insight and might open the way to new therapeutic approaches.

Increase of bradykinin in plasma of patients undergoing cardiopulmonary bypass: the importance of lung exclusion.

STUDY OBJECTIVES: Hemodynamic complications including hypotensive episodes are frequently associated with cardiopulmonary bypass (CPB) and can be attributed to a generalized inflammatory response in which bradykinin may be a mediator. The purpose of this study was to determine the plasma levels of bradykinin-(1-9)nonapeptide in patients during CPB and the physiologic elimination of bradykinin by the lungs. DESIGN: Prospective, observational study. SETTING: University hospital, cardiac surgery unit. PATIENTS AND METHODS: Intra-arterial BP was monitored and serial blood samples were obtained from 27 patients undergoing CPB for cardiac surgery. We measured plasma bradykinin and parameters of coagulation, fibrinolysis, complement, contact system, and the cytokine tumor necrosis factor (TNF). RESULTS: Mean arterial pressure fell progressively until the end of CPB (- 18 mm Hg, p = 0.001) but returned to baseline by the end of surgery. The venous bradykinin level, normal in basal conditions (median, 1.90 fmol/mL), was increased (p = 0.001) from 15 min after the beginning of CPB (5.71 fmol/mL) to the end of the operation (7.07 fmol/mL), with a peak at the end of CPB (9.81 fmol/mL; p = 0.0001); it was normal at recovery 24 h later (2.81 fmol/mL). Bradykinin plasma levels fell 60% across the lung when the pulmonary circulation was fully restored while the patients were still receiving CPB. Activated-factor XII, thrombin-antithrombin complexes, prothrombin fragment F1 + 2, plasmin-antiplasmin complexes, C(3)a, and TNF increased significantly after the beginning of the surgical procedure, rising further during CPB, and remained elevated until the end of surgery, but they all returned to normal within 24 h. Changes in plasma bradykinin levels were not correlated with any of the other variables. CONCLUSIONS: During CPB, there is a progressive increase of plasma bradykinin that is at least partially due to reduced catabolism as a consequence of shunting the lungs. The increase in bradykinin may contribute to the fall in BP.

Bradykinin in the ascitic fluid of patients with liver cirrhosis.

Bradykinin, a nonapeptide with vasodilatory and permeabilizing activity, is generated through the cleavage of high-M(r) ('high-molecular-weight') kininogen by kallikrein, and its generation is facilitated by plasmin. In the ascitic fluid of patients with cirrhosis, there is massive cleavage of high-M(r) kininogen and activation of fibrinolysis, but bradykinin has never been measured directly. In the ascitic fluid of 24 patients with cirrhosis, we measured bradykinin-(1-9)-nonapeptide levels by RIA after liquid-phase and subsequent HPLC extraction, and those of its catabolic product bradykininin-(1-5)-pentapeptide by ELISA after liquid-phase extraction. Cleaved high-M(r) kininogen, activated factor XII and plasmin-antiplasmin complexes were measured in ascitic fluid and plasma. Plasma renin activity (PRA) was also determined. As a control, we also analysed plasma from 24 healthy subjects matched for sex and age with the patients. In the ascitic fluid from patients with cirrhosis, the median bradykinin-(1-9) concentration was 3.3 fmol/ml (range 0.2-29.0 fmol/ml), and the median bradykinin-(1-5) concentration was 210 fmol/ml (range 58-7825 fmol/ml). The levels of bradykinin-(1-5) in ascitic fluid were higher in patients with refractory ascites [median 1091 fmol/ml (range 58-7825 fmol/ml)] than in patients with responsive ascites [134 fmol/ml (72-1084 fmol/ml)] (P=0.010). Ascitic fluid levels of bradykinin-(1-9) were not related to the severity of ascites. PRA was higher in patients with refractory ascites [23.0 ng x h(-1) x ml(-1) (7.9-80.0 ng.h(-1).ml(-1))] than in patients with responsive ascites [6.9 ng x h(-1) x ml(-1) (0.9-29.4 ng x h(-1) x ml(-1))] (P=0.002). In ascitic fluid, 48% (19-68%) of high-M(r) kininogen was cleaved, and plasmin-antiplasmin complexes were more concentrated than in plasma (P=0.0001). In conclusion, in the ascitic fluid of patients with cirrhosis, both bradykinin-(1-9) and bradykinin-(1-5) are present, with cleavage of high-M(r) kininogen and activation of fibrinolysis. The highest levels of the long-lived metabolite bradykinin-(1-5) were found in the ascitic fluid of patients with refractory ascites and high PRA. Activation of the kinin system may therefore be involved in decompensating cirrhosis, but a cause-effect relationship remains to be established.

Treatment of Raynaud's phenomenon with intravenous prostaglandin E1alpha-cyclodextrin improves endothelial cell injury in systemic sclerosis.

OBJECTIVE: To evaluate the efficacy and safety of prostaglandin (PG) E1alpha-cyclodextrin for Raynaud's phenomenon (RP) secondary to systemic sclerosis (SSc) and its effect on variables of immune activation and endothelial injury in SSc such as tumor necrosis factor-alpha (TNF-alpha), soluble interleukin 2 receptor (sIL-2R), circulating intercellular adhesion molecule-1 (cICAM-1), von Willebrand factor (vWF), and tissue-type plasminogen activator (t-PA). METHODS: We studied 36 women with SSc, 24 of them given three 60 microg intravenous PGE1alpha-cyclodextrin infusions on 5 consecutive days at 6 week intervals during the winter. RP symptoms and healing of digital lesions were evaluated. Twenty age matched healthy women were the controls. TNF-alpha, sIL-2R, cICAM-1, vWF, and t-PA were measured after the first and last infusion of PGEE1alpha-cyclodextrin and correlated with clinical features. RESULTS: RP symptoms improved in 87% of the patients. The benefit of each 5 day cycle lasted 4 or more weeks in 75%. PGE1alpha-cyclodextrin reduced the daily frequency of RP symptoms by 20% (p < 0.05), 41% (p < 0.005), and 53% (p < 0.0005) from baseline after the 1st, 2nd, and 3rd infusions, respectively. The severity of the attacks was reduced to a limited degree. In 12 of the 14 patients with digital lesions, these healed completely. Ten patients had mild side effects during treatment (headache, increased intestinal motility, flushing). TNF-alpha, sIL-2R, cICAM-1, vWF, and t-PA plasma concentrations were significantly higher in patients with SSc than controls (p < 0.05, p < 0.001). TNF-alpha, sIL-2R, and cICAM-1 were higher in diffuse SSc and patients with lung involvement. The plasma levels of cICAM-1 and t-PA were significantly reduced after the 1st infusion of PGE1alpha-cyclodextrin (both p < 0.005) and further reduced after the last (p < 0.0005 and p < 0.005). CONCLUSION: PGE1alpha-cyclodextrin reduces RP symptoms and plasma levels of the markers of endothelial injury in SSc, suggesting that an improvement of endothelial dysfunction contributes to its prolonged therapeutic effect.

[The kinin system: biological mechanisms and clinical implications]. / Sistema delle chinine: meccanismi biologici ed implicazioni cliniche.

The discovery of the kinin system is not recent, but its study in clinical field has been done only in the last years. This system is composed by substrates (kininogens) and plasma and tissue kallikreins are the specific activators of these substrates producing two vasoactive peptides called bradykinin and kallidin. The biologic effects of kinins are mediated by specific receptors called B1 and B2. The activation of this system is particularly important in blood pressure regulation and in inflammatory reactions. The kinin system is involved in many clinical situations including respiratory allergic reactions, septic shock, hypertension and its treatment, hypotensive transfusion reactions, heart diseases, pancreatitis, hereditary and acquired angioedema, Alzheimer's disease and liver cirrhosis with ascites. The study of the kinin system in clinical field, which had been limited by methodological difficulties, has now received an important stimulus by the recent availability of specific and sensitive methods of dosage.

Plasma bradykinin levels in human chronic congestive heart failure.

Induction of congestive heart failure by high-frequency pacing has been reported to increase plasma levels of immunoreactive kinins in dogs. In the present study, we evaluated plasma bradykinin levels in human heart failure. Utilizing a recently developed method, we specifically measured plasma levels of bradykinin-(1-9) nonapeptide in 21 patients with chronic congestive heart failure [New York Heart Association (NYHA) stages III and IV). At the same time, we measured plasma atrial natriuretic peptide levels and plasma renin activity, and, as a marker of inflammation, plasma levels of tumour necrosis factor. In addition, 18 healthy subjects matched for gender and age served as normal controls. Plasma bradykinin concentrations were not higher in patients with chronic congestive heart failure (median 2.1 fmol/ml) than in healthy subjects (2.6 fmol/ml). In contrast, plasma atrial natriuretic peptide levels were clearly higher (patients, 63 fmol/ml; controls, 24 fmol/ml; P<0.0001), despite diuretic treatment and in the presence of high plasma renin activity (patients, 13.0 ng x h(-1) x ml(-1); controls, 0.3 ng x h(-1) x ml(-1); P<0.0001). Tumour necrosis factor was elevated in heart failure patients in NYHA class IV only (27 pg/ml, compared with 21 pg/ml in controls; P=0.013). Bradykinin, atrial natriuretic peptide and plasma renin activity levels were not correlated with the severity of the disease, as assessed by NYHA classification. These results indicate that a rather selective cytokine activation, without concomitant stimulation of the kallikrein-kinin system, occurs in human chronic congestive heart failure.

Activation of the contact system in cerebrospinal fluid of patients with Alzheimer disease.

Several converging lines of evidence suggest that beta-amyloid and inflammation may be linked in the pathogenesis of Alzheimer disease (AD), but the mechanism of beta-amyloid neurotoxicity is unclear. In this study, by demonstrating that high molecular weight kininogen may be massively cleaved in the cerebrospinal fluid (CSF) of patients with AD, we provide evidence of the potential involvement of the contact system in the inflammatory processes taking place in this disease. In the CSF of patients with neuroimmune inflammatory disease (multiple sclerosis, chronic inflammatory demyelinating polyneuropathy), there was no evidence of increased cleavage of high molecular weight kininogen, suggesting that this finding may be characteristic of the Alzheimer brain. The data obtained from in vitro experiments seem to indicate that the cleavage of high molecular weight kininogen in vivo may be the result of the interaction of beta-amyloid with factor XII and of kallikrein generation. The actual relevance of such a phenomenon remains to be established in vivo. However, the demonstration that the contact system may be activated in the brains of Alzheimer patients points to the potential involvement of the kallikrein-kinin system in the inflammatory process of this disease.