Oxandrolone treatment of childhood hereditary angioedema.

BACKGROUND: The virilizing effects of danazol, stanozolol, and methyltestosterone significantly restrict the usefulness of these agents in the treatment of children with hereditary angioedema (HAE). Oxandrolone is a synthetic anabolic steroid with limited virilizing effects that has been used in a variety of pediatric conditions and has an acceptable safety profile. OBJECTIVE: To report the effective use of oxandrolone in a 6-year-old boy with recurrent, life-threatening episodes of angioedema. METHODS: Oxandrolone was administered at a dose of 0.1 mg/kg per day. Symptoms and laboratory findings were evaluated by parental report and laboratory analysis of serum C1 esterase inhibitor and C4 levels, respectively. RESULTS: Oxandrolone therapy resulted in a marked reduction in clinical episodes and normalization of serum complement levels; cessation of oxandrolone therapy resulted in recurrence of symptoms and decreased complement levels. However, early signs of virilization were noted. CONCLUSIONS: Oxandrolone treatment was associated with significant clinical and laboratory evidence of a therapeutic effect in a prepuberal boy with HAE. It is imperative to treat HAE with the lowest dose of oxandrolone that controls life-threatening episodes of angioedema.

Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors.

Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 micro/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 microM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 microM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 microM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM).

Increased expression of C1-inhibitor mRNA in patients with hereditary angioedema treated with Danazol.

The attenuated androgen Danazol can partially reverse the biochemical defect and prevent angioedema in patients with inherited C1-inhibitor (C1-INH) deficiency (hereditary angioedema, HAE). Though its clinical effectiveness is independent from significant increase of C1-INH plasma levels, its mechanism of action remains unknown. Since angioedema is a local phenomenon, it could be controlled by restoring tissue levels of C1-INH. We measured the expression of C1-INH mRNA in peripheral blood mononuclear cells (PBMCs) of 13 patients with HAE type 1 (seven untreated and asymptomatic, and six on Danazol at the minimal effective dose) and of eight normal controls. mRNA levels were quantitated by computerized optical densitometry of reverse transcriptase-PCR products, normalized for the amount of glyceraldehyde-3-phosphate-dehydrogenase and expressed as percent of normal pooled RNAs. Each determination represented the mean of three separate experiments. Measurement of C1-INH mRNA in two patients before and after 1 month of Danazol 400 mg per day demonstrated a post-treatment increase of 15 and 21%, respectively. When HAE patients and controls were analyzed as groups, C1-INH mRNA levels of patients untreated and asymptomatic (median 73%, range 65-78) were significantly lower (P=0.001) compared to controls (median 101%, range 87-121) and to patients on Danazol (median 91%, range 82-96); the difference among the last two groups was not statistically significant. Our data demonstrate that minimal effective doses of Danazol increase the expression of C1-INH mRNA in PBMC of HAE patients even in the absence of a significant increase of C1-INH plasma levels.

Purification of proplatelet formation (PPF) stimulating factor: thrombin/antithrombin III complex stimulates PPF of megakaryocytes in vitro and platelet production in vivo.

In this study, the protein which stimulates proplatelet formation (PPF) of megakaryocytes was purified from normal human plasma using 7 steps procedures. Two different protease inhibitors were identified based on their amino acid sequences, i.e. antithrombin III (AT III) and C1 inhibitor. They were included in high density lipoprotein (HDL). HDL was necessary for AT III to be active in PPF in vitro. The biological effects of the AT III/HDL or thrombin-AT III (TAT)/HDL were studied in vitro. PPF of murine megakaryocytes was stimulated by negative control (BSA) (1.8 +/- 0.3%), AT III (2.0 +/- 0.4%), HDL (1.2 +/- 0.9%), AT III/HDL (14.8 +/- 2.1%) or TAT/HDL (23.3 +/- 3.5%), respectively. TAT/HDL also had a synergistic effect with the mpl ligand, judging by the acetylcholinesterase (AchE) expression of murine megakaryocytes (2.7 fold increase). In vivo subcutaneous administration of AT III alone or TAT for 3 days significantly stimulated thrombocytosis (136% and 144%, respectively, p<0.05) and AT III/HDL showed rapid and further stimulation (150%, p <0.01). These results and the previous studies indicate that megakaryocytopoiesis is regulated by the mpl ligand, while a protease/protease inhibitor complex such as TAT, which is involved in the coagulation cascade associated with platelet consumption, might be one of the regulators in platelet production.

A monoclonal antibody raised against human beta-factor XIIa which also recognizes alpha-factor XIIa but not factor XII or complexes of factor XIIa with C1 esterase inhibitor.

A monoclonal antibody (mAb 2/215) against human beta-factor XIIa (beta-FXIIa), was shown by equilibrium binding studies to have a high affinity for alpha-factor XIIa (alpha-FXIIa) (Kd 1.8 nM) and beta-FXIIa (Kd 0.65 nM) but no detectable reaction with FXII zymogen or alpha-FXIIa:C1 esterase inhibitor (C1-INH) complex. Surface plasmon resonance studies showed that the mAb 2/215 bound to immobilized alpha-FXIIa with high affinity (KD 3.93 +/- 1.46 x 10(-11) M). Western blots employing mAb 2/215 indicated that human plasma contained small amounts of alpha-FXIIa but no beta-FXIIa. mAb 2/215 did not inhibit the amidolytic activity of beta-FXIIa and protected beta-FXIIa from inhibition by C1-INH. The recovery by ELISA,employing mAb 2/215 as the capture antibody, of alpha-FXIIa added to plasma was 11.3%, 42% after inhibition of alpha-FXIIa with 3:4dichloroisocoumarin, and 82% when 0.5% Triton-X100 was added to the assay. Gel filtration showed that the majority of plasma alpha-FXIIa existed as a complex (Mr approximately 170,000). This distinctive mAb increases the capacity to study the contact system in health and disease.

[Angioedema is caused by a defect in C1-inhibitor synthesis]. / Obrzek naczynioruchowy zalezny od defektu syntezy C1-inhibitora.

Deficit of the first component of complement inhibitor (C1-inhibitor, C1-inh) may clinically be manifested as angioedema. The disease is characterized by episodic swellings of mucosa and subcutaneous tissue at different locations of the body. Laryngeal swelling can be life-threatening. The major mediators of edema are discussed to be bradykinin and C2b derived peptides. These mediators increase capillary permeability. Antifibrinolytic agents (aminocaproic acid, tranexamic acid) and attenuated androgens (danazol or stanazolol) are used for prophylaxis. Prolonged use both of them might result in more or less severe side effects. In experiments in vitro it has been shown that IFN-gamma, IL-1, IL-6 have a stimulatory effect on C1-inh synthesis. We want to verify the practical use of probiotics as natural inductors of IFN-gamma synthesis for elevating C1-inh level.

Heparin coating of extracorporeal circuits inhibits contact activation during cardiac operations.

OBJECTIVE: Heparin coating reduces complement activation on the surface of extracorporeal circuits. In this study we investigated its effect on activation of the contact system in 30 patients undergoing coronary artery bypass grafting with the use of a heparin-coated (Duraflo II, Baxter Healthcare Corp., Edwards Division, Santa Ana, Calif.; n = 15) or an uncoated extracorporeal circuit (n = 15). METHODS: Plasma markers that reflect activation of contact (kallikrein-C1-inhibitor complexes), coagulation (prothrombin fragments F1 + 2), or fibrinolytic (plasmin-alpha 2-antiplasmin complexes) systems were determined before and during the operation. The generation of kallikrein-C1-inhibitor complexes was reduced by 62% (p = 0.06) after the onset of cardiopulmonary bypass and by 43% (p = 0.026) after the cessation of bypass in the group in which a heparin-coated circuit was used compared with the group in which the circuit was uncoated. Generation was reduced by 58% (p = 0.06) when the ratio of kallikrein-C1-inhibitor to prekallikrein after onset of bypass was considered. We detected significant increases in F1 + 2 levels in both groups and increases in plasmin-alpha 2-antiplasmin complexes in the heparin-coated group at cessation of bypass, but no intergroup differences were observed. Thus use of heparin-coated extracorporeal circuits during cardiac operations reduces formation of kallikrein-C1-inhibitor complexes when compared with use of uncoated circuits. The heparin coating is not accompanied by similar reductions in coagulation or fibrinolysis, suggesting that thrombin and plasmin formation during cardiopulmonary bypass occurs mainly independently of the contact system activation.

Functional characterization of two different Kupffer cell populations of normal rat liver.

BACKGROUND: Kupffer cells of the liver represent the largest population of tissue macrophages. Small and large Kupffer cells were distinguished in normal liver, leading to the suggestion that they have different functions. This study intends to further characterize small and large Kupffer cells of normal rat liver in vivo and in vitro. METHODS: Sections of rat liver were investigated by double-staining immunofluorescence with the monoclonal antibodies ED1 and ED2. Isolated nonparenchymal liver cells were separated according to size to obtain small and large Kupffer cells. In culture, phagocytosis was studied by zymosan ingestion and cell proliferation by incorporation of 3H-thymidine. Synthesis of the proteins C1-inhibitor, apolipoprotein E and interleukin-1 was studied by endogenous labeling of newly synthesized proteins, immunoprecipitation and sodium dodecylsulfate-polyacrylamide gel electrophoresis. RESULTS: ED1+ ED2+ Kupffer cells were located in the liver along the sinusoids. ED1+ ED2+ cells were found mainly located around the central vein and portal vessels. By counterflow elution, small ED1+ ED2- cells were separated from larger ED1+ ED2+ cells and cultured. The larger cells abundantly synthesized C1-inhibitor and apolipoprotein E, while the small cells synthesized only trace amounts of these proteins. Interferon-gamma increased C1-inhibitor synthesis in small (5-fold) and large cells (1.5-fold). 3H-thymidine incorporation was 11-fold higher in small than in large cells. However, lipopolysaccharide-induced pro-interleukin-1 alpha and pro-interleukin-1 beta synthesis and phagocytic activity were similar in both populations. CONCLUSIONS: The data demonstrate two different populations of mononuclear phagocytes in normal rat liver well distinguished by immunocytochemical and functional markers.

The value of C1 esterase inhibitor in patients with aspirin-sensitive urticaria.

The aim of the study was to evaluate the concentration and activity of C1 esterase inhibitor (C1 INH) in patients with aspirin-sensitive urticaria. C1 INH deficiency is the basis of hereditary angioneurotic edema. The study was performed on 32 subjects with aspirin-sensitive urticaria. The value of C1 INH in examined patients was the same as in the control group. There seems to be no coexistence of aspirin-sensitive urticaria and C1 esterase inhibitor deficiency.

The effect of WSEWS pentapeptide and WSEWS-specific monoclonal antibodies on constitutive and IL-6 induced acute-phase protein production by a human hepatoma cell line, HEPG-2.

Interleukin-6 receptor (IL-6R) is a member of the cytokine receptor superfamily characterised by the obligatory presence of WSXWS (Trp-Ser-X-Trp-Ser) sequence motif near the transmembrane domain. To more clearly understand the role of this motif, we treated the HepG2 hepatoma cell line with synthetic WSEWS peptide (E is glutamic acid) and checked the spontaneous and IL-6-induced production of acute-phase protein fibrinogen and C1-inhibitor (C1-INH). The peptide revealed a definitely stimulatory effect both on the constitutive synthesis of C1-INH and on the IL-6-induced fibrinogen synthesis of HepG2 cells. Monoclonal antibody specific for WSEWS pentapeptide was stimulatory for the spontaneous secretion of both fibrinogen and C1-INH. However, the IL-6-induced elevations of these acute-phase proteins were oppositely regulated, since the anti-WSEWS monoclonal antibody was inhibitory on the production of fibrinogen induced by IL-6 but strongly augmented the IL-6 induced production of C1-INH. Our study indicates that the WSEWS motif is critical in the effect of IL-6 on the acute-phase protein production influencing either the ligand binding by the WSEWS-containing receptor molecule or the signal transduction.

[Inhibition of Cls activity by methylprednisolone].

From a clinical study it was found that serum ClINH activity increased 6 hours after methylprednisolone (MP) pulse therapy in all the patients we studied with connective tissue diseases. Furthermore plasma ClINH-Cls complex decreased after pulse therapy. These phenomena lead us to investigate the effect of MP on Cls. 1) When a constant amount of Cls (8 micrograms/ml) was incubated with several concentrations of MP (2-50 mg/ml), the Cls activity of consuming C4 hemolysis was inhibited by MP in a dose-dependent manner. 2) MP inhibited consumption of C2 as well as C4 by Cls in a dose-dependent manner, even when MP had been removed by dialysis following incubation with Cls. These experimental data suggested that the trace amounts of Cls generated by immune complexes could be inhibited by long circulation of MP even at low concentrations in vivo, and resulted in a decrease of ClINH consumption and ClINH-Cls complex formation. These results indicated that the inhibition of Cls activity is one of the most important mechanisms in the process of the anti-inflammatory effect of MP in vivo.

Level of plasma prekallikrein and its inhibitors in reactors and nonreactors during intravenous enhancement with contrast media.

Complex contact activation systems may play a major role in the side effects of i.v. contrast media (CM). This is why quantitative measurements of several factors (plasma prekallikrein, hematocrit (hct), alpha-2-macroglobulin, alpha-1-antitrypsin, and C1-esterase inhibitor) were determined prior to and following the injection of CM during body CT examination in 5 patient groups, each (n = 10) receiving one of 5 different CM, including ioxaglate, meglumine iodamide, metrizamide, iohexol, and meglumine diatrizoate. The initial plasma prekallikrein level was available from 45 patients and was statistically lower in reactors (mean 90.6 mumol TAMe/ml/h; n = 13) than in nonreactors (mean 107 mumol TAMe/ml/h; n = 32) (p = 0.006), but there was no statistically significant difference in the decrease of plasma prekallikrein before and at 5 min after the injection for those 2 groups. The initial plasma C1-esterase inhibitor level was lower in reactors, while the plasma alpha-2-macroglobulin level was higher in that group than in nonreactors. The results indicate that the measurement of plasma prekallikrein combined with plasma C1-esterase inhibitor and alpha-2-macroglobulin measurement could be useful when predicting which patients are prone to CM reactions.

Autoimmune angioedema: a new role for autoantibody in disease pathogenesis.

Angioedema may be due to hereditary forms of Cl-Inh deficiency, but recently an autoimmune form of angioedema has been described in which the mechanism is novel. While the peripheral blood monocytes of patients with autoimmune angioedema produce a normal, functionally active, 105 KD Cl-Inh in normal quantities, the Cl-Inh isolated from the patient's plasma exists in a dysfunctional lower molecular weight (96 KD) performance. Rather than bind and biologically inactivate the enzyme, a relatively common phenomenon in autoimmune disease, the autoimmune angioedema cleave the Cl-Inh molecule. The following sequence of events is proposed: structural and functionally normal Cl-Inh is synthesised and secreted, this secreted inhibitor is complexed by autoantibody and following enzyme interaction, denatured 96 KD Cl-Inh is proposed. This process depletes the pool of normal, functional Cl-Inh to critical levels and predisposes patients to episodes of oedema.