Understanding COVID-19 outcome: Exploring the prognostic value of soluble biomarkers indicative of endothelial impairment.

Host proteins released by the activated endothelial cells during SARS-CoV-2 infection are implicated to be involved in coagulation and endothelial dysfunction. However, the underlying mechanism that governs the vascular dysfunction and disease severity in COVID-19 remains obscure. The study evaluated the serum levels of Bradykinin, Kallikrein, SERPIN A, and IL-18 in COVID-19 (N-42 with 20 moderate and 22 severe) patients compared to healthy controls (HC: N-10) using ELISA at the day of admission (DOA) and day 7 post-admission. The efficacy of the protein levels in predicting disease severity was further determined using machine learning models. The levels of bradykinins and SERPIN A were higher (P ≤ 0.001) in both severe and moderate cases on day 7 post-admission compared to DOA. All the soluble proteins studied were found to elevated (P ≤ 0.01) in severe compared to moderate in day 7 and were positively correlated (P ≤ 0.001) with D-dimer, a marker for coagulation. ROC analysis identified that SERPIN A, IL-18, and bradykinin could predict the clinical condition of COVID-19 with AUC values of 1, 0.979, and 1, respectively. Among the models trained using univariate model analysis, SERPIN A emerged as a strong prognostic biomarker for COVID-19 disease severity. The serum levels of SERPIN A in conjunction with the coagulation marker D-dimer, serve as a predictive indicator for COVID-19 clinical outcomes. However, studies are required to ascertain the role of these markers in disease virulence.

An antibody against HK blocks Alzheimer's disease peptide ß-amyloid-induced bradykinin release in human plasma.

Bradykinin is a proinflammatory factor that mediates angioedema and inflammation in many diseases. It is a key player in some types of hereditary angioedema and is involved in septic shock, traumatic injury, Alzheimer's disease (AD), and stroke, among others. Activation of the plasma contact system leads to elevated levels of plasma kallikrein, which cleaves high molecular weight kininogen (HK) to release bradykinin. Drug development for bradykinin-meditated pathologies has focused on designing inhibitors to the enzymes that cleave HK (to prevent bradykinin release) or antagonists of endothelial bradykinin receptors (to prevent downstream bradykinin action). Here we show a strategy to block bradykinin generation by using an HK antibody that binds to HK, preventing its cleavage and subsequent bradykinin release. We show that this antibody blocks dextran sodium sulfate-induced HK cleavage and bradykinin production. Moreover, while the pathogenic AD peptide ß-amyloid (Aß)42 cleaves HK and induces a dramatic increase in bradykinin production, our HK antibody blocked these events from occurring. These results may provide strategies for developing treatments for bradykinin-driven pathologies.

A mechanism for hereditary angioedema with normal C1 inhibitor: an inhibitory regulatory role for the factor XII heavy chain.

The plasma proteins factor XII (FXII) and prekallikrein (PK) undergo reciprocal activation to the proteases FXIIa and kallikrein by a process that is enhanced by surfaces (contact activation) and regulated by the serpin C1 inhibitor. Kallikrein cleaves high-molecular-weight kininogen (HK), releasing the vasoactive peptide bradykinin. Patients with hereditary angioedema (HAE) experience episodes of soft tissue swelling as a consequence of unregulated kallikrein activity or increased prekallikrein activation. Although most HAE cases are caused by reduced plasma C1-inhibitor activity, HAE has been linked to lysine/arginine substitutions for Thr309 in FXII (FXII-Lys/Arg309). Here, we show that FXII-Lys/Arg309 is susceptible to cleavage after residue 309 by coagulation proteases (thrombin and FXIa), resulting in generation of a truncated form of FXII (δFXII). The catalytic efficiency of δFXII activation by kallikrein is 15-fold greater than for full-length FXII. The enhanced rate of reciprocal activation of PK and δFXII in human plasma and in mice appears to overwhelm the normal inhibitory function of C1 inhibitor, leading to increased HK cleavage. In mice given human FXII-Lys/Arg309, induction of thrombin generation by infusion of tissue factor results in enhanced HK cleavage as a consequence of δFXII formation. The effects of δFXII in vitro and in vivo are reproduced when wild-type FXII is bound by an antibody to the FXII heavy chain (HC; 15H8). The results contribute to our understanding of the predisposition of patients carrying FXII-Lys/Arg309 to angioedema after trauma, and reveal a regulatory function for the FXII HC that normally limits PK activation in plasma.

Targeted LC-MS/MS platform for the comprehensive determination of peptides in the kallikrein-kinin system.

The kallikrein-kinin system (KKS) is involved in many physiological and pathophysiological processes and is assumed to be connected to the development of clinical symptoms of angioedema or COVID-19, among other diseases. However, despite its diverse role in the regulation of physiological and pathophysiological functions, knowledge about the KKS in vivo remains limited. The short half-lives of kinins, their low abundance and structural similarities and the artificial generation of the kinin bradykinin greatly hinder reliable and accurate determination of kinin levels in plasma. To address these issues, a sensitive LC-MS/MS platform for the comprehensive and simultaneous determination of the four active kinins bradykinin, kallidin, des-Arg(9)-bradykinin and des-Arg(10)-kallidin and their major metabolites bradykinin 2-9, bradykinin 1-7 and bradykinin 1-5 was developed. This platform was validated according to the bioanalytical guideline of the US Food and Drug Administration regarding linearity, accuracy, precision, sensitivity, carry-over, recovery, parallelism, matrix effects and stability in plasma of healthy volunteers. The validated platform encompassed a broad calibration curve range from 2.0-15.3 pg/mL (depending on the kinin) up to 1000 pg/mL, covering the expected concentrations in disease states. No source-dependent matrix effects were identified, and suitable stability of the analytes in plasma was observed. The applicability of the developed platform was proven by the determination of endogenous levels in healthy volunteers, whose plasma kinin levels were successfully detected in the low pg/mL range. The established platform facilitates the investigation of kinin-mediated diseases (e.g. angioedema, COVID-19) and enables the assessment of the impact of altered enzyme activities on the formation or degradation of kinins.

COVID-19: imbalance of multiple systems during infection and importance of therapeutic choice and dosing of cardiac and anti-coagulant therapies.

The renin-angiotensin-aldosterone system and its metabolites play an important role in homeostasis of body, especially the cardiovascular system. In this study, we discuss the imbalance of multiple systems during the infection and the importance of therapeutic choice, dosing, and laboratory monitoring of cardiac and anti-coagulant therapies in COVID-19 patients. The crosstalk between angiotensin, kinin-kallikrein system, as well as inflammatory and coagulation systems plays an essential role in COVID-19. Cardiac complications and coagulopathies imply the crosstalks between the mentioned systems. We believe that the blockage of bradykinin can be a good option in the management of COVID-19 and CVD in patients and that supportive treatment of respiratory and cardiologic complications is needed in COVID-19 patients. Ninety-one percent of COVID-19 patients who were admitted to hospital with a prolonged aPTT were positive for lupus anticoagulant, which increases the risk of thrombosis and prolonged aPTT. Therefore, the question that is posed at this juncture is whether it is safe to use the prophylactic dose of heparin particularly in those with elevated D-dimer levels. It should be noted that timing is of high importance in anti-coagulant therapy; therefore, we should consider the level of D-dimer, fibrinogen, drug-drug interactions, and risk factors during thromboprophylaxis administration. Fibrinogen is an independent predictor of resistance to heparin and should be considered before thromboprophylaxis. Alteplase and Futhan might be a good choice to assess the condition of heparin resistance. Finally, the treatment option, dosing, and laboratory monitoring of anticoagulant therapy are critical decisions in COVID-19 patients.

Increased plasma bradykinin level is associated with cognitive impairment in Alzheimer's patients.

Alzheimer's disease (AD) is characterized by the presence of proteinaceous brain deposits, brain atrophy, vascular dysfunction, and chronic inflammation. Along with cerebral inflammation, peripheral inflammation is also evident in many AD patients. Bradykinin, a proinflammatory plasma peptide, is also linked to AD pathology. For example, bradykinin infusion into the hippocampus causes learning and memory deficits in rats, and blockade of the bradykinin receptor lessens cognitive impairment in AD mouse models. Even though it has been hypothesized that plasma bradykinin could contribute to inflammation in AD, the level of plasma bradykinin and its association with beta-amyloid (Aß) pathology in AD patients had not been explored. Here, we assessed plasma bradykinin levels in AD patients and age-matched non-demented (ND) control individuals. We found significantly elevated plasma bradykinin levels in AD patients compared to ND subjects. Additionally, changes in plasma bradykinin levels were more profound in many AD patients with severe cognitive impairment, suggesting that peripheral bradykinin could play a role in dementia most likely via inflammation. Bradykinin levels in the cerebrospinal fluid (CSF) were reduced in AD patients and exhibited an inverse correlation with the CSF Aß40/Aß42 ratio. We also report that bradykinin interacts with the fibrillar form of Aß and co-localizes with Aß plaques in the post-mortem human AD brain. These findings connect the peripheral inflammatory pathway to cerebral abnormalities and identify a novel mechanism of inflammatory pathology in AD.

Evidence for bradykinin release in chronic spontaneous urticaria.

BACKGROUND: Chronic spontaneous urticaria (CSU) is characterized by recurrent itchy weals and/or angioedema and is believed to be driven by mast cell activation. It was shown that excessive mast cell activation during anaphylaxis initiates contact activation, resulting in bradykinin release. Evidence for bradykinin release was never demonstrated in CSU. OBJECTIVE: To study biomarkers of bradykinin release in CSU. METHODS: Plasma samples of CSU patients were collected during routine visits at the outpatient clinic. Cleaved high molecular weight kininogen (cHK) was used as a biomarker for bradykinin release. cHK, factor XIIa-C1-inhibitor (FXIIa-C1-INH), kallikrein-C1-INH, plasmin-antiplasmin (PAP) complexes and soluble urokinase-type plasminogen activator receptor (suPAR) levels were determined by ELISA. Clinical data and data on tryptase levels were collected from medical records. cHK levels were compared to previously determined levels in hereditary angioedema (HAE). RESULTS: One hundred seventeen samples from 88 CSU patients and 28 samples from healthy controls were analysed. Median cHK level in CSU was 9.1% (range: 1.4%-21.5%), significantly increased compared to healthy controls (median 6.0% range: 0%-19.9%; P = .0005) and comparable to HAE (n = 46, median 10.3%, range 0%-44.3%, P > .9999). cHK levels normalized in patients during disease remission (median 6.5% range 1.5%-20.8%) but were not dependent on the presence of angioedema, acute angioedema attacks or response to antihistamines. Surprisingly, cHK levels were inversely correlated to serum tryptase (r = -0.65 P = .0137). C1-INH complexes and suPAR levels were not elevated in patients compared to healthy controls. PAP-complex levels in patients were elevated compared to healthy controls but there was no correlation between PAP-complex and cHK levels. CONCLUSIONS: cHK levels are elevated in symptomatic CSU patients compared to healthy controls, indicating increased bradykinin production. Increased cHK levels are not limited to patients with angioedema. CLINICAL RELEVANCE: If elevated bradykinin generation has clinical implications in the pathology of CSU is open to debate.

Pharmacogenetics of angiotensin-converting enzyme inhibitor-induced angioedema.

Angioedema is a rare adverse effect of the commonly used angiotensin-converting enzyme inhibitors (ACEi) and is reported to occur with a prevalence of 0.1%-0.7%. Although most ACEi-induced angioedema (ACEi-A) cases are mild, severe cases requiring intensive care and even resulting in death have been reported in the literature. The mechanisms underlying ACEi-A are not yet fully understood, but bradykinin and/or substance P accumulation resulting from inhibition of ACE is believed to play a crucial role. ACEi-A occurs at variable frequencies across different racial groups, suggesting a genetic association with the development of ACEi-A. To date, one genome-wide association study and several candidate gene studies have been published on the association of genetic variation with ACEi-A. Genetic associations reported have been attributed to several distinct mechanisms: (a) genes coding for alternative enzymes responsible for the degradation of bradykinin and/or substance P in the diminution of ACE activity (b) ACE gene function, (c) bradykinin receptor genes, (d) genes implicated in immune and inflammation regulation and (e) genes in the fibrinolytic and coagulation pathway. Despite several plausible genetic associations, there are currently no genetic variants with sufficient effect to be clinically useful. The low incidence of ACEi-A suggests that a combination of genomic approaches with the capability to detect potentially important variants might be required to shed light on the mechanism of this adverse reaction. Additionally, many non-genetic risk factors associated with ACEi-A suggest the potential contribution of epigenetic dysregulation.

Flow-mediated vasodilation assay indicates no endothelial dysfunction in hereditary angioedema patients with C1-inhibitor deficiency.

BACKGROUND: Hereditary angioedema with C1 inhibitor deficiency (C1-INH-HAE) is a rare, potentially life-threatening disorder characterized by recurrent edematous attacks. The edema formation is the consequence of interaction of bradykinin and various vasoactive peptides with endothelium. Besides these agents, danazol, a modified testosterone derivative used in these patients to prevent edematous attacks, can also affect the function of the endothelium, because it shifts the blood lipid profile to a pro-atherogenic phenotype. OBJECTIVE: To assess the endothelial function in C1-INH-HAE patients and in healthy matched controls. METHODS: To evaluate the endothelial function, we used the flow-mediated dilation method measured in the region of the brachial artery in 33 C1-INH-HAE patients and in 30 healthy matched controls. Laboratory measurements of standard biochemical parameters were performed on computerized laboratory analyzers. RESULTS: No difference was found in endothelial function (reactive hyperemia, RH) between patients (median, 9.0; 25%-75% percentile, 6.3-12.9) and controls (median, 7.37; 25%-75% percentile, 4.52-9.93). Although we found elevated cardiovascular risk (high body mass index and low-density lipoprotein/high-density lipoprotein ratio) in danazol-treated C1-INH-HAE patients, RH values did not differ between danazol-treated and nontreated patients. Furthermore, risk factors correlated with the endothelial function only in healthy controls and patients not treated with danazol. CONCLUSION: In summary, our results did not indicate any signs of endothelial dysfunction in C1-INH-HAE patients. Moreover, the normal endothelial function in danazol-treated patients with pro-atherogenic lipid profile suggests that elevated bradykinin level or other factor(s) involved in the pathogenesis of edematous attacks may have a protective role against endothelial dysfunction and atherosclerosis.

Activation of bradykinin B2 receptor induced the inflammatory responses of cytosolic phospholipase A2 after the early traumatic brain injury.

Phospholipase A2 is a known aggravator of inflammation and deteriorates neurological outcomes after traumatic brain injury (TBI), however the exact inflammatory mechanisms remain unknown. This study investigated the role of bradykinin and its receptor, which are known initial mediators within inflammation activation, as well as the mechanisms of the cytosolic phospholipase A2 (cPLA2)-related inflammatory responses after TBI. We found that cPLA2 and bradykinin B2 receptor were upregulated after a TBI. Rats treated with the bradykinin B2 receptor inhibitor LF 16-0687 exhibited significantly less cPLA2 expression and related inflammatory responses in the brain cortex after sustaining a controlled cortical impact (CCI) injury. Both the cPLA2 inhibitor and the LF16-0687 improved CCI rat outcomes by decreasing neuron death and reducing brain edema. The following TBI model utilized both primary astrocytes and primary neurons in order to gain further understanding of the inflammation mechanisms of the B2 bradykinin receptor and the cPLA2 in the central nervous system. There was a stronger reaction from the astrocytes as well as a protective effect of LF16-0687 after the stretch injury and bradykinin treatment. The protein kinase C pathway was thought to be involved in the B2 bradykinin receptor as well as the cPLA2-related inflammatory responses. Rottlerin, a Protein Kinase C (PKC) δ inhibitor, decreased the activity of the cPLA2 activity post-injury, and LF16-0687 suppressed both the PKC pathway and the cPLA2 activity within the astrocytes. These results indicated that the bradykinin B2 receptor-mediated pathway is involved in the cPLA2-related inflammatory response from the PKC pathway.

Recurrent Angioedema: Occurrence, Features, and Concomitant Diseases in an Italian Single-Center Study.

BACKGROUND: Angioedema (AE) is a potentially life-threatening condition with hereditary (HAE), acquired (AAE), or iatrogenic causes. A careful workup allows for the identification of the etiology of attacks and the appropriate management. In this cohort study, based on a clinical practice setting, we aimed at investigating clinical and laboratory findings concerning different features of patients with recurrent AE who were referred to a single, tertiary-level center for HAE. METHODS: Clinical and laboratory data of patients fulfilling the criteria for C1-inhibitor-deficient HAE (C1-INH-HAE), C1-INH-AAE, angiotensin-converting enzyme inhibitor-related AE (ACEI-RA), and idiopathic AAE (I-AAE) were evaluated. Descriptive statistics were analyzed by means of the Mann-Whitney U test. The Fisher exact test was used for group comparisons. RESULTS: Patients were diagnosed with type 1 HAE (n = 14), type 2 HAE (n = 1), C1-INH-AAE (n = 8), ACEI-RA (n = 16), or I-AAE (n = 26). We included only patients with concomitant autoimmune diseases from the I-AAE group (n = 8, aut-I-AAE). Age at disease onset and at diagnosis was younger in type 1 HAE than in all the other groups. The diagnostic delay was longer in type 1 HAE than in ACEI-RA. C4 and C1q levels were lower in C1-INH-AAE than in type 1 HAE, ACEI-RA, and aut-I-AAE. Both HAE and C1-INH-AAE showed lower C1-INH antigen and function compared to the other groups. Peripheral attacks were more frequent in type 1 HAE, while airway, abdominal, and oral attacks were prevalent in C1-INH-AAE. CONCLUSION: Investigating the clinical and laboratory features of recurrent AE without wheals represents a major topic for facilitating early diagnosis and improving treatment strategies for this heterogeneous and misdiagnosed condition.

Deficiency of plasminogen activator inhibitor 2 in plasma of patients with hereditary angioedema with normal C1 inhibitor levels.

BACKGROUND: Hereditary angioedema with normal C1 inhibitor levels (HAE-N) is associated with a Factor XII mutation in 30% of subjects; however, the role of this mutation in the pathogenesis of angioedema is unclear. OBJECTIVE: We sought evidence of abnormalities in the pathways of bradykinin formation and bradykinin degradation in the plasma of patients with HAE-N both with and without the mutation. METHODS: Bradykinin was added to plasma, and its rate of degradation was measured by using ELISA. Plasma autoactivation was assessed by using a chromogenic assay of kallikrein formation. Plasminogen activator inhibitors (PAIs) 1 and 2 were also measured by means of ELISA. RESULTS: PAI-1 levels varied from 0.1 to 4.5 ng/mL (mean, 2.4 ng/mL) in 23 control subjects, from 0.0 to 2 ng/mL (mean, 0.54 ng/mL) in patients with HAE-N with a Factor XII mutation (12 samples), and from 0.0 to 3.7 ng/mL (mean, 1.03 ng/mL) in patients with HAE-N without a Factor XII mutation (11 samples). PAI-2 levels varied from 25 to 87 ng/mL (mean, 53.8 ng/mL) in control subjects and were 0 to 25 ng/mL (mean, 4.3 ng/mL) in patients with HAE-N with or without the Factor XII mutation. Autoactivation at a 1:2 dilution was abnormally high in 8 of 17 patients with HAE-N (4 in each subcategory) and could be corrected by supplemental C1 inhibitor in 4 of them. Bradykinin degradation was markedly abnormal in 1 of 23 patients with HAE-N and normal in the remaining 22 patients. CONCLUSIONS: Bradykinin degradation was normal in all but 1 of 23 patients with HAE-N studied. By contrast, there was a marked abnormality in PAI-2 levels in patients with HAE-N that is not seen in patients with C1 inhibitor deficiency. PAI-1 levels varied considerably, but a statistically significant difference was not seen. A link between excessive fibrinolysis and bradykinin generation that is estrogen dependent is suggested.

Hereditary angioedema with normal C1 inhibitor and factor XII mutation: a series of 57 patients from the French National Center of Reference for Angioedema.

Hereditary angioedema (HAE) is a rare disease associated with either a quantitative or qualitative deficiency in C1-inhibitor (C1-INH) or normal C1-INH. HAE with normal C1-INH is associated in 20% of cases with mutations in the gene for factor XII (FXII) or FXII-HAE. A recent review described 41 families, including 14 German and 15 Spanish families. We have constructed a register of French patients and their characteristics. A national survey was launched through the French National Center of Reference for Angioedema (CREAK) to study the clinical, biological and therapeutic characteristics of patients with HAE linked to a mutation of FXII gene. Fifty-seven patients were identified from 24 different families. In most cases they were young women (mean age at diagnosis: 31 years, mean age at first symptom: 21 years, female/male ratio: 76%). Twenty-one per cent of the patients experienced angioedema attacks only during pregnancy or when on oestrogen contraception. Sixty-three per cent had attacks at all times, but they were more severe during these same periods. Male carriers of the mutation were more frequently asymptomatic than females (P = 0·003). C1-INH concentrate and icatibant were both effective for treating attacks. The prophylactic use of tranexamic acid led to a 64% decrease in the number of attacks. This is one of the largest series reported of HAE patients with FXII mutation. The therapeutic management appeared to be identical to that of HAE with C1-INH deficiency.

Use of deuterium labeling by high-temperature solid-state hydrogen-exchange reaction for mass spectrometric analysis of bradykinin biotransformation.

RATIONALE: Studies of molecular biodegradation by mass spectrometry often require synthetic compounds labeled with stable isotopes as internal standards. However, labeling is very expensive especially when a large number of compounds are needed for analysis of biotransformation. Here we describe an approach for qualitative and quantitative analysis using bradykinin (BK) and its in vitro degradation metabolites as an example. Its novelty lies in the use of deuterated peptides which are obtained by a high-temperature solid-state exchange (HSCIE) reaction. METHODS: Deuterated and native BK were analyzed by positive electrospray ionization high-resolution mass spectrometry (ESI-HRMS) using an Orbitrap Fusion mass spectrometer. High-energy collision-induced dissociation (HCD) experiments were performed on [M+H](+) and [M+2H](2+) ions in targeted-MS(2) mode with adjusted normalized HCD value. RESULTS: After the HSCIE reaction, each amino acid residue of the deuterated peptide contained deuterium atoms and the average degree of substitution was 5.5 atoms per the peptide molecule. The deuterated peptide demonstrated the same chromatographic mobility as the unlabeled counterpart, and lack of racemization during substitution with deuterium. Deuterium-labeled and unlabeled BKs were incubated with human plasma and their corresponding fragments BK(1-5) and BK(1-7), well known as the major metabolites, were detected. CONCLUSIONS: Quantitative assays demonstrated applicability of the heavy peptide for both sequencing and quantification of generated fragments. Applicability of the HSCIE deuterated peptide for analysis of routes of its degradation has been shown in in vitro experiments. Copyright © 2016 John Wiley & Sons, Ltd.

Involvement of Renin-Angiotensin System in Damage of Angiotensin-Converting Enzyme Inhibitor Captopril on Bone of Normal Mice.

This study was performed to investigate the effect of angiotensin-converting enzyme inhibitor, captopril, on bone metabolism and histology, and the action of captopril on the components of the skeletal renin-angiotensin system (RAS) and bradykinin receptor in normal male mice. The mice were orally administered captopril (10 mg/kg) for 4 weeks with vehicle-treated mice as normal control. The histology of trabecular bone at the distal femoral end was determined by hematoxylin & eosin, Safranin O and Masson-Trichrome staining. The captopril-treated mice showed a decreased level of testosterone (p<0.05) and procollagen type I N-terminal propeptide (p<0.05) in serum as compared to those in the control group. Captopril has detrimental effects on trabecular bone as demonstrated by the loss of cancellous bone mass and network connections as well as changes to the chondrocytes zone. The expression of angiotensin-converting enzyme (p<0.05), renin receptor (p<0.01), angiotensin II (p<0.05) and bradykinin receptor 2 (p<0.05) was significantly up-regulated following the captopril treatment. Thus, the potential underlying mechanism of the damage of captopril on bone can be attributed the increased activity of local bone RAS and the activation of bradykinin receptor.

The effects of an oral preparation containing hyaluronic acid (Oralvisc®) on obese knee osteoarthritis patients determined by pain, function, bradykinin, leptin, inflammatory cytokines, and heavy water analyses.

The purpose of this study was to determine the effects of an oral preparation containing hyaluronic acid on osteoarthritic knee joint pain and function as well as changes in inflammatory cytokines, bradykinin, and leptin. We also used heavy water to determine the turnover rates of glycosaminoglycans in synovial fluid. This was a double-blind, randomized, placebo-controlled study of 40 subjects over a period of 3 months. Visual analog scale, Western Ontario McMaster pain, and WOMAC function scores were recorded. Serum and synovial fluid were measured by enzyme-linked immunosorbent assays for inflammatory cytokines, bradykinin, and leptin. In 20 subjects, terminal heavy water ingestion was used for spectral analyses of serum and joint fluid samples. There were statistically significant improvements in pain and function. Both serum and synovial fluid samples showed significant decreases for a majority of inflammatory cytokines, leptin, and bradykinin in the oral hyaluronic acid preparation group. Heavy water analyses revealed a significant decrease in hyaluronic acid turnover in the synovial fluid of the treatment group. A preparation containing hyaluronic acid and other glycosaminoglycans holds promise for a safe and effective agent for the treatment for patients with knee osteoarthritis and who are overweight. Further studies will be required to see whether this is a disease-modifying agent.

Effect of Bushenwenyanghuayu decoction on nerve growth factor and bradykinin/bradykinin B1 receptor in a endometriosis dysmenorrhea mouse model.

OBJECTIVE: To observe the effects of Bushenwenyanghuayu decoction (BD), a Traditional Chinese Medicine (TCM), on the serum concentration of nerve growth factor (NGF) and bradykinin (BK), and protein and mRNA levels of NGF and bradykinin B1 receptor (BKB1R) in a mouse model of endometriosis dysmenorrhea. METHODS: Seventy-five experimental female BALB/c mice were randomly divided into five groups, 15 mice each: sham, model, BD high dose (61.67 g/kg), BD low dose (15.42 g/kg), and gestrinone (0.4 mg/kg) groups. All the mice except for those in the sham group underwent auto-transplantation surgery and were gavaged estradiol valerate (0.5 mg/kg, daily for 12 days) after surgery. On the 12th day, 1 h after administration, writhing response was induced by intraperitoneal injection of oxytocin at 2 U/mouse. The writhing frequency and latency were recorded and the volume of the ectopic foci was measured. The concentration of serum NGF and BK was detected by enzyme-linked immunosorbent assay, the protein expression of NGF and BKB1R was tested by immunohistochemistry and western blotting, and NGF and BKB1R mRNAs were detected by real-time PCR. RESULTS: Compared with the model group, the volume of the ectopic foci in the treatment groups was significantly lower (P < 0.01), the writhing frequency was decreased (P < 0.05), and the writhing latency was prolonged (P < 0.01). Compared with the sham group, serum NGF and BK levels in the model group were significantly increased (P < 0.01). There were positive correlations for writhing frequency among the NGF and BK groups (P < 0.01). The serum NGF and BK levels were significantly lower in the treatment groups than the model group (P < 0.05). The protein expression of NGF, BKB1R was significantly decreased in the treatment groups compared with the model group (P < 0.01). NGF and BKB1R mRNA expression was significantly decreased in the treatment groups compared with the model group (P < 0.01). CONCLUSION: NGF and BK/BKB1R may play an important role in the development of endometriosis-associated dysmenorrhea, and BD was found to inhibit the development of endometriosis and relieve dysmenorrhea by influencing NGF and BK/ BKB1R mRNA and protein levels.

Modulation of the kallikrein/kinin system by the angiotensin-converting enzyme inhibitor alleviates experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Bradykinin is the end-product of the kallikrein/kinin system, which has been recognized as an endogenous target for combating CNS inflammation. Angiotensin-converting enzyme (ACE) inhibitors influence the kallikrein/kinin system and reportedly have immunomodulatory characteristics. The objectives of this study were to determine whether bradykinin is involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and whether bradykinin control by the ACE inhibitor could be a therapeutic target in MS. The ACE inhibitor enalapril (1·0 or 0·2 mg/kg/day) was administered orally to EAE mice and the serum levels of bradykinin and cytokines in EAE mice were analysed. As a result, the administration of enalapril increased serum bradykinin levels, decreased the clinical and pathological severity of EAE and attenuated interleukin-17-positive cell invasion into the CNS. Additionally, bradykinin receptor antagonist administration reduced the favourable effects of enalapril. Our results suggest that bradykinin is involved in the pathomechanism underlying CNS inflammation in EAE, possibly through inhibiting cell migration into CNS. Control of the kallikrein/kinin system using ACE inhibitors could be a potential therapeutic strategy in MS.