Targeting the Protective Arm of the Renin-Angiotensin System to Reduce Systemic Lupus Erythematosus Related Pathologies in MRL-lpr Mice.

Patients with Systemic Lupus Erythematosus (SLE) suffer from a chronic inflammatory autoimmune disease that results from the body's immune system targeting healthy tissues which causes damage to various organ systems. Patients with lupus are still in need of effective therapies to treat this complex, multi-system disease. Because polymorphisms in ACE are associated with the activity of SLE and lupus nephritis and based on well-documented renal-protective effects of Renin Angiotensin System (RAS)-modifying therapies, ACE-I are now widely used in patients with SLE with significant efficacy. Our research explores alternate ways of modifying the RAS as a potential for systemic therapeutic benefit in the MRL-lpr mouse model of SLE. These therapeutics include; angiotensin (1-7) [A(1-7)], Nor-Leu-3 Angiotensin (1-7) (NorLeu), Losartan (ARB), and Lisinopril (ACE-I). Daily systemic treatment with all of these RAS-modifying therapies significantly reduced the onset and intensity in rash formation and swelling of the paw. Further, histology showed a corresponding decrease in hyperkeratosis and acanthosis in skin sections. Important immunological parameters such as decreased circulating anti-dsDNA antibodies, lymph node size, and T cell activation were observed. As expected, the development of glomerular pathologies was also attenuated by RAS-modifying therapy. Improved number and health of mesenchymal stem cells (MSCs), as well as reduction in oxidative stress and inflammation may be contributing to the reduction in SLE pathologies. Several studies have already characterized the protective role of ACE-I and ARBs in mouse models of SLE, here we focus on the protective arm of RAS. A(1-7) in particular demonstrates several protective effects that go beyond those seen with ACE-Is and ARBs; an important finding considering that ACE-Is and ARBs are teratogenic and can cause hypotension in this population. These results offer a foundation for further pharmaceutical development of RAS-modifying therapies, that target the protective arm, as novel SLE therapeutics that do not rely on suppressing the immune system.

Diabetes and COVID-19: evidence, current status and unanswered research questions.

Patients with diabetes who get coronavirus disease 2019 (COVID-19) are at risk of a severe disease course and mortality. Several factors especially the impaired immune response, heightened inflammatory response and hypercoagulable state contribute to the increased disease severity. However, there are many contentious issues about which the evidence is rather limited. There are some theoretical concerns about the effects of different anti-hyperglycaemic drugs. Similarly, despite the recognition of angiotensin converting enzyme 2 (ACE2) as the receptor for severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), and the role of ACE2 in lung injury; there are conflicting results with the use of angiotensin converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARB) in these patients. Management of patients with diabetes in times of restrictions on mobility poses some challenges and novel approaches like telemedicine can be useful. There is a need to further study the natural course of COVID-19 in patients with diabetes and to understand the individual, regional and ethnic variations in disease prevalence and course.

Essential oil of the leaves of Eugenia sulcata preserve myocardial contractility and does not present immunotoxicity

The essential oil of the leaves of Eugenia sulcata, in the Myrtaceae family, has a demonstrated antihypertensive effect, but its effects on heart muscle and its toxicity have not yet been elucidated. Little chemical or biological data are available for E. sulcata, whether emphasizing the beneficial effects or the pharmacological security of this species. This study aims to evaluate myocardial contractility and to analyze angiotensin converting enzyme (ACE) and myosin ATPase activities associated with use of this essential oil. In addition, we evaluated the immunotoxicity of E. sulcata essential oil. Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) were treated daily for 30 days (10 mg/kg of oil) to evaluate the isometric force of the papillary muscle, ACE measured by fluorimetry, and myosin ATPase activities by inorganic phosphate. Lymphocyte cultures were used to evaluate cytotoxicity, DNA damage, and mutagenicity of the essential oil. The results demonstrate that the treatment did not change the cardiac contraction force and did not alter the functioning of the sarcoplasmic reticulum, extrusion of the membrane calcium, or modify the membrane calcium channels or ß-adrenergic receptor activity. Tetanic contractions were potentiated in the SHR animals. Myosin ATPase activity was also increased in the SHR animals. Cardiac ACE activity was reduced in both animal strains, and the serum ACE was reduced only in the SHR animals. The essential oil did not cause cytotoxicity or mutagenicity and presented low DNA damage. Our results demonstrated that the essential oil does not change myocardial contractility and does not present relevant immunotoxicity

Epidemiology of ACE Inhibitor Angioedema Utilizing a Large Electronic Health Record.

BACKGROUND: Angiotensin-converting enzyme inhibitors (ACEIs) are a common cause of drug-induced angioedema in the United States. Most epidemiologic ACEI angioedema data are from large multicenter clinical trials. OBJECTIVE: The objective of this study was to identify the incidence of and risk factors for ACEI angioedema using a large integrated electronic health record (EHR). METHODS: We conducted a retrospective cohort study of all ACEI prescriptions in the outpatient setting of a large academic center between January 1, 2000, and September 30, 2008. We determined frequency, timing, and risk factors for ACEI angioedema within 5 years of prescription. All data were derived from EHR sources, with angioedema defined by EHR reactions of angioedema, swelling, edema, or lip, eye, face, tongue, throat or mouth swelling. RESULTS: Among 134,945 patients prescribed an ACEI, 0.7% (n = 888) developed angioedema during the subsequent 5 years. Sex was similar but patients who developed ACEI angioedema were younger (61.5 vs 62.7 years, P = .007). Patients with ACEI angioedema were more likely to have a history of nonsteroidal anti-inflammatory drug allergy compared with patients who did not develop angioedema (7.1% vs 4.2%, P < .001). We identified a 0.07% incidence of ACEI angioedema within 1 month of prescription and a 0.23% incidence during the first year. Incidence of angioedema was relatively constant annually over the subsequent 4 years (0.10% to 0.12%). CONCLUSIONS: The incidence of ACEI angioedema within a large EHR is consistent with large clinical trial data. We observed a persistent and relatively constant annual risk; however, angioedema risk factors and underlying genetic and pathophysiological mechanisms require further study.

Cofactors and comorbidities in patients with aspirin/NSAID hypersensitivity.

Hypersensitivity reactions to aspirin and other NSAIDs occur in individuals genetically predisposed and exhibit different clinical manifestations, especially respiratory, cutaneous, and generalised. Five different phenotypes define distinct clinical pictures: aspirin-exacerbated respiratory disease, aspirin/NSAID cutaneous disease, NSAID-induced urticaria, angio-oedema and anaphylaxis, single NSAID reactions, and delayed reactions. They are observed more frequently in middle-aged women, and in atopic individuals. While ASA/NSAID hypersensitivity shares comorbidities with asthma, chronic rhinosinusitis, nasal polyposis, chronic urticaria and angio-oedema, ASA and other NSAIDs can also be cofactors for other clinically relevant conditions, especially food-dependent exercise-induced anaphylaxis, angio-oedema induced by angiotensin-converting enzyme inhibitors, and oral mite anaphylaxis. Awareness on these relationships is required for the correct diagnosis, classification, and treatment of affected patients.

Blockade of the renin-angiotensin system prevents acute and immunologically relevant colitis in murine models.

BACKGROUND: Blockade of the renin-angiotensin system (RAS) has been shown to alleviate inflammatory processes in the gastrointestinal tract. The aim of this study was to determine if blockade of the RAS would be effective in an immunologically relevant colitis model, and to compare outcome with an acute colitis model. METHODS: A losartan analog, CCG-203025 (C23H26ClN3O5S) containing a highly polar sulfonic acid moiety that we expected would allow localized mucosal antagonism with minimal systemic absorption was selected as an angiotensin II type 1a receptor antagonist (AT1aR-A). Two colitis models were studied: (1) Acute colitis was induced in 8- to 10-week-old C57BL/6J mice by 2.5 % dextran sodium sulfate (DSS, in drinking water) for 7 days. (2) IL10-/-colitis Piroxicam (200 ppm) was administered orally in feed to 5-week-old IL-10-/-mice (C57BL/6J background) for 14 days followed by enalaprilat (ACE-I), CCG-203025 or PBS administered transanally for 14 days. RESULTS: In the DSS model, weight loss and histologic score for CCG-203025 were better than with placebo. In the IL10-/-model, ACE-I suppressed histologic damage better than CCG-203025. Both ACE-I and CCG-203025 reduced pro-inflammatory cytokines and chemokines. CONCLUSIONS: This study demonstrated the therapeutic efficacy of both ACE-I and AT1aR-A for preventing the development of both acute and immunologically relevant colitis.

Development of an enzymatic assay for the detection of neutralizing antibodies against therapeutic angiotensin-converting enzyme 2 (ACE2).

Therapeutic proteins have the potential to elicit immune responses in animals and humans (Mire-Sluis et al., 2004; Yu et al., 2006; Shankar et al., 2008). Contributors to the response could include product related factors such as chemical modifications, impurities that co-purify with product, contaminants, formulation, aggregates, and clinical factors such as dose concentration, dosing frequency, route of drug administration, rate of administration, patient underlying disease, concomitant medication, and genetic status among others (Patten and Schellekens, 2003). Further, an immune response triggered by a therapeutic enzyme may neutralize the endogenous counterpart resulting in a decrease or depletion of the therapeutic and endogenous enzymes imposing safety concerns for patients. Therefore, monitoring of anti-drug antibody (ADA) and neutralizing antibody (NAb) responses to both the recombinant therapeutic enzyme and endogenous enzyme is important during early development and subsequent clinical studies. Testing considerations for NAb detection against therapeutic enzymes have been published mostly for lysosomal storage diseases (Wang et al., 2008). NAb cross-reactivity to the endogenous counterpart has also been characterized (Sominanda et al., 2010). Here, we describe an enzymatic NAb assay which detects neutralizing antibodies to both recombinant and endogenous angiotensin-converting enzyme 2 (ACE2). NAb assay sensitivity was optimized by selecting the assay incubation time as 20 min with an enzyme concentration of 0.5 µg/mL. Four anti-ACE2 antibodies out of a commercial panel of 18 were found to have neutralizing capabilities based upon their ability to abrogate ACE2 enzymatic activity. We demonstrated assay specificity by small peptide inhibitors specific for ACE or ACE2. DX600, an ACE2 specific inhibitor did not cross-react with ACE. Conversely, captopril, an inhibitor of ACE did not inhibit ACE2. The assay specificity for ACE2 neutralizing antibodies was further demonstrated by the lack of reactivity of two species control antibodies and 14 anti-ACE2 antibodies. Moreover, we demonstrated assay specificity to human endogenous ACE2 from human epithelial cells. Three human cell lines (Calu-3, Caco-2, Huh-7) were evaluated for the cell surface expression of ACE2 by flow cytometry and Western blot. Subsequently, whole cell lysates, cell culture supernatant, and live cells were evaluated in the assay. Results demonstrated that Calu-3 had elevated levels of ACE2 compared to Caco-2 or Huh-7. Calu-3 also demonstrated elevated ACE2 enzymatic activity in all three sources and could be inhibited by the ACE2 specific inhibitor DX600 as well as the neutralizing antibodies for the recombinant ACE2. Thus, we describe here a method to detect NAb against a therapeutic enzyme and assess NAb cross-reactivity to the native endogenous enzyme. The approach of method development described here could be applied for the assessment of NAb responses to other enzymatic therapeutics.

Immunotargeting of the pulmonary endothelium via angiotensin-converting-enzyme in isolated ventilated and perfused human lung.

Vascular immunotargeting of catalase via angiotensin-converting-enzyme (ACE) attenuated lung ischemia reperfusion injury in the rat. As this might be a promising modality for extension of the viability of human lung grafts for transplantation we tested the hypothesis whether anti-ACE antibodies are suitable for human lung protection within the model of isolated perfused and ventilated human lung resections. Right after surgery for lung cancer, human lung specimens were isolated, ventilated and perfused under physiological conditions with 500 µg of either mouse monoclonal antibodies (mAb) to human ACE (9B9, I2H5, 3G8) or non-immune mouse IgG (as a negative control) followed by wash-out perfusion. Perfusion pressure, pH and lung weight gain were measured before and during perfusion. After mAb perfusion and wash-out perfusion period lung tissue was tested for the uptake of mAbs by immunohistochemistry and by enzyme-capture technique. Furthermore, antibody concentration and ACE shedding were measured within the perfusate. We found that ACE activity in tumor and normal lung tissue did not differ between the groups perfused with different mAbs. However, ACE activity in normal lung tissue (17.0 ± 6.0 U/g) was significantly higher compared to tumor tissue (6.0 ± 3.0; p < 0.01). Absolute retaining of mAbs was with 1.3 ± 1.1% of injected dose per gram of tissue in normal lung tissue, 0.7 ± 0.7% of injected dose per gram of tumor tissue and was significantly higher compared to non-immune mouse IgG (0.1 ± 0.1%/g; p < 0.01). Anti-ACE mAbs concentration in the perfusate dropped significantly to 47 ± 11% (p < 0.001) at 40 min of perfusion. No significant difference between different anti-ACE mAbs in the depletion from perfusate has been observed. mAb 9B9 showed the most intense immunostaining (i.e., most significant lung uptake) after each experiment in normal and tumor lung tissue compared to mAbs i2H5 and 3G8 (p < 0.01). These results validate the possibility of immunotargeting of pulmonary endothelium in the human lung tissue by anti-ACE mAbs under in vivo conditions. Furthermore, the model might be useful to investigate targeted therapies in lung cancer without side effects for the patient.

Enalapril treatment discloses an early role of angiotensin II in inflammation- and oxidative stress-related muscle damage in dystrophic mdx mice.

Inhibitors of angiotensin converting enzymes (ACE) are clinically used to control cardiomyopathy in patients of Duchenne muscular dystrophy. Various evidences suggest potential usefulness of long-term treatment with ACE inhibitors to reduce advanced fibrosis of dystrophic muscle in the mdx mouse model. However, angiotensin II is known to exert pro-inflammatory and pro-oxidative actions that might contribute to early events of dystrophic muscle degeneration. The present study has been aimed at evaluating the effects of an early treatment with enalapril on the pathology signs of exercised mdx mouse model. The effects of 1 and 5 mg/kg enalapril i.p. for 4-8 weeks have been compared with those of 1 mg/kg α-methyl-prednisolone (PDN), as positive control. Enalapril caused a dose-dependent increase in fore limb strength, the highest dose leading to a recovery score similar to that observed with PDN. A dose-dependent reduction of superoxide anion production was observed by dihydroethidium staining in tibialis anterior muscle of enalapril-treated mice, approaching the effect observed with PND. In parallel, a significant reduction of the activated form of the pro-inflammatory Nuclear Factor-kB has been observed in gastrocnemious muscle. Histologically, 5 mg/kg enalapril reduced the area of muscle necrosis in both gastrocnemious muscle and diaphragm, without significant effect on non-muscle area. In parallel no significant changes have been observed in both muscle TGF-ß1 and myonuclei positive to phosphorylated Smad2/3. Myofiber functional indices were also monitored by microelectrodes recordings. A dose-dependent recovery of macroscopic chloride conductance has been observed upon enalapril treatment in EDL muscle, with minor effects being exerted in diaphragm. However a modest effect, if any, was found on mechanical threshold, a functional index of calcium homeostasis. No recovery was observed in creatine kinase and lactate dehydrogenase. Finally the results suggest the ability of enalapril to blunt angiotensin-II dependent activation of pro-inflammatory and pro-oxidant pathways which may be earlier events with respect to the pro-fibrotic ones, and may in part account for both functional impairment and muscle necrosis. The PDN-like profile may corroborate the combined use of the two classes of drugs in DMD patients so to potentiate the beneficial effects at skeletal muscle level, while reducing both spontaneous and PDN-aggravated cardiomyopathy.

Pre-ischaemic conditioning of the pulmonary endothelium by immunotargeting of catalase via angiotensin-converting-enzyme antibodies.

BACKGROUND: Alleviation of oxidative stress via targeted delivery of catalase to the pulmonary endothelium by conjugation of angiotensin-converting-enzyme (ACE) monoclonal antibodies attenuates lung injury in an in vivo model of warm lung ischaemia and reperfusion. This study evaluates treatment of lung allografts with conjugates of anti-ACE antibody with catalase (9B9-CAT) in the setting of hypothermic preservation and reports the effect on ischaemia/reperfusion injury in this model. METHODS: Rats were injected 1h prior to lung harvesting with mouse immunoglobulin G (IgG) (negative controls), catalase only (CAT) or anti-ACE mAb 9B9 conjugated with catalase (9B9-CAT). Lungs were flushed with low-potassium dextran (LPD) solution, excised and stored at 4 degrees C for 4 and 8h. Grafts were isolated and directly reperfused at 37 degrees C for up to 180 min. Peak inspiratory pressure (PIP), pulmonary arterial pressure (PAP) and lung weight were measured during reperfusion. Anti-oxidative capacity and catalase activity were measured in frozen lung tissue and inflammatory parameters were detected during reperfusion in perfusate solution. RESULTS: Cold ischaemia of 8h significantly increased lung weight gain, PIP and PAP in non-immune mouse IgG and CAT-treated lungs than in 9B9-CAT-treated lungs (p<0.005). Significantly higher catalase activity and anti-oxidative status were found in the lung tissue of animals conditioned with 9B9-CAT after 4 and 8h of cold storage than in animals treated with catalase (CAT) alone or in animals treated with non-immune mouse IgG (p<0.01). CONCLUSION: These results validate immunotargeting by anti-ACE mAb conjugated with catalase as a prospective and specific strategy to augment anti-oxidative defence of the pulmonary endothelium during lung transplantation. Vascular immunotargeting of anti-oxidative enzymes could limit reactive oxygen species mediated ischaemia-reperfusion (I/R) injury of the lung and has the potential to become a promising modality for extension of the viability of banked transplantation tissue.

Hereditary angioedema with normal c1 inhibition.

Until recently, it was assumed that hereditary angioedema was a disease resulting exclusively from a genetic deficiency of the C1 inhibitor. In 2000, families with hereditary angioedema, normal C1 inhibitor activity, and protein in plasma were described. Since then, numerous patients and families with this condition have been reported. Most of the patients were women. In many of the affected women, oral contraceptives, hormone replacement therapy containing estrogens, and pregnancies triggered the clinical symptoms. In some families, mutations in the coagulation factor XII (Hageman factor) gene were detected in the affected persons.

[Characteristics of monoclonal antibody binding with the C domain of human angiotensin converting enzyme].

Binding of a panel of eight monoclonal antibodies (mAbs) with the C domain of angiotensin converting enzyme (ACE) to human testicular ACE (tACE) (corresponding to the C domain of the somatic enzyme) was studied and the inhibition of the enzyme by the mAb 4E3 was found. The dissociation constants of complexes of two mAbs, IB8 and 2H9, with tACE were 2.3 +/- 0.4 and 2.5 +/- 0.4 nM, respectively, for recombinant tACE and 1.6 +/- 0.3 nM for spermatozoid tACE. Competition parameters of mAb binding with tACE were obtained and analyzed. As a result, the eight mAbs were divided into three groups, whose binding epitopes did not overlap: (1) 1E10, 2B11, 2H9, 3F11, and 4E3; (2) 1B8 and 3F10; and (3) IB3. A diagram demonstrating mAb competitive binding with tACE was proposed. Comparative analysis of mAb binding to human and chimpanzee ACE was carried out, which resulted in revealing of two amino acid residues, Lys677 and Pro730, responsible for binding of three antibodies, 1E10, 1B8, and 3F10. It was found by mutation of Asp616 located close to Lys677 that the mAb binding epitope 1E10 contains Asp616 and Lys677, whereas mAbs 1B8 and 3F10 contain Pro730.

Determination of antihypertensive peptides from an izumi shrimp hydrolysate.

The izumi shrimp (Plesionika izumiae Omori, 1971) is an unused resource which can be caught off the southern coast of Tokushima Prefecture. We have previously found that an izumi shrimp hydrolysate significantly inhibited the age-associated spontaneous increase in blood pressure in stroke-prone spontaneously hypertensive rats. In this present study, two angiotensin I-converting enzyme inhibitory peptides were isolated from an izumi shrimp hydrolysate by using high-performance liquid chromatography, and their amino acid sequences were determined to be Val-Trp-Tyr-His-Thr and Val-Trp. A single oral administration of synthetic Val-Trp-Tyr-His-Thr or Val-Trp significantly decreased the blood pressure in stroke-prone spontaneously hypertensive rats. The antigenicity and allergenicity of the izumi shrimp hydrolysate against BALB/c mice were very low. These results demonstrate that the angiotensin I-converting enzyme inhibitory peptides isolated from the izumi shrimp hydrolysate had an anti-hypertensive effect on rats.

Pediatric angioedema.

Angioedema is a self-limited nonpitting edema generally affecting the deeper layers of the skin and mucous membranes. It is the result of increased vascular permeability causing the leakage of fluid into the skin in response to potent vasodilators released by immunologic mediators. Two main pathways are thought to be implicated in angioedema. The mast cell pathway in which preformed mediators, such as histamine, rapidly formed mediators, leukotrienes and prostaglandins, are released from mast cells through IgE or direct activation. This is the most common pathway among children, with food, medications, and infections commonly implicated. The second pathway is the kinin pathway, most notably affected by angiotensin-converting enzyme (ACE) inhibitors and hereditary forms of angioedema, which ultimately results in the formation of bradykinin, a potent vasodilator. Angioedema is being encountered with increasing frequency, particularly among children and is important to recognize and treat for its life-threatening associated manifestations such as anaphylaxis and airway obstruction. Although angioedema is still not fully understood, we have broadened our understanding of the possible causes and clinical course of angioedema. An understanding of these potential causes and mechanisms by which angioedema can occur may guide the clinician in determining the need for diagnostic testing and the extent of treatment.

Fine epitope mapping of monoclonal antibody 5F1 reveals anticatalytic activity toward the N domain of human angiotensin-converting enzyme.

Angiotensin I-converting enzyme (ACE, peptidyl dipeptidase, EC 3.4.15.2) is a key enzyme in cardiovascular pathophysiology. A wide spectrum of monoclonal antibodies to different epitopes on the N and C domains of human ACE has been used to study different aspects of ACE biology. In this study we characterized the monoclonal antibody (mAb) 5F1, developed against the N domain of human ACE, which recognizes both the catalytically active and the denatured forms of ACE. The epitope for mAb 5F1 was defined using species cross-reactivity, synthetic peptide (PepScan technology) and phage display library screening, Western blotting, site-directed mutagenesis, and protein modeling. The epitope for mAb 5F1 shows no overlap with the epitopes of seven other mAbs to the N domain described previously and is localized on the other side of the N domain globule. The binding of mAb 5F1 to ACE is carbohydrate-dependent and increased significantly as a result of altered glycosylation after treatment with alpha-glucosidase-1 inhibitor, N-butyldeoxynojirimycin (NB-DNJ), or neuraminidase. Out of 17 species tested, mAb 5F1 showed strict primate ACE specificity. In addition, mAb 5F1 recognized human ACE in Western blots and on paraffin-embedded sections. The sequential part of the epitope for mAb 5F1 is created by the N-terminal part of the N domain, between residues 1 and 141. A conformational region of the epitope was also identified, including the residues around the glycan attached to Asn117, which explains the sensitivity to changes in glycosylation state, and another stretch localized around the motif 454TPPSRYN460. Site-directed mutagensis and inhibition assays revealed that mAb 5F1 inhibits ACE activity at high concentrations due to binding of residues on both sides of the active site cleft, thus supporting a hinge-bending mechanism for substrate binding of ACE.

Monoclonal Antibodies 1G12 and 6A12 to the N-domain of human angiotensin-converting enzyme: fine epitope mapping and antibody-based detection of ACE inhibitors in human blood.

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N- and C-), each bearing a Zn-dependent active site. ACE inhibitors are among the most prescribed drugs in the treatment of hypertension and cardiac failure. Fine epitope mapping of two monoclonal antibodies (mAb), 1G12 and 6A12, against the N-domain of human ACE, was developed using the N-domain 3D-structure and 21 single and double N-domain mutants. The binding of both mAbs to their epitopes on the N-domain of ACE is significantly diminished by the presence of the C-domain in the two-domain somatic tissue ACE and further diminished by the presence of sialic acid residues on the surface of blood ACE. The binding of these mAbs to blood ACE, however, increased dramatically (5-10-fold) in the presence of ACE inhibitors or EDTA, whereas the effect of these compounds on the binding of the mAbs to somatic tissue ACE was less pronounced and even less for truncated N-domain. This implies that the binding of ACE inhibitors or removal of Zn2+ from ACE active centers causes conformational adjustments in the mutual arrangement of N- and C-domains in the two-domain ACE molecule. As a result, the regions of the epitopes for mAb 1G12 and 6A12 on the N-domain, shielded in somatic ACE by the C-domain globule and additionally shielded in blood ACE by sialic acid residues in the oligosaccharide chains localized on Asn289 and Asn416, become unmasked. Therefore, we demonstrated a possibility to employ these mAbs (1G12 or 6A12) for detection and quantification of the presence of ACE inhibitors in human blood. This method should find wide application in monitoring clinical trials with ACE inhibitors as well as in the development of the approach for personalized medicine by these effective drugs.

Repeated massive tongue swelling due to the combined use of estramustine phosphate and angiotensin-converting enzyme inhibitor.

A 70-year-old man presenting with a chief complaint of tongue swelling had been diagnosed with prostate cancer 1 year earlier. He had been on an oral angiotensin-converting enzyme inhibitor (ACE) inhibitor for hypertension for 20 years. Two months before the first of 4 episodes of tongue swelling within a period of 40 days, he had been prescribed oral estramustine phosphate (EMP) for the prostate cancer. He was admitted to our hospital for the evaluation after massive swelling of the tongue and epiglottis which necessitated tracheotomy. Food allergies, allergic reactions to environmental factors, and hereditary angioneurotic edema were excluded. Massive swelling of the tongue and epiglottis disappeared completely after EMP was discontinued. We concluded that angioedema was induced by EMP used concurrently with the ACE inhibitor.

A natural carrier effect and the generation of specific antibodies to biologically active peptides.

Production of specific antibodies to haptens, especially antipeptides, without interference by carrier protein, is desirable. The bradykinin-potentiating peptides (BPPs) are a family of pyroglutamyl proline-rich oligopeptides with strong antihypertensive properties. In this work, the production of antibodies to BPPs by use of an efficient immunization protocol in mice genetically modified for the high antibody responsiveness (H(III) line) is described. Although it was possible to induce antibody production by single-dose administration of free BPPs, higher antibody titers were obtained in mice preimmunized with carrier protein before administration of peptides conjugated to this carrier. Interestingly, both mouse groups had a higher titer of IgG(1) than IgG(2a) isotypes, regardless of prior immunization with the carrier protein. However, a lower titer of IgG(2a) was observed in unprimed mice. A single band of about 27kDa corresponding to the BPP precursor protein was recognized by these antibodies in the cytosol of the Bothrops jararaca venom gland. This work proposes an efficient immunization protocol based on classic studies described for the hapten-carrier effect for generating specific antibodies against biologically active peptides.

Randomized double-blind placebo-controlled study of an angiotensin immunotherapeutic vaccine (PMD3117) in hypertensive subjects.

Immunization against components of the renin-angiotensin system offers a potential alternative to daily medication in some patients with hypertension or heart failure. Our primary objective was to determine whether a sustained antibody titre to Ang I (angiotensin I) can be achieved in hypertensive patients. The secondary objective was to determine whether the antibodies block the renin system. Patients (n=27) with essential hypertension responsive to an ACEi (angiotensin-converting enzyme inhibitor) or ARB (angiotensin blocker) were randomly assigned to receive three or four injections of the Ang I vaccine PMD3117 or aluminium hydroxide (Alhydrogel trade mark ) over a 6 week period. Antibody titre was measured prior to each injection and every 30 days until disappearance. Indices of renin blockade were changes in renin and aldosterone (blood and urine) and a within-patient comparison of the pre- and post-vaccination rise in 24 h ambulatory blood pressure after 2 weeks of withdrawal of ACEi or ARB. The anti-(Ang I) antibody titre rose from the second injection in both regimes and peaked on day 64. Median half-life was 85 (95% CI, 44 and 153) days (where CI is confidence interval). Vaccination did not influence blood pressure, but significantly blunted the fall in plasma renin following withdrawal of ACEi or ARB. At 42 days after the first injection, aldosterone excretion was decreased by PMD3117 to 6 (95% CI, 1 and 31)% of values in patients receiving Alhydrogel trade mark (P=0.012). In patients with essential hypertension, PMD3117 generated a prolonged antibody response to Ang I. Biochemical measurements show evidence of blockade of the renin system, but higher titres will be required to achieve a decrease in blood pressure.