Loss-of-function mutations in the gene encoding filaggrin underlie a Japanese family with food-dependent exercise-induced anaphylaxis.

BACKGROUND: Food-dependent exercise-induced anaphylaxis (FDEIA) is a serious food allergy in which anaphylaxis develops when exercise is performed within several hours after food intake. The precise mechanism underlying allergic sensitization in FDEIA has been an important issue but remains poorly understood. OBJECTIVES: We aimed to elucidate the pathomechanism including the route of allergen sensitization involved in FDEIA. METHODS: A Japanese family with wheat-dependent exercise-induced anaphylaxis (WDEIA), a specific form of FDEIA, were clinically examined. Mutation analysis of the gene encoding filaggrin (FLG) was also performed. RESULTS: Two of the family members were confirmed as WDEIA on the basis of their medical history and positive provocation test results. Notably, the two affected individuals in the family had concomitant ichthyosis vulgaris. Mutation analysis of FLG revealed that they carry one or more loss-of-function mutations that have not been described in the Japanese population. CONCLUSION: These results indicate that FLG mutations might be involved in the pathogenesis of WDEIA in the present case.

Highly prevalent SERPINB7 founder mutation causes pseudodominant inheritance pattern in Nagashima-type palmoplantar keratosis.

BACKGROUND: Nagashima-type palmoplantar keratosis (NPPK) is a distinct autosomal recessive genodermatosis characterized by diffuse transgressive palmoplantar keratoderma (PPK). Very recently, putative loss-of-function mutations in SERPINB7, which encodes a member of the serine protease inhibitor superfamily and is abundantly expressed in the epidermis, have been identified as a cause of NPPK. OBJECTIVES: To confirm further the role of SERPINB7 mutations in the pathogenesis of NPPK. METHODS: We analysed 10 Japanese families with NPPK using Sanger and/or whole-exome sequencing. RESULTS: We identified one novel and three recurrent null mutations in SERPINB7. In all the families, the NPPK trait was inherited in an autosomal recessive manner; in one of the families, there was pseudodominant inheritance, which had not been described in NPPK. CONCLUSIONS: These data clearly provide further evidence that NPPK is caused by loss-of-function mutations in SERPINB7.

Comprehensive screening for a complete set of Japanese-population-specific filaggrin gene mutations.

Mutations in FLG coding profilaggrin cause ichthyosis vulgaris and are an important predisposing factor for atopic dermatitis. Until now, most case-control studies and population-based screenings have been performed only for prevalent mutations. In this study, we established a high-throughput FLG mutation detection system by real-time PCR with a set of two double-dye probes and conducted comprehensive screening for almost all of the Japanese-population-specific FLG mutations (ten FLG mutations). The present comprehensive screening for all ten FLG mutations provided a more precise prevalence rate for FLG mutations (11.1%, n = 820), which seemed high compared with data of previous reports based on screening for limited numbers of FLG mutations. Our comprehensive screening suggested that population-specific FLG mutations may be a significant predisposing factor for hay fever (odds ratio = 2.01 [95% CI: 1.027-3.936, P < 0.05]), although the sample sizes of this study were too small for reliable subphenotype analysis on the association between FLG mutations and hay fever in the eczema patients and the noneczema individuals, and it is not clear whether the association between FLG mutations and hay fever is due to the close association between FLG mutations and hay fever patients with eczema.

A novel splice site mutation in NCSTN underlies a Japanese family with hidradenitis suppurativa.

BACKGROUND: Hidradenitis suppurativa (HS) is a chronic follicular occlusive disease with characteristic recurrent draining sinuses, skin abscesses and disfiguring scars, mainly involving the axilla, groin, perianal and perineal regions. While most HS cases are nonfamilial, familial cases showing autosomal dominant inheritance have been reported. Recently, loss-of-function mutations in the genes encoding γ-secretase have been identified as a cause of familial HS in the Chinese and British populations. OBJECTIVES: To identify mutations in the genes encoding γ-secretase in Japanese patients with familial and nonfamilial HS. METHODS: Two affected and three unaffected individuals from a Japanese family with familial HS and nine patients with nonfamilial HS were recruited. We conducted mutation analysis of the γ-secretase genes in Japanese patients with familial and nonfamilial HS. RESULTS: A novel splice site mutation in the nicastrin gene NCSTN, one of the six key component genes encoding γ-secretase, was identified in the patients with familial HS. Neither unaffected individuals in the family nor 100 ethnically matched control alleles carry this mutation. None of the nine patients with nonfamilial HS carry nonsense, frameshift or splice site mutations in this gene. CONCLUSIONS: A novel splice site mutation, c.582+1delG, in NCSTN was identified in the familial patients with HS. We also reveal for the first time that a γ-secretase gene mutation is not linked to the development of nonfamilial HS. These results would further pave the way to a better understanding of the contribution of γ-secretase and other genes to the pathogenesis of HS and to the development of a new therapeutic strategy for HS.

Increased granzyme B production from peripheral blood mononuclear cells in patients with acute coronary syndrome.

OBJECTIVES: To clarify the role of granzyme B in acute coronary syndrome. DESIGN AND SETTING: Granzyme B is a member of the serine esterase family released from cytotoxic lymphocytes and plays an important role in cellular apoptosis by activating intracellular caspases. Granzyme B expression was compared between patients with stable and unstable angina pectoris (UAP). PATIENTS: 173 patients with coronary artery disease (CAD) were enrolled. 84 patients were found to have stable angina pectoris (SAP) and 89 patients to have UAP. METHODS: Peripheral blood was drawn from the patients. Peripheral blood mononuclear cells (PBMCs) isolated by gradient centrifugation were cultured at a density of 2x106 cells/ml for 24 hours. The supernatants were collected 24 hours after incubation and the granzyme B level was measured by enzyme-linked immunosorbent assay. Polychromic flow cytometric analysis was performed to evaluate the expression of granzyme B in the cells. RESULTS: Granzyme B production from PBMCs of UAP patients was significantly higher than from those of patients with SAP (39.1 (SEM 6.6) versus 17.0 (SEM 1.8) pg/ml, p<0.05). Granzyme B production from PBMCs increased with the increasing TIMI risk score in UAP patients. The percentage of granzyme B-positive lymphocytes to CD3-positive lymphocytes in UAP patients was significantly higher than in SAP (32.1% (SEM 1.6%) versus 18.4% (SEM 0.9%), p<0.01). CONCLUSIONS: These results suggest that granzyme B might play an important role in triggering acute coronary events by inducing apoptosis and the degradation of atherosclerotic coronary plaques.

Clear cell sarcoma associated with multiple pulmonary metastases with structural and numerical karyotypic changes.

Cytogenetic analysis was performed on a specimen from a pulmonary metastasis of clear cell sarcoma originated on the right ankle of a 53-year-old Japanese woman. It revealed near-triploid with several numerical changes including abnormalities in the copy number of chromosomes 7, 8, and 22, and structural abnormalities of chromosome 22 and others. This is a minor case which showed the multiple abnormalities of chromosomes in the absence of a t (12;22) translocation.

Effects of therapeutic doses of human atrial natriuretic peptide on load and myocardial performance in patients with congestive heart failure.

The benefits of atrial natriuretic peptide (ANP) in patients with congestive heart failure (CHF) have been demonstrated. However, the myocardial actions of ANP remain unclear. Using relatively load-insensitive left ventricular pressure-volume analysis, the myocardial and load-altering actions of ANP in patients with moderate CHF were studied. After obtaining steady-state data using micromanometers and conductance catheters, ANP was infused in 9 patients with CHF at 0.01 and 0.1 microg/kg/min for 30 minutes, respectively. Hemodynamic variables, plasma ANP, and cyclic guanosine monophosphate (cGMP) levels were determined before and 30 minutes after each ANP infusion. ANP at 0.01 microg/kg/min increased plasma ANP and cGMP levels from 73 +/- 34 to 139 +/- 34 pg/ml and from 4 +/- 1 to 8 +/- 2 pmol/ml, respectively. ANP infusion caused a significant decrease in end-systolic pressure without any changes in heart rate. End-diastolic pressure was significantly decreased but there was no significant change in left ventricular end-diastolic volume. The time constant for isovolumetric relaxation was decreased. ANP infusion at 0.1microg/kg/min caused further decreases in end-systolic pressure, end-diastolic pressure and volume, and the time constant for isovolumetric relaxation (p <0.05) without any changes in heart rate. The slope of the end-systolic pressure-volume relation was increased from 1.3 +/- 0.2 to 1.6 +/- 0.3 mm Hg/ml (p <0.05), indicating increased contractility. Plasma ANP and cGMP levels were increased to 422 +/- 44 pg/ml and 16 +/- 3 pmol/ml, respectively. Thus, ANP infusion increased cGMP generation, decreased afterload and preload, and improved left ventricular systolic and diastolic function.

Tissue factor expression in coronary circulation as a prognostic factor for late restenosis after coronary angioplasty.

We investigated changes in blood coagulation in the coronary circulation after percutaneous transluminal coronary angioplasty (PTCA) and its clinical significance. We examined 43 patients with ischemic heart disease who underwent elective PTCA of isolated stenotic lesions in the left coronary artery. Ten patients underwent PTCA alone, 15 received percutaneous transluminal rotational atherectomy (PTRA) and 18 stent implantation. Blood samples were drawn from the coronary sinus before and immediately after PTCA, as well as 4 and 24 h later. Plasma levels of tissue factor (TF), thrombin-antithrombin III complex (TAT) and prothrombin fragment 1+2 (F 1+2) were measured by enzyme-linked immunosorbent assay. Follow-up coronary angiography was performed 6 months after PTCA. Minimal luminal diameter was assessed by quantitative coronary angiography to evaluate late loss index. TF, TAT and F 1+2 levels in the coronary sinus blood showed significant increases 24 h after PTCA. A significant positive correlation was found between changes in TF levels 24 h after PTCA and late loss index 6 months after the procedure. TF levels in the coronary sinus blood were significantly higher in patients with late restenosis than in those without restenosis. These results suggest that TF expression in the coronary circulation after PTCA is a prognostic factor for late restenosis.

Chemokine expression in coronary circulation after coronary angioplasty as a prognostic factor for restenosis.

Recent studies have clarified the significance of chemokines in cardiovascular diseases, such as development of atherosclerosis, atheromatous plaque rupture and restenosis after coronary angioplasty. We investigated changes in chemokine expression in the coronary circulation induced by percutaneous transluminal coronary angioplasty (PTCA) and their clinical significance. We examined 40 patients with angina pectoris who underwent elective PTCA for isolated stenotic lesions of the left coronary artery. Eight patients received PTCA only, 14 percutaneous transluminal rotational atherectomy and 18 stent implantation. Venous blood samples were obtained from the coronary sinus before, and immediately after as well as 4 and 24 h after PTCA. Plasma levels of interleukin (IL)-8, macrophage-colony stimulating factor (M-CSF) and monocyte chemoattractant protein-1 (MCP)-1 were measured by enzyme-linked immunosorbent assay. Plasma levels of M-CSF in the coronary sinus blood showed significant increases 4 and 24 h after PTCA. On the other hand, plasma MCP-1 levels did not change significantly during a 24-h observation period after PTCA. Immunoreactive IL-8 was not detected in any patients before or after PTCA. A significant positive correlation was found between plasma M-CSF levels 24 h after PTCA and late loss index 6 months after the procedure. Plasma levels of M-CSF 24 h after PTCA were significantly higher in patients with than in those without late restenosis. PTCA induced increases in plasma levels of M-CSF in the coronary circulation. Increased M-CSF expression may be involved in neointima formation at injured vessels through activation of mononuclear phagocytes.

Torsades de pointes ventricular tachycardia induced by mosapride and flecainide in the presence of hypokalemia.

We report a 68-year-old man who developed torsades de pointes ventricular tachycardia induced by combined use of mosapride and flecainide. He had a permanent pacemaker (DDD mode) implanted because of sick sinus syndrome (bradytachy syndrome) 6 years earlier. The patient had started taking mosapride for upper abdominal discomfort 2 weeks earlier. On admission, ECG showed prolongation of the QTc interval from 0.48 to 0.56 seconds and self-terminating torsades de pointes occurred. We considered that this proarrhythmia was induced by mosapride in combination with antiarrhythmic agents.

Release of endothelin 1 and angiotensin II induced by percutaneous transluminal coronary angioplasty.

Endothelial injury plays critical roles in acute and chronic complications after percutaneous transluminal coronary angioplasty (PTCA). We investigated coronary endothelial injury and the release of vasoactive substances induced by PTCA. We examined 44 patients with ischemic heart disease who underwent elective PTCA to isolated stenotic lesions in left coronary arteries. Eleven patients received balloon angioplasty (BA), 14 percutaneous transluminal rotational atherectomy (PTRA), and 19 stent implantation. Blood samples were drawn from the coronary sinus immediately before and after as well as 4 hr and 24 hr after PTCA. Plasma levels of endothelin (ET) 1, angiotensin (ANG) II, von Willebrand factor (vWF), and thrombomodulin (TM) were measured. Seven control subjects who underwent diagnostic coronary angiography (CAG) were used as controls. In all patients, ET-1 levels in the coronary sinus blood significantly increased immediately after PTCA. ANG II levels and vWF activity showed significant increases 4 hr after PTCA. Changes in levels of these markers were similar among the BA, PTRA, and stent groups. TM levels were elevated in all groups of patients, including those simply undergoing diagnostic CAG. Changes in ET-1, ANG II, and vWF levels in the coronary sinus reflect coronary endothelial injury induced by PTCA.

Assessment of coagulation and platelet activation in coronary sinus blood induced by transcatheter coronary intervention for narrowing of the left anterior descending coronary artery.

Influences of recently developed methods for coronary intervention on hemostasis in the coronary circulation are unclear. The objective of this study was to investigate changes in coagulation and platelet activation in the coronary circulation induced by percutaneous transluminal coronary angioplasty (PTCA). We studied 35 patients with coronary heart disease who underwent elective PTCA to isolated stenotic narrowing of left coronary arteries. Seven patients received only PTCA, 12 underwent percutaneous transluminal rotational atherectomy (PTRA), and 16 underwent stent implantation. Blood samples were drawn from the coronary sinus immediately before and after as well as 4 and 24 hours after PTCA. Plasma levels of tissue factor (TF), thrombin-antithrombin III complex, plasminogen activator inhibitor (PAI)-1, tissue plasminogen activator (t-PA), beta-thromboglobulin, and platelet factor 4 were measured by enzyme-linked immunosorbent assay. In all patients, TF levels in the coronary sinus blood showed significant increases 4 and 24 hours after PTCA and thrombin-antithrombin III complex levels showed significant increases 24 hours after PTCA. PAI-1 showed significant increases 24 hours after PTCA and t-PA showed significant increases 4 and 24 hours after PTCA. Changes in levels of these markers by PTCA were similar among the 3 groups. In PTRA, levels of beta-thromboglobulin and platelet factor 4, markers of platelet activation, increased immediately after the procedure and returned to baseline levels after 4 hours. PTCA induced increases in blood coagulation and fibrinolysis in the coronary circulation. PTRA caused a marked but transient activation of platelets. These changes may contribute to acute complications during the procedure.

Stimulus-specific alteration of the relationship between cytosolic Ca(2+) transients and nitric oxide production in endothelial cells ex vivo.

1. To investigate the quantitative relationship between elevation in the intracellular Ca(2+) concentration ([Ca(2+)](i)) and nitric oxide (NO) production, the changes in [Ca(2+)](i) and NO production were determined in parallel, using fluorimetry of fura-2 and 2, 3-diaminonaphthalene, respectively, in endothelial cells ex vivo of pig aortic valves. 2. The extent of [Ca(2+)](i) elevation was quantitatively assessed by two parameters: the level of peak [Ca(2+)](i) elevation and the area under the [Ca(2+)](i) curve during treatment (the integrated [Ca(2+)](i) elevation). The amount of NO production was expressed as a percentage of that obtained with 10 microM ATP for 3 min. 3. ATP, bradykinin, thrombin, and ionomycin were used as stimulation to induce NO production, and all these caused [Ca(2+)](i) increases and NO production in a concentration-dependent manner. 4. The relationships between the peak [Ca(2+)](i) and NO production or between the integrated [Ca(2+)](i) elevation and NO production were well described by a straight line. However, the slope value of the linear relationship in both cases varied with the type of stimulation, with thrombin giving the greatest value, followed by ATP, bradykinin and ionomycin. 5. These data suggest that in endothelial cells ex vivo: (1) [Ca(2+)](i) elevation regulates NO production, but (2) the peak [Ca(2+)](i) elevation- or the integrated [Ca(2+)](i) elevation-NO production relationships varies depending on the type of agonists. Our results thus demonstrate the presence of the agonists-dependent modulation of the relationship between [Ca(2+)](i) elevation and NO production in endothelial cells ex vivo.

Interleukin 6 expression in coronary circulation after coronary angioplasty as a risk factor for restenosis.

OBJECTIVE: To investigate changes in cytokine expression in the coronary circulation induced by percutaneous transluminal coronary angioplasty (PTCA). METHODS: The study involved 32 patients with ischaemic heart disease who underwent elective PTCA for isolated stenotic lesions of the left coronary artery. Ten patients had plain old balloon angioplasty, 10 had percutaneous transluminal rotational atherectomy, and 12 had stent implantation. Blood samples were drawn from the coronary sinus before and immediately after PTCA. Plasma concentrations of interleukin 6 (IL-6), platelet derived growth factor (PDGF), monocyte chemoattractant protein 1 (MCP-1), and macrophage coronary stimulating factor (M-CSF) were measured. The patients were scheduled for follow up angiography six months after PTCA. Late loss index was calculated using quantitative coronary angiography. RESULTS: IL-6 concentrations in coronary sinus blood increased immediately after PTCA (p < 0.001), but there was no change in PDGF, MCP-1, or M-CSF. There was a positive correlation between changes in IL-6 concentrations immediately after PTCA and late loss index six months after PTCA (r = 0.73, p < 0.001). IL-6 concentrations in coronary sinus blood were higher in patients with late restenosis than in those without restenosis (p < 0.001). CONCLUSIONS: PTCA induces IL-6 production in the coronary circulation. This may induce subsequent inflammatory responses in injured vessels and play an important role in late restenosis after PTCA.

Inhibition of angiotensin converting enzyme cannot prevent increases in angiotensin II production in coronary circulation.

OBJECTIVE: To determine whether inhibition of angiotensin converting enzyme (ACE) can prevent angiotensin II production in the coronary circulation induced by percutaneous transluminal coronary angioplasty (PTCA) in patients with myocardial ischaemia. DESIGN, PATIENTS: 41 patients who underwent elective PTCA and six control subjects who received diagnostic coronary angiography were studied. Patients were divided into two groups according to the chronic administration of ACE inhibitors (group A, 15 patients treated with ACE inhibitors; group B, 26 patients without ACE inhibitors). Blood samples were drawn through catheters placed in the aorta and coronary sinus before and 24 hours after PTCA. RESULTS: Mean levels of ACE activity in the aorta were significantly lower in patients in group A than in group B. However, mean angiotensin II concentrations in the aorta were not significantly different between the two groups. Differences in basal angiotensin II concentrations between the coronary sinus and aorta, which reflected basal angiotensin II production in the coronary circulation, were not significant among group A, group B, and control subjects. The production of angiotensin II in the coronary circulation was significantly increased 24 hours after PTCA in both group A and group B to the same extent. No significant changes were observed in control subjects 24 hours after diagnostic coronary angiography. CONCLUSIONS: This study revealed that inhibition of ACE activity by ACE inhibitors could not prevent increases in angiotensin II production in the coronary circulation induced by PTCA.

Proteolysis and phosphorylation-mediated regulation of thrombin receptor activity in in situ endothelial cells.

The regulatory mechanism of thrombin receptor responsiveness in in situ endothelial cells was investigated by evaluating elevations of cytosolic Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded porcine aortic valvular strips. Once stimulated with thrombin, endothelial cells did not respond to the second thrombin stimulation within 90 min. However, applying thrombin receptor activating peptide (TRAP7) at 15 min after the thrombin stimulation caused [Ca(2+)](i) elevation, which was smaller than that seen without preceding stimulation. After 90 min, response to TRAP7 recovered to the control level. When stimulated with TRAP7, the subsequent responses to thrombin and TRAP7 were attenuated at 15 min, and fully recovered after 90 min. Staurosporine partially prevented the TRAP7-induced desensitization. The recovery of responsiveness was inhibited completely by calyculin-A and partially by okadaic acid. Proteolysis and phosphorylation thus play an important role in thrombin receptor desensitization in in situ endothelial cells. Both cleaved and uncleaved receptors were desensitized through phosphorylation in part by staurosporine-sensitive kinase, and restored the responsiveness through dephosphorylation by type 1 phosphatase. The mechanism of regulation of thrombin receptor activity in in situ endothelial cells differed from those reported in cultured endothelial cells. We suggest that the cell-specific regulatory mechanism may be altered by culture conditions.