Total atrial conduction time provides novel information in prediction for stroke in patients with sinus rhythm.

The total atrial conduction time (TACT) measured by echocardiography predicts the risk of atrial fibrillation (AF). This study aimed to investigate whether adding the TACT to the revised Framingham stroke risk profile (rFSRP) improves the efficacy of predicting stroke incidence in patients without prior stroke or known AF. The TACT was measured in 376 consecutive patients > 18 years (58.5 ± 16.3 years; 46% male) receiving echocardiography without any prior history of stroke or AF. The primary endpoint was the occurrence of ischemic stroke, and the secondary endpoint was any documentation of AF during the 2 years of follow-up. During the follow-up period, ischemic strokes occurred in 10 patients (2.65%), and AF in 22 patients (5.85%). The TACT was significantly longer in those who later had a stroke compared with those who did not (169.4 vs. 142.7 ms, p < 0.001). Both rFSRP and TACT predicted the risk for stroke incidence. The univariate model showed that the TACT was a predictor of ischemic stroke incidence (p < 0.001; hazard ratio of 1.94 for every 10 ms; 95% confidence interval, 1.49-2.54). The addition of TACT to rFSRP significantly improved the area under the receiver operating characteristic curve (0.79 vs. 0.85, p = 0.001). Stroke risk prediction was significantly improved by the addition of TACT to rFSRP. The utility of the TACT should be further investigated in large-scale randomized clinical trials.

Potentially inappropriate medications in elderly Japanese patients: effects of pharmacists' assessment and intervention based on Screening Tool of Older Persons' Potentially Inappropriate Prescriptions criteria ver.2.

WHAT IS KNOWN AND OBJECTIVES: The Screening Tool of Older Persons' Potentially Inappropriate Prescriptions (stopp) criteria were updated in 2014 (stopp criteria ver.2), but few studies have evaluated the usefulness of stopp criteria in elderly patients. This prospective observational study evaluated the prevalence of potentially inappropriate medications (PIMs), and the efficacy of hospital pharmacists' assessment and intervention based on stopp criteria ver.2. METHODS: The study was conducted at three medical units of Kobe University Hospital between April 2015 and March 2016. Pharmacists assessed and detected PIMs based on stopp criteria ver.2 and considered the patient's intention to change the prescription at the time of admission of each patient. If the pharmacists judged that benefits outweighed risks of prescription change and the patients consented to change the medications, they recommended the doctor to change the prescription. If there was a risk of exacerbation of disease by the change of medications and the pharmacists judged it to be difficult to adjust medications during hospitalization or the patients did not consent to change the medications, they did not recommend to change it. The pharmacists and the doctors discussed and finally decided whether to change the PIMs or not. The number of patients prescribed PIMs, the number and contents of PIMs, and the number of medications changed after pharmacists' intervention were calculated. RESULTS: Totally, 822 new inpatients aged ≥65 years prescribed ≥1 daily medicine were included. Their median (interquartile range) age was 75·0 (71·0-80·0) years, and 54·9% were male. According to the criteria, 346 patients (42·1%) were prescribed ≥1 PIMs. Patients prescribed PIMs took significantly more medications than others: 10·0 (7·0-13·0) vs. 6·0 (4·0-9·0), P < 0·001. The total number of PIMs was 651%, 47·6% of which (n = 310) were recommended the doctors to change, and 292 of 651 PIMs (44·9%) were finally discontinued/changed after pharmacists' assessment and intervention. PIMs related to benzodiazepines, including Z-drugs, were most frequent, with a detailed classifications as follows (changed/total): (i) benzodiazepines for 4 or more weeks (75/205), (ii) drugs that predictably increase the risk of falls in older people (benzodiazepines) (30/67) and (iii) drugs that predictably increase the risk of falls in older people (hypnotic Z-drugs) (15/31). CONCLUSION: Over 40% elderly patients were prescribed PIMs, and pharmacists' assessments and interventions based on stopp criteria ver.2 were useful in detecting and correcting prescription of PIMs.

Development of a detection algorithm for statin-induced myopathy using electronic medical records.

WHAT IS KNOWN AND OBJECTIVE: Demonstration of the utility of electronic medical records (EMRs) for pharmacovigilance (PV) has been highly anticipated. Analysis using appropriately selected EMRs should enable accurate estimation of adverse drug event (ADE) frequencies and thus promote appropriate regulatory actions. Statin-induced myopathy (SIM) is a clinically important ADE, but pharmacoepidemiological methodology for detecting this ADE with high predictability has not yet been established. This study aimed to develop a detection algorithm, highly selective for SIM using EMRs. METHODS: We collected EMRs on prescriptions, laboratory tests, diagnoses and medical practices from the hospital information system of Kobe University Hospital, Japan, for a total of 5109 patients who received a statin prescription from April 2006 to March 2009. The current algorithm for extracting SIM-suspected patients consisted of three steps: (i) event detection: increase in creatine kinase (CK) and subsequent statin discontinuation, (ii) filtration by exclusion factors (disease diagnosis/medical practices) and (iii) refinement by the time course of CK values (baseline, event and recovery). A causal relationship between the event and statin prescription (probable/possible/unlikely) was judged by review of patient medical charts by experienced pharmacists. The utility of the current algorithm was assessed by calculating the positive predictive value (PPV). In a comparative analysis, subjects screened in step 1 were extracted by the diagnostic term/code for 'myopathy/rhabdomyolysis', and the PPV of this diagnostic data approach was also estimated. RESULTS AND DISCUSSION: Five subjects with suspected SIM were identified using our proposed algorithm, giving a frequency of 0·1% for the adverse event. Review of the medical charts revealed that the causal association of SIM with statin use was judged as 'Likely (probable/possible)' for all five suspected patients; thus, the PPV was estimated as 100% (95% confidence interval: 56·6-100%). The higher utility of the current algorithm compared with the diagnostic data approach was also shown by assessing the PPV (100 vs. 33·3%). WHAT IS NEW AND CONCLUSION: We report on a detection algorithm with high predictability for SIM using EMRs. Combined use of exclusion criteria for disease, medical practice data and time course of CK values contributes to better prediction of SIM. The utility of the proposed algorithm should be further confirmed in a larger study.

Role of CMR-derived atrial deformation analysis in the prediction of atrial fibrillation recurrence rate after pulmonary vein isolation.

PURPOSE: Left atrial (LA) dimensions have been identified as anatomical predictors for atrial fibrillation (AF) recurrence after pulmonary vein isolation (PVI). The value of LA function compared to pure LA anatomical risk prediction for AF recurrence after PVI is not well investigated. Cardiovascular magnetic resonance (CMR) is able to simultaneously assess LA anatomical, tissue and functional markers within one examination. The hypothesis of this explorative study was that CMR-derived LA strain has incremental value for the prediction of AF recurrence after PVI. METHOD: Fifty-two patients with paroxysmal or persistent AF were retrospectively enrolled for CMR (1.5T) prior to PVI. Strain-analysis was derived from standard cine images in 4-, 3- and 2-chamber view. LA function was divided into LA reservoir strain and strain rate (εs and SRs), LA conduit (εe and SRe) and LA booster pump function (εa and SRa). The primary endpoint was recurrence of AF within one year after PVI. RESULTS: Twelve patients (23 %) presented with AF recurrence. There was no difference in age, LA size as well as LA sphericity index between the groups. Patients with AF recurrence (68.3 ± 5.5 years, 66 % male) showed significantly reduced LA booster pump function compared to the patients without AF recurrence (66.3 ± 10.5 years, 50 % male) (εa: p = 0.015; SRa: p = 0.036). In binomial logistic regression analyses, the only predictor for AF recurrence after PVI was εa (p = 0.033). CONCLUSIONS: In this descriptive study, impaired LA booster pump function predicted AF recurrence one year after PVI. Compared to further LA strain and anatomical parameters, LA booster pump might serve as additional predictor of AF recurrence.

Different reactions of aortic and venular endothelial cell monolayers to histamine on macromolecular permeability: role of cAMP, cytosolic Ca2+ and F-actin.

Endothelial cells assume a central role in the one process that the permeation of microvessels is accelerated in case of inflammation. We studied the effect of histamine on endothelial permeability, [Ca2+]i, cAMP and F-actin, using same origin aortic and venular cultured endothelial monolayers. When HUVEC were treated with histamine (10(-7)-10(-5) M), permeability of FITC-dextran (molecular weight 70,000) and [Ca2+]i were increased, while cAMP content was unchanged, and F-actin content was reduced. When bovine vein-derived endothelial cells were treated with histamine, [Ca2+]i was increased via H1 receptors, but permeability and F-actin content were not altered. When human aorta-derived endothelial cells were, [Ca2+]i was increased via H1 receptors and cAMP content was increased via H2 receptors, while permeability and F-actin content were not changed. When bovine aorta-derived endothelial cells were, cAMP and F-actin content were increased, while permeability was reduced. These findings suggest that endothelial cells derived from different tissues clearly showed the different reactions to histamine, the increase in [Ca2+]i led to the increase in endothelial permeability, while the increase in cAMP levels led to the reduction in permeability, and finally, F-actin regulated endothelial macromolecular permeability.

Bruceine B, a potent inhibitor of leukocyte-endothelial cell adhesion.

Leukocyte adhesion to vascular endothelial cells is an essential step in the development of inflammatory diseases. We have searched for inhibitors of leukocyte-endothelial cell adhesion that could be used as anti-inflammatory drugs and found that bruceine B (0.2 microgram/ml; 0.44 microM) inhibited human neutrophil or T cell adhesion to tumor necrosis factor-alpha (TNF) stimulated human umbilical vein endothelial cells (HUVEC). The inhibition of neutrophil adhesion to TNF-stimulated HUVEC by bruceine B was not derived from cytotoxic effects, as determined by measurement of the level of lactate dehydrogenase (LDH) activity in conditioned medium. The effect of bruceine B on neutrophil adhesion to HUVEC was not seen when the neutrophils were preincubated with bruceine B. However, inhibitory effects were evident when the HUVEC were preincubated with bruceine B. Bruceine B also inhibited neutrophil adhesion to lipopolysaccharide-stimulated HUVEC and T cell adhesion to TNF-stimulated HUVEC. These findings suggest that bruceine B may have anti-inflammatory activity.

Isolation and properties of tumor-derived endothelial cells from rat KMT-17 fibrosarcoma.

Rat KMT-17 fibrosarcoma-derived endothelial cells were isolated by Percoll gradient centrifugation with an attaching-speed separation technique, and their properties in culture were examined. The primary cultured tumor-derived endothelial cells (TEC) showed angiotensin-converting enzyme activity, positivity for Factor VIII-related antigen staining, and typical capillary-like formation on Matrigel. The primary cultured TEC monolayer showed greater permeability than normal tissue-derived endothelial cell (aorta, vena cava and epididymal fat capillary) monolayers on FITC-dextran diffusion (molecular weight 70,000). Leukocyte adhesion to TEC was reduced compared to that to fat-derived capillary endothelial cells. These characteristics resembled those of tumor vascular endothelium, and were observed both in the primary and first-passage cell cultures, but not in the fourth-passage cell cultures. Our findings indicate that primary or subcultured TEC are applicable for studies of the physiological characteristics of tumor endothelial cells.

Beta2-adrenergic receptor genotype-related changes in cAMP levels in peripheral blood mononuclear cells after multiple-dose oral procaterol.

PURPOSE: To evaluate the beta2-adrenergic receptor (beta2AR) genotype frequency in the Japanese population and the relationship between beta2AR genotype at amino acid position 16 (beta2AR-16) and desensitization to beta2-agonist ex vivo. METHODS: The beta2AR genotypes at amino acid positions 16, 27, and 164 of 92 healthy Japanese subjects were determined by polymerase chain reaction-restriction fragment-length polymorphism. The relationship between the beta2AR-16 genotype and the desensitization to beta2-agonist was examined in 10 male subjects ex vivo. Procaterol tablet (HCl salt, 50 microg, Meptin) was given orally for 5 days, and peripheral blood was obtained before and after 5 days of consecutive medications followed by the assessment of the intracellular cAMP levels in peripheral blood mononuclear cells after incubation with or without procaterol hydrochloride (0-1000 ng/mL). RESULTS: Allele frequency was Arg16:Gly16 = 46%:54%, Gln27: Glu27 = 92%:8%, and Thr164:Ile164 = 100%:0%, respectively. The cAMP levels were increased by incubation with procaterol hydrochloride, and the increase was suppressed after 5 days of consecutive medications. The suppression was more significant in the homozygote for Gly16 than the homozygote for Arg16. CONCLUSIONS: The desensitization to beta2-agonist was associated more frequently with the mutation at beta2AR-16 (Gly16).

Effect of tumour cell-conditioned medium on endothelial macromolecular permeability and its correlation with collagen.

Conditioned medium prepared from mouse melanoma B16 cells (B16-CM) increases the macromolecular permeability of bovine aortic, venous and human umbilical vein endothelial monolayer. Collagen, which is synthesised by endothelial cells, has an important function in regulating the permeability of endothelial monolayer. Briefly, low collagen content leads to hyperpermeable structure of the endothelial monolayer. In the present studies, we examined the relationship between the increase of endothelial permeability and content of synthesised collagen of endothelial cells cultured with B16-CM. The B16-CM reduced endothelial collagen content but did not digest collagen directly. Matrix metalloproteinase inhibitor, 1,10-phenanthroline, inhibited the increase in permeability due to addition of B16-CM. These data suggest that B16-CM acts on endothelial cells, stimulating the digestion of endothelial collagen, and that the reduced content of collagen leads to the hyperpermeability of the endothelial monolayer.

Suppression of solid tumor growth by a monoclonal antibody against tumor vasculature in rats: involvement of intravascular thrombosis and fibrinogenesis.

We have reported that immunization of rat tumor-derived endothelial cells (TEC) isolated from KMT-17 solid tumors results in the generation of several monoclonal antibodies (MAbs). TES-23, one of these MAbs, recognizes a naturally occurring 80-kDa antigen expressed on endothelial cells of tumor blood vessels. To determine whether such MAbs can suppress solid tumor growth in vivo by impairment of endothelial cells in tumors following direct binding, we tested the biodistribution of (125)I-labeled TES-23 in rats bearing KMT-17 solid tumors. We also examined the effect of treatment using unconjugated TES-23 on tumor growth and histo-pathological changes in tumor tissues. Biodistribution studies showed localization of TES-23 into tumor tissues 60 min after intravenous injection. TES-23 suppressed significantly the growth of KMT-17 solid tumors following administration for 5 days. Histo-pathological examination showed that TES-23 caused degeneration, apoptosis and/or necrosis and denudation of endothelial cells in viable tumor areas following local aggregation and adhesion of lymphocytes, with subsequent intravascular thrombus formation by platelets and fibrin. Our results indicate that TES-23, which recognizes TEC, can target endothelial cells of solid tumor vasculature directly, resulting in growth suppression in vivo by reduction of blood flow due to intravascular thrombosis. Our results also suggest that targeting tumor vasculature is a potentially attractive approach for the treatment of solid tumors.

Effective cancer targeting using an anti-tumor tissue vascular endothelium-specific monoclonal antibody (TES-23).

Immunoconjugate targeting of solid tumors has not been routinely successful because the endo-thelial cells of blood vessels act as a physical barrier against the transport of macromolecules, such as antibodies. In the present study, we attempted to achieve tumor vascular targeting with an anti-tumor tissue endothelium-specific monoclonal antibody (TES-23). TES-23, an IgG1 monoclonal antibody raised against rat KMT-17 fibrosarcoma-derived endothelial cells, was covalently conjugated with neocarzinostatin (NCS) in a previous study. The TES-23-NCS conjugate induced tumor hemorrhagic necrosis, and showed marked anti-tumor effects against rat KMT-17 fibrosarcoma. This result prompted us to investigate whether this approach would be applicable to various other types of solid tumors. One hour after injection of (125)I-labeled TES-23 into BALB / c mice bearing Meth-A fibrosarcoma and Colon 26 adenocarcinoma, the tumor accumulation of TES-23 was greater than that of the control IgG. In the present study, we report the anti-tumor effects of this monoclonal antibody in mice bearing Meth-A fibrosarcoma. Mice treated with the immunoconjugate showed improved survival with no side effects. This result indicates that common antigens may be found in different kinds of tumor endothelial cells, and that TES-23 might recognize these antigens.

Identification of tumor vascular antigens by monoclonal antibodies prepared from rat-tumor-derived endothelial cells.

We have reported the isolation and specific in vitro properties of tumor-derived endothelial cells (TEC) from rat KMT-17 fibrosarcomas transplanted into rats. To develop antibody-based tumor vascular targeting therapy for solid tumors, we have generated monoclonal antibodies (MAbs) using passive immunization of outside-out membrane vesicles of rat epididymal-fat-pad-derived capillary endothelial cells (FCEC) followed by active immunization of those of rat TEC. The MAbs produced were screened against TEC and FCEC. Of all cultured hybridomas, 75 (3.3%) of the secreted MAbs preferentially recognized TEC. We selected a total of 7 MAbs which detected antigens highly abundant in TEC, although 5 of the 7 MAbs were weakly positive for FCEC in cell-ELISA and FACS analyses. The antigens recognized by these MAbs, with the exception of MAb TES-7, were present on endothelial cells of tumor blood vessels in KMT-17 fibrosarcoma tissues, as shown by immunohistochemical analysis. Antigens of 40- and 80-kDa were recognized by MAbs TES-1, 7, 17, 21 and 26 and by MAbs TES-23 and 27 respectively. Although the function of these antigens, which are preferentially expressed on rat tumor-derived endothelial cells, is still unknown, we believe that future studies of such antigens will help elucidate the role of endothelial cells in tumor vasculature. Our results indicate that MAbs may provide a novel tool for the development of antibody-based therapy targeting tumor vasculature.

Tumor vascular targeting using a tumor-tissue endothelium-specific monoclonal antibody as an effective strategy for cancer chemotherapy.

In this study, we attempted to develop tumor vascular targeting with a tumor tissue endothelium-specific monoclonal antibody. TES-23, which strongly and selectively recognizes tumor tissue endothelial cells, was chemically conjugated with Neocarzinostatin (NCS), and the anti-tumor effect was examined. The immunoconjugate, TES-23-NCS, showed, through the use of tumor hemorrhagic necrosis, a marked anti-tumor effect on KMT-17 tumors in rats at a dosage of 17 micrograms/kg (NCS equivalent) without any side effects, probably due to specific tumor vascular injury. By contrast, TES-23 alone (107 micrograms/kg), NCS alone (17 micrograms/kg), and Mopc-NCS (Mopc, 107 micrograms/kg; NCS, 17 micrograms/kg), the immunoconjugate of control antibody, did not have any anti-tumor activities. By tissue distribution analysis, TES-23 and TES-23-NCS showed high accumulation in KMT-17 tumors 1 h after intravenous administration. Moreover TES-23 also accumulated in Sarcoma-180 tumors in mice 1 h after intravenous administration. These results suggest that TES-23 may be a candidate for a potential tumor vascular targeting agent that is applicable to a wide variety of tumor types.

Antibody-based therapy targeting tumor vascular endothelial cells suppresses solid tumor growth in rats.

We have developed a new approach to antibody-based therapy of solid tumors by targeting tumor vascular endothelial cells (EC) which are essential for the growth of solid tumors. We investigated the effect of an antibody against tumor-derived endothelial cells (TEC) on the growth of solid tumors in rats. Intravenous administration of TES-23, a monoclonal antibody generated by TEC isolated from rat KMT-17 solid tumors, at 1 mg/rat/day for 5 days resulted in significant suppression of KMT-17 tumor growth. Histopathological analysis of tumors administered with TES-23 showed that adhesion of lymphocytes to EC followed by denudation of EC in the viable tumor area. In contrast, little obvious toxicity was observed in most of the rat organs examined. These findings suggest that the concept of an antibody-based therapy with targeting tumor vascular EC would be promising in treatment of solid tumors.

Specific binding of TES-23 antibody to tumour vascular endothelium in mice, rats and human cancer tissue: a novel drug carrier for cancer targeting therapy.

The tissue distribution of anti-tumour vascular endothelium monoclonal antibody (TES-23) produced by immunizing with plasma membrane vesicles from isolated rat tumour-derived endothelial cells (TECs) was assessed in various tumour-bearing animals. Radiolabelled TES-23 dramatically accumulated in KMT-17 fibrosarcoma, the source of isolated TECs after intravenous injection. In Meth-A fibrosarcoma, Colon-26 adenocarcinoma in BALB/c mice and HT-1080 human tumour tissue in nude mice, radioactivities of 125I-labelled TES-23 were also up to 50 times higher than those of control antibody with little distribution to normal tissues. The selective recognition of TES-23 to TECs was competitively blocked by preadministration of unlabelled TES-23 in vivo. Furthermore, immunostaining of human tissue sections showed specific binding of TES-23 on endothelium in oesophagus cancers. These results indicate that tumour vascular endothelial cells express common antigen in different tumour types of various animal species. In order to clarify the efficacy of TES-23 as a drug carrier, an immunoconjugate, composed of TES-23 and neocarzinostatin, was tested for its anti-tumour effect in rats bearing KMT-17 fibrosarcomas. The immunoconjugate (TES-23-NCS) caused marked regression of the tumour, accompanied by haemorrhagic necrosis. Thus, from a clinical view, TES-23 would be a novel drug carrier because of its high specificity to tumour vascular endothelium and its application to many types of cancer.