A genome-wide association study identifies new loci for ACE activity: potential implications for response to ACE inhibitor.

Because angiotensin-converting enzyme (ACE) activity is implicated widely in biological systems, we aimed to identify its novel quantitative trait loci for the purposes of understanding ACE activity regulation and pharmacogenetics relating to ACE inhibitor (ACEI). We performed a two-stage genome-wide association study: (1) from 400 young-onset hypertension (YOH) subjects and (2) a confirmation study with an additional 623 YOH subjects. In the first stage, eight single nucleotide polymorphisms (SNPs) of the ACE structural gene and one SNP of ABO genes were significantly associated with ACE activity. SNP rs4343 in exon17 near the well-known insertion/deletion polymorphism had the strongest association. We confirmed in the second stage that three SNPs: rs4343 in ACE gene (P=3.0 x 10⁻²5), rs495828 (P=3.5 x 10⁻8) and rs8176746 (P=9.3 x 10⁻5) in ABO gene were significantly associated with ACE activity. We further replicated the association between ABO genotype/blood types and ACE activity in an independent YOH family study (428 hypertension pedigrees), and showed a potential differential blood pressure response to ACEI in subjects with varied numbers of ACE-activity-raising alleles. These findings may broaden our understanding of the mechanisms controlling ACE activity and advance our pharmacogenetic knowledge on ACEI.

Neoadjuvant therapy with interleukin-12-loaded polylactic acid microspheres reduces local recurrence and distant metastases.

BACKGROUND: We previously demonstrated that the intratumoral injection of biodegradable polylactic acid microspheres that were loaded with interleukin (IL)-12 can induce a systemic antitumor immunity. We sought to investigate the clinical potential as neoadjuvant therapy. METHODS: Mice were inoculated with 5 x 10(7) Line-1 cells subcutaneously. Six days later, a single intratumoral injection of IL-12- or BSA-loaded microspheres were given; 14 days later, autopsy was performed to document metastases. Mice were inoculated with 5 x 10(7) Line-1 cells and 10 days later either treated with IL-12- or BSA-loaded microspheres or resected. Treated tumors were resected 6 days after treatment. Mice were observed 45 days for local recurrence before autopsy. RESULTS: Intratumoral injection of IL-12 microspheres resulted in significant suppression of tumor growth compared with controls (599 +/- 255 mm(3) vs 1591 +/- 372 mm(3); P =.001) and pulmonary metastases (0.4 vs 3.8 nodules per mouse; P =.003). Given before the operation, IL-12-loaded microspheres both decreased the local recurrence rate (100% to 40%) and pulmonary metastases (5.2 vs 0.6 nodules per mouse; P =.06). Earlier resection did not improve local recurrence or distant metastases. CONCLUSIONS: Intratumoral injection of IL-12-loaded polylactic acid microspheres promotes the development of systemic antitumor immunity that can eradicate micrometastases. As a neoadjuvant therapy, this can result in decreased local and distant recurrence.

Human inflammatory cells within the tumor microenvironment of lung tumor xenografts mediate tumor growth suppression in situ that depends on and is augmented by interleukin-12.

The human tumor microenvironment includes a mixture of tumor cells, inflammatory cells, fibroblasts, and endothelial cells, all of which are tethered to an extracellular matrix. It has been difficult to study the dynamic interactions of these cells in human tumors in situ for obvious ethical and logistical considerations that prohibit experimental manipulations of tumors while still in patients. Fresh tissue from human lung tumor biopsy implanted into SCID mice was shown to remain viable, and the histologic appearance of the tumor microenvironment was maintained in the tumor xenografts for at least 3 months. In this study, the authors established that the inflammatory cells within human tumor xenografts can suppress tumor growth, and that this suppression is a result, in part, of endogenously produced interleukin-12 (IL-12) because IL-12 neutralizing antibodies enhance the growth of the tumor xenografts. The tumor-inhibitory activity of the inflammatory leukocytes is also enhanced by the local and sustained release of human recombinant IL-12 into the tumor microenvironment from cytokine-loaded biodegradable microspheres. Neither the anti-IL-12 neutralizing antibody nor the delivery of exogenous IL-12 from microspheres had any effect on tumor xenografts in the absence of the inflammatory leukocytes. In conclusion, the inflammatory cells within the tumor microenvironment of human lung tumor xenografts are functional and can suppress tumor growth, and the dynamic effects of the inflammatory cells can be modulated by exogenous cytokines.

Incidence and predictors of isolated systolic hypertension and isolated diastolic hypertension in Taiwan.

BACKGROUND AND PURPOSE: The significance of isolated systolic hypertension (ISH) has been well documented, particularly in the elderly. However, isolated diastolic hypertension (IDH) has not been formally recognized as a unique hypertension entity. This study compared the ages of onset and characteristics of ISH and IDH. METHODS: The Cardiovascular Disease Risk Factors Two-Township Study (CVDFACTS) is an ongoing longitudinal study of the risk factors for and pathogenesis of cardiovascular disease in two Taiwanese townships, Chu-Dung (a Hakka community) and Pu-Tzu (a Fukienese community); participating patients were included in our study. Among the 3,357 subjects who were aged at least 20 years, free of hypertension, and had complete data at baseline, 2,374 subjects were followed. The average duration of follow-up was 3.23 years and the follow-up rate was 71%. Data regarding smoking, alcohol consumption, health and socioeconomic background, blood pressure, and body mass index were collected. Clinical and hemostatic profiles were assessed. RESULTS: ISH (systolic blood pressure, SBP > or = 140 mmHg and diastolic blood pressure, DBP < or = 90 mmHg) incidence increased with age in general (men: 0 per 1,000 person-years at age 20-34 yr, 1.9 at age 35-49, 14.3 at age 50-64, 40.9 at age 65-74, and 73.3 at age 75+ yr; women: 0 per 1,000 person-yr at age 20-34 yr, 3.6 at age 35-49, 17.8 at age 50-64, 64.9 9 at age 65-74, and 33.5 at age 75+ yr), but peak incidence of IDH (DBP > or = 90 mmHg and SBP < or = 140 mmHg) occurred between 35 and 49 years (men: 8.9 per 1,000 person-yr at age 20-34 yr, 14.5 at age 35-49, 12.3 at age 50-64, 2.7 at age 65-74, and 0 at age 75+ yr; women: 1.7 per 1,000 person-yr at age 20-34, 4.2 at age 35-49, 3.7 at age 50-64, 0 at age 65-74, and 0 at age 75+ yr). Significant predictors for ISH were older age (men: hazard ratio, HR = 8.25 at 45-64 yr and HR = 22.91 at 65+ yr; women: HR = 34.11 at 45-64 yr and HR = 97.98 at 65+ yr), diabetes (HR = 2.57) and elevated fibrinogen (HR = 1.49) in men, and shorter clotting time in women (HR = 1.23). Significant predictors for IDH were elevated body mass index (men: HR = 4.03; women: HR = 7.4), and higher glucose (HR = 1.46) and uric acid concentrations (HR = 1.94) in men. CONCLUSIONS: The results of this study indicate that ISH and IDH have different age incidence patterns and predictors, and suggest that the pathogenesis of ISH and IDH may be different.

In situ tumor vaccination with interleukin-12-encapsulated biodegradable microspheres: induction of tumor regression and potent antitumor immunity.

An alternative technology for the local and sustained delivery of cytokines to tumors for cancer immunotherapy was evaluated and shown here to induce tumor regression, suppression of metastasis, and development of systemic antitumor immunity. Treatment of tumor-bearing BALB/c mice with a single intratumoral injection of biodegradable polylactic acid microspheres loaded with recombinant interleukin-12 (IL-12) promoted complete regression of the primary tumor and prevented the metastatic spread to the lung. Mice that experienced tumor regression after being treated rejected a subsequent challenge with live tumor cells, which indicated the development of systemic antitumor immunity. In situ tumor vaccination, ie., injection of IL-12 microspheres into existing tumors, was superior to vaccination of mice with mixtures of tumor cells (live or irradiated) and IL-12 microspheres in inducing systemic antitumor immunity. The sustained release of IL-12 from the microspheres was superior to bolus injection of free IL-12, and intratumoral delivery of microspheres was more effective than other routes of administration. These studies establish the utility of biodegradable polymer microspheres as a clinically feasible alternative to systemic cytokine therapy and cytokine gene-modified cell vaccines for the treatment of neoplastic disease.

Cytokines delivered by biodegradable microspheres promote effective suppression of human tumors by human peripheral blood lymphocytes in the SCID-Winn model.

A new technology for the local and sustained delivery of immunostimulatory molecules to the tumor environment for cancer immunotherapy was evaluated. The ability of cytokines delivered by biodegradable microspheres to promote the antitumor activity of human peripheral blood lymphocytes (PBL) was tested in a human PBL, human tumor, and SCID mouse (SCID-Winn) model. Co-engraftment of human recombinant IL-12-loaded microspheres with human PBL and tumors in SCID mice promoted complete tumor suppression in as many as 100% of the mice, whereas microspheres loaded with polyethyleneglycol-interleukin-2 suppressed but did not eliminate the growth of tumor xenografts. Control microspheres (loaded with bovine serum albumin) in the presence of human PBL or cytokine-loaded microspheres in the absence of human PBL had no tumor-suppressive effect. Coincident with the enhancement of the human PBL-mediated antitumor activity in mice treated with IL-12-loaded microspheres was the production and release of human IFN-gamma indicating that IL-12 released from the microspheres results in the activation of the engrafted human PBL. The results establish that biodegradable microspheres represent an effective tool for the local and sustained delivery of cytokines to the tumor environment for cancer immunotherapy.

Interleukin-12 delivered by biodegradable microspheres promotes the antitumor activity of human peripheral blood lymphocytes in a human head and neck tumor xenograft/SCID mouse model.

BACKGROUND: The role of cytokines in tumor regression is now well established. The major limitation for the clinical use of cytokines is the lack of a simple and effective protocol for the local and sustained delivery of cytokines to the tumor milieu. This study reports suppression of human head and neck squamous cell carcinoma (HNSCC) by human peripheral blood lymphocytes (HuPBL) following local, sustained delivery of interleukin-12 (IL-12) to tumors with biodegradable microspheres in a human/SCID mouse chimeric model. Materials and Methods Nondisrupted biopsy pieces (120 mg) of primary HNSCC were implanted s.c. into severe combined immunodeficient (SCID) mice and were expanded by serial passage in mice. Tumors were then titrated with different doses of allogeneic HuPBL by coengraftment of tumor pieces and HuPBL into the subcutis of SCID mice to determine whether the HuPBL possessed antitumor activity (the SCID/Winn model). The lymphocyte subsets that were responsible for the suppression of tumor engraftment were identified by selective depletion of the CD4+, CD8+, and CD56+ cells from the HuPBL prior to engraftment into mice. Attempts were then made to augment the antitumor activity of the HuPBL either by repeated intralesional bolus injections of recombinant human IL-12 (0.5 microg x 10 doses) or with a single dose of IL-12-loaded microspheres ( approximately 1.65 microg IL-12/mg microspheres, 2 mg microspheres/mouse). RESULTS: Successful engraftment of HNSCC was observed in 12 of 19 different patient samples. Normal histological architecture of tumor was maintained up to four serial passages in the SCID mice. After the first tumor engraftment, but not in subsequent passages, human immunoglobulin produced by plasma cells present in the tumor infiltrating lymphocyte population was detected in the mouse sera. Allogeneic human PBL displayed antitumor cytotoxic activity in a cell dose-dependent fashion when coengrafted with the tumors passaged in SCID mice. Lymphocyte subset depletion studies established that tumor suppression was dependent on both the CD8+ T lymphocytes and the CD56+ natural killer cells. Treatment of tumors with a single intralesional injection of IL-12-loaded microspheres was highly effective, resulting in the complete suppression of tumor engraftment in 50% of the mice. In contrast, treatment of tumors with repeated bolus IL-12 injections suppressed tumor engraftment only transiently and did not result in complete tumor rejection in any of the mice. CONCLUSION: The coengraftment of HNSCC and allogeneic lymphocytes into SCID mice provides a viable model with which to evaluate immunotherapeutic strategies for human cancer. The use of biodegradable microspheres for local sustained delivery of cytokines to augment lymphocyte mediated antitumor immunity within the tumor microenvironment provides a safer and simpler alternative to current cytokine immunotherapy protocols.

Cytokine immunotherapy of cancer with controlled release biodegradable microspheres in a human tumor xenograft/SCID mouse model.

A novel biodegradable poly(lactic acid) microsphere formulation was evaluated for in vivo cytokine immunotherapy of cancer in a human tumor xenograft/ severe combined immunodeficiency (SCID) mouse model. Co-injection of interleukin-2 (IL-2)-loaded microspheres with tumor cells into a subcutaneous site resulted in the complete suppression of tumor engraftment in 80% of animals. In contrast, bovine-serum-albumin(BSA)-loaded particles or bolus injections of poly(ethylene glycol)/IL-2 were ineffective in preventing tumor growth. The antitumor effect of IL-2 released by the microspheres was shown to be mediated by the mouse natural killer cells. This is the first evidence that the rejection of human tumor xenografts can be provoked by the sustained in vivo delivery of IL-2 from biodegradable microspheres. The use of poly(lactic acid) microspheres to deliver cytokines to the tumor environment could provide a safer and simpler alternative to gene therapy protocols in the treatment of cancer.

Biologically erodable microspheres as potential oral drug delivery systems.

Biologically adhesive delivery systems offer important advantages over conventional drug delivery systems. Here we show that engineered polymer microspheres made of biologically erodable polymers, which display strong adhesive interactions with gastrointestinal mucus and cellular linings, can traverse both the mucosal absorptive epithelium and the follicle-associated epithelium covering the lymphoid tissue of Peyer's patches. The polymers maintain contact with intestinal epithelium for extended periods of time and actually penetrate it, through and between cells. Thus, once loaded with compounds of pharmacological interest, the microspheres could be developed as delivery systems to transfer biologically active molecules to the circulation. We show that these microspheres increase the absorption of three model substances of widely different molecular size: dicumarol, insulin and plasmid DNA.

Incidence of NIDDM and the effects of gender, obesity and hyperinsulinaemia in Taiwan.

Our aim is to determine non-insulin-dependent diabetes mellitus (NIDDM) incidence in Taiwan and examine its relation to obesity and hyperinsulinaemia in Chinese men and women. A total of 995 men and 1195 women aged 35-74 years free from diabetes in two townships in Taiwan were followed up with a second examination. At baseline general and metabolic data were recorded, and detailed anthropometric parameters and plasma glucose and insulin were assessed. World Health Organisation (WHO) criteria of fasting glucose 7.8 mmol/l or greater was utilized for defining diabetes. The age-standardized incidence rate based on the United States population in 1970 was 9.3/1000 (CI 5.8-12.8) in men and 9.3/1000 (CI 6.2-12.4) in women and the based on the WHO population in 1976 was 8.9/1000 (CI .5-12.3) in men and 8.9/1000 (CI 5.9-11.9) in women for the Chinese who had a mean BMI slightly greater than 24 (kg/m2). The predictability of the plasma glucose level was greater than that of the insulin level and the obesity indices. NIDDM incidence increased approximately threefold with each 0.67 mmol/l increase in plasma glucose level in men and women. The present study demonstrated the essential relationship of not only BMI but also central obesity indices (such as subscapular and waist circumference) to the incidence of NIDDM among men and women and a stronger relationship between NIDDM incidence and obesity in women than in men. The predictive effects of obesity indices and fasting plasma insulin values on NIDDM risk were independent of each other in men. Obesity and hyperinsulinaemia each without the presence of the other can lead to an increased risk of NIDDM. In women the NIDDM incidence increased more than additively in those with both obesity and hyperinsulinaemia compared to those with single obesity or hyperinsulinaemia. A slightly higher incidence of NIDDM in Taiwan than in western countries was found. The importance of obesity is indicated for predicting NIDDM in the community. Hyperinsulinaemia was found to play a significant role in predicting NIDDM incidence independent of obesity in men and synergistically with obesity in women.

Controlled release of platelet-derived growth factor using ethylene vinyl acetate copolymer (EVAc) coated on stainless-steel wires.

Controlled delivery of bioactive molecules to modulate or control biological processes has a number of clinical applications. The present study reports a delivery system which was designed to deliver growth factors locally to a fracture site. Ethylene vinyl acetate copolymer (EVAc), bovine serum albumin (BSA) and platelet-derived growth factor (PDGF-BB) were combined and coated onto a stainless-steel Kirschner wire (K-wire). A K-wire can be used as an intramedullary nail in small animal fractures, such as the rat. PDGF-BB stimulates thymidine uptake in human bone cell cultures when released from the K-wire delivery system. BSA release was modified by providing a final coating on the K-wire of 10% pure EVAc at the end of the fabrication. Electron microscopic examination of the surface of the rods revealed different surface pores on the K-wires coated with pure EVAc. Differences in porosity and tortuosity may account, in part, for the different release kinetics observed.

[Barium intoxication: a case report].

Barium intoxication, a rare cause of hypokalemia, can sometimes result in respiratory paralysis and ventricular tachyarrhythmia. Herein, we report one such case. A 29-year-old man swallowed barium-contaminated fried flour-coated sweet potatoes. Then, abdominal discomfort, vomiting, diarrhea, progressive muscular weakness, apnea and ventricular tachycardia developed and laboratory data revealed profound hypokalemia. He regained his health after mechanical ventilation, anti-arrhythmic agent and aggressive potassium chloride supplement. Analysis of blood, urine and contaminated flour showed the presence of barium carbonate. Barium intoxication is a medical emergency which requires rapid therapy to prevent mortality.

[Treatment with new surgical method for chronic atrial fibrillation in 5 cases with mitral valve disease].

Chronic atrial fibrillation is the most common arrhythmia in patients with mitral valve disease, it not only deteriorates cardiac output, but also promotes a 5.6 times higher incidence of thromboembolic events. Nevertheless, the exact mechanism of atrial fibrillation is still poorly understood. Moreover, the long-term results of treatment, either by drugs or cardioversion, are almost always disappointing. Recent investigations suggest that atrial fibrillation is probably due to intraatrial multiple reentrant waves. It is also well-known from animal experiments that for sustenance of the fibrillation a critical mass of atrium is necessary. Based on these data, a new surgical method has been carefully designed to reduce atrial mass and to prevent sustenance of fibrillation. Between July and December 1988, five patients, one male and four females, with mitral valve disease and chronic atrial fibrillation were selected for this new surgical method. The age distribution ranged from 25 to 57 years, with a mean of 36 +/- 13 years. All of them had chronic atrial fibrillation which had been documented for more than one year. The operation was performed using the cardiopulmonary bypass and cardioplegia myocardial protection techniques. The left atrium was opened via the interatrial sulcus. After the mitral valve was repaired, cryosurgery (-60 degrees C for 120 sec) was applied along the borders of the left atrial free-wall. The left atrium was separated by these cryolesions into two sections, each about half the mass of the original and both sections were connected with a few atrial fibers located behind the coronary sinus. No surgical mortality or complications were encountered.(ABSTRACT TRUNCATED AT 250 WORDS)

Reappraisals of atrioventricular node reentrant tachycardia: lessons learned from surgical treatment.

The exact site of reentrant circuit involved in the atrioventricular node reentrant tachycardia was questioned. Seven patients (6 females and 1 male), aged 21 to 64 years (mean = 40 +/- 17 years), with refractory nodal reentry, underwent surgical treatment. The associated cardiac diseases included rheumatic valvar disease in two and an atrial septal defect. Electrophysiologic studies before surgery showed dual nodal pathways in 4 patients. Right atrial endocardial mapping was performed and the earliest retrograde atrial activation during tachycardia was mapped to the apex of the triangle of Koch in 6 patients and near the orifice of coronary sinus in one. Perinodal dissection was performed according to the location of earliest retrograde atrial activity. Care was taken to preserve as much of the atrioventricular node and its arterial supply as was possible. Immediately after surgery, conduction in an antegrade direction recovered and the tachycardia could no longer be reproduced. There was no surgical mortality or morbidity. At 10 to 26 months of follow-up, all patients remain free of tachycardia without antiarrhythmic drugs. Four patients underwent repeated electrophysiologic studies at 2 weeks to 6 months after surgery. Dual nodal pathways were no longer demonstrated. It is concluded that the perinodal atrial tissue plays a part in the atrioventricular nodal reentry, and that surgical dissection is a simple and effective treatment for patients with refractory atrioventricular node reentrant tachycardia.

[Cavernous hemangioma of the heart: report of a case].

Cavernous hemangioma of the heart is a very rare disease. A 57-year-old male patient was admitted due to frequent onset of dull chest pain, which had been occurring for about 1 year. The pain was not related to exercise and was not relieved with nitroglycerin. On echocardiographic examination, a tumor was shown in the outflow tract of the right ventricle and was confirmed with computer tomography. He underwent open heart surgery for resection of the tumor. After a median sternotomy and opening of the pericardial cavity, a reddish-brown-colored tumor, 3 cm in diameter, was found protruding from the epicardial layer of the right ventricular outflow tract. The tumor involved all layers of the ventricle and could be resected only with the help of the cardiopulmonary bypass technique. The defect in the right ventricle was repaired with a woven dacron patch. The patient recovered without incident after the operation, and experienced no chest pain during 7 months of follow-up. Histology showed it to be a cavernous hemangioma.

Time course of the ventricular arrhythmias following coronary reperfusion in dogs.

The time course of ventricular arrhythmia following reperfusion was investigated in 12 dogs undergoing a 2-hour coronary artery occlusion followed by reperfusion. At days 1, 3 and 7 following coronary reperfusion, the cardiac rhythm was monitored with a 24-hour Holter electrocardiographic recorder. Six dogs with sham operations were also studied with Holter monitoring at the same time periods as a control group. This showed that, following coronary reperfusion, all 12 dogs (group A) developed frequent premature ventricular complexes (PVCs) at day 1, with a total count (beats per hour, bph) of 1,806 +/- 390 (mean +/- SD). The PVC count decreased to 298 +/- 96 at day 3 (p less than 0.001) and 0.4 +/- 0.1 at day 7 (p less than 0.001). The incidence of spontaneous sustained ventricular tachycardia (VT) at day 1 was 100%; 1 of them deteriorated to ventricular fibrillation (VF). At day 3, 9 of the remaining 11 dogs (82%) had recurrent VTs (p greater than 0.05 vs day 1) which were of shorter duration and a slower rate. One of the 11 dogs died at day 5 and the remaining 10 dogs survived to day 7, with all the VTs subsiding (p less than 0.001 vs day 1 or 3). In the control group, only isolated PVCs were observed at day 1 (7 +/- 3 bph, p less than 0.001 vs group A) and no spontaneous VT or VF was noted. We conclude that spontaneous ventricular arrhythmias were observed in all dogs with coronary reperfusion following a 2-hour coronary artery occlusion and the arrhythmias could subside in 1 week of those survived. Only rarely did VT deteriorate to VF.