Inhibition of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway in rheumatoid synovial fibroblasts using small molecule compounds.

Janus kinase (JAK) inhibitors have been developed as anti-inflammatory agents and have demonstrated clinical efficacy in rheumatoid arthritis (RA). We investigated if JAK-3-selective inhibition alone could disrupt cytokine signalling in rheumatoid synovial fibroblasts. In-vitro studies were performed using synovial fibroblasts isolated from patients with RA. Levels of activated JAK and signal transducer and activator of transcription (STAT) proteins were detected by immunoblot analysis. Target-gene expression levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) or real-time PCR. The JAK inhibitors CP-690,550 and INCB028050 both suppressed activation of JAK-1/-2/-3 and downstream STAT-1/-3/-5, as well as the expression levels of target proinflammatory genes (MCP-I, SAA1/2) in oncostatin-M (OSM)-stimulated rheumatoid synovial fibroblasts. In contrast, the JAK-3-selective inhibitor, PF-956980, suppressed STAT-1/-5 activation but did not affect STAT-3 activation in OSM-stimulated rheumatoid synovial fibroblasts. In addition, PF-956980 significantly suppressed MCP-1 gene expression, but did not block SAA1/2 gene expression in OSM-stimulated rheumatoid synovial fibroblasts. These data suggest that JAK-3-selective inhibition alone is insufficient to control STAT-3-dependent signalling in rheumatoid synovial fibroblasts, and inhibition of JAKs, including JAK-1/-2, is needed to control the proinflammatory cascade in RA.

Is driving a car a risk for Legionnaires' disease?

Legionnaires' disease (LD) is a major cause of severe community-acquired pneumonia but the source and mode of transmission are not always apparent, especially in sporadic cases. We hypothesized that LD can be acquired from the air-conditioning systems of motor cars. Swabs were taken from the evaporator compartments of the air-conditioning system of scrapped cars. Healthy subjects who were mainly employees of regional transportation companies were tested for antibody to Legionella pneumophila serogroups 1-6; they also completed a questionnaire. Legionella species were detected in 11/22 scrapped cars by the loop-mediated isothermal amplification method. The prevalence of microplate agglutination titres > or =1:32 was significantly higher in subjects who sometimes used car air-conditioning systems. Although we did not prove a direct link between Legionella spp. in the car evaporator and LD, our findings point to a potential risk of car air-conditioning systems in LD, which needs further investigation.

Specific-wavelength visible light irradiation inhibits bacterial growth of Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: The effects of laser irradiation on Porphyromonas gingivalis have been reported, but the results are still controversial regarding the efficiency because of the differences of the light sources and irradiation conditions. The aim of this study was to determine the wavelength and irradiation conditions under which the most effective inhibitory effect on P. gingivalis growth was seen without any photosensitizers. MATERIAL AND METHODS: Using an Okazaki large spectrograph, monochromatic light spectra ranging from 400 to 700 nm were evaluated to determine which spectra effectively inhibited bacterial growth. Moreover, using a monochromatic 405-nm irradiating device, the effects of various irradiating conditions on P. gingivalis growth were examined. RESULTS: Growth of bacteria irradiated at 400 nm and 410 nm was significantly suppressed compared with a nonirradiated control, whereas wavelengths of 430 nm and longer produced no significant inhibition. A constant energy density of 15 J/cm2 was found to be enough to show an inhibitory effect. Significant inhibition of bacterial growth was found after only 1 min at 50 mW/cm2 irradiation. CONCLUSION: These results indicate that P. gingivalis growth is specifically suppressed by 405-nm light irradiation, suggesting that visible blue light irradiation is a promising means for eradicating periodontopathogenic bacteria from periodontal lesions.

Side population cells expressing ABCG2 in human adult dental pulp tissue.

AIM: To investigate the presence of side population (SP) cells by the Hoechst exclusion method in human adult dental pulp tissue. METHODOLOGY: Human adult dental pulp-derived cells were generated from third molar teeth. The cells were stained with Hoechst 33342 and sorted into SP cells or non-SP cells [main population (MP) cells]. Both cell types were compared with cell growth and RT-PCR analyses. RESULTS: SP cells that express ABCG2, Nestin, Notch-1 and alpha-smooth muscle actin were found at frequencies ranging from 0.67% to 1.02%. This SP profile disappeared in the presence of verapamil. These SP cells expressed dentine sialophosphoprotein and dentine matrix protein-1 when cultured in osteogenic medium. CONCLUSION: Human adult dental pulp tissue contains SP cells that differentiate into odontoblast-like cells.

Osteopetrosis complicated by osteomyelitis of the mandible: a case report including characterization of the osteopetrotic bone.

A case of a 53-year-old Japanese man with osteopetrosis complicated by osteomyelitis of the mandible is presented. The patient experienced frequent exacerbations and remissions of osteomyelitis of the mandible, despite undergoing several surgical debridements and sequesterectomies with appropriate antimicrobial therapy, for 3 years. Finally, the patient underwent mandibular segmental resection followed by reconstruction with a titanium reconstruction plate. Fifty-one months after surgery there is no evidence of recurrent osteomyelitis of the mandible, suggesting that a more radical surgical approach is preferable for patients with severe complications resulting from osteopetrosis. Also presented here are the histopathological and biochemical features of the osteopetrotic bone. The osteopetrotic cortical bone was morbidly sclerotic with compact and irregular laminations. Degradation of osteocytes in the osteopetrotic bone was due to hypoxia and lack of nutrition resulting from osteosclerosis. There were no significant differences between osteopetrotic and normal bone according to X-ray diffraction, Fourier transform infrared spectroscopy, collagen content or mineral content. Micro-Vickers hardness measurements showed that osteopetrotic bone was significantly harder than normal bone, and the standard deviation of hardness was greater in osteopetrotic bone. Such a loss of integrity in osteopetrotic bone is considered to be a primary reason for the greater risk of a variety of complications such as pathological fracture and refractory osteomyelitis.

Flow-mediated dilation in brachial artery in the second half of pregnancy and prediction of pre-eclampsia.

Endothelial injury and increased vascular reactivity are involved in the pathogenesis of pre-eclampsia (pregnancy-induced hypertension). To investigate whether flow-mediated dilation (endothelium-dependent dilation) and the reactive hyperemic response can predict pre-eclampsia, we prospectively measured flow-mediated dilation and the Doppler flow velocity pattern (V, cm/s) in the brachial artery using high-resolution ultrasound in 43 pregnant women (32+/-5 years old) in the second half of their pregnancy, and compared the findings with traditional risk factors. Regarding the Doppler flow velocity pattern, the pulsatility index (PI)=(systolic V-diastolic V)/mean V and resistance index (RI)=(systolic V-diastolic V)/systolic V were calculated. For the flow-mediated dilation, the per cent diameter changes were determined based on those from baseline to hyperemic conditions. Nine women suffered from pre-eclampsia and 34 women remained normotensive. Only flow-mediated dilation was found to be significantly lower in the subsequently developed pre-eclampsia patients (1.6+/-1.0% in subsequently developed pre-eclampsia patients vs 11.0+/-4.5% in normotensive patients, P<0.05). Neither the other traditional factors nor the Doppler flow velocity pattern were significantly different between the subsequently developed pre-eclampsia and normotensive patients. If a normal cutoff value of 3.0% obtained from age-matched 14 nonpregnant women (32+/-7 years old) in our laboratory was used, the positive predictive value of flow-mediated dilation (<3.0%) for subsequent pre-eclampsia is 90% and the negative predictive value is 100%. In conclusion, flow-mediated dilation in brachial artery can be a simple and noninvasive modality to predict pre-eclampsia.

Novel mutations in the a3 subunit of vacuolar H(+)-adenosine triphosphatase in a Japanese patient with infantile malignant osteopetrosis.

A case of infantile malignant osteopetrosis is described. The patient died from respiratory hemorrhage at 7 months of age despite treatment that included high doses of active vitamin D and administration of interferon-gamma. A postmortem examination revealed the presence of many osteoclasts in the bone, which lacked ruffled borders. This observation was consistent with the histology of bone reported in Atp6i-knockout mice, which lack the gene encoding the a3 subunit of vacuolar-type H(+)-adenosine triphosphatase (ATPase). Sequence analysis of the TCIRG1 gene encoding the a3 subunit revealed two novel mutations: a deletion/insertion mutation in exon 9 and a T-to-C transition at the splice donor site of intron 19. The former mutation caused a frame shift and premature stop codon. The latter was associated with abnormal splicing, which was confirmed by sequencing the products amplified by reverse transcription-polymerase chain reaction (RT-PCR), using total RNA from the liver specimen as template. Although several mutations in the TCIRG1 gene in infantile malignant osteopetrosis have been reported in other populations, this is the first case of a Japanese patient with a mutation identified in this gene. These results support the important role of the subunit in the function of the proton pump.

Assessment of left ventricular volume by an ambulatory radionuclide monitoring system during head-up tilt in patients with unexplained syncope: relation to autonomic activity assessed by heart rate variability.

BACKGROUND: Decreased left ventricular volume during head-up tilt plays an important role in triggering syncope in patients with neurally mediated syncope. However, precise changes in left ventricular volume during head-up tilt have not been well investigated. This study was conducted to test the hypothesis that the decline in left ventricular volume during tilt could trigger ventricular mechanoreceptor activation. METHODS AND RESULTS: To investigate the mechanisms of tilt-induced syncope, we measured the temporal changes in left ventricular volume, ejection fraction, cardiac output, and heart rate variability indices during head-up tilt in 25 patients with syncope of undetermined etiology. Eleven patients had a cardioinhibitory response (CI group), 7 patients showed a vasodepressor response (VD group), and 7 patients demonstrated a negative response (NG group). Before syncope, ejection fraction increased most in the CI group, the left ventricular end-diastolic volume declined most in the VD group (VD group, -11.0% +/- 3.3%; CI group, -2.8% +/- 4.8%; NG group, -3.4% +/- 2.2%; P <.005), and the high-frequency spectra increased most in the CI group (CI group, 25.0% +/- 21.0%; VD group, -4.1% +/- 11.7%; NG group, -5.3% +/- 12.7%; P <.01). The vasodepressor response was dependent on left ventricular volume, whereas the cardioinhibitory response was related to the vagal activity reflected by high-frequency spectra. CONCLUSIONS: The precise evaluation of left ventricular volume by an ambulatory radionuclide monitoring system combined with a heart rate variability analysis is considered useful for clarifying the pathophysiology of neurally mediated syncope.

New thermo-couple copper constantan catheter for measuring regional coronary blood flow and evaluating metabolism.

A new thermodilution catheter with a thermo-couple copper constantan and latex balloon in the tip has recently been developed. This device has two orifices. The left anterior descending (LAD) and/or left circumflex (LCX) flows were measured using a Doppler flow wire. The correlation between the LAD and GCV flow as well as between the LCX and CS-GCV flow were studied in closed chest animals. The flow distributions were evaluated by colored microspheres that were infused into the LAD and LCX. The regional lactate extraction ratio was measured by GCV and/or CS blood sampling performed either with or without balloon inflation before and during pacing in either LAD or LCX occluded animals. The correlations between the LAD and GCV flow, as well as between the LCX and CS-GCV flow were significant (r=0.96, r=0.93, n=30, p<0.001). The flow distribution by color microspheres in the GCV was 75+/-10% for the LAD, whereas in CS it was 70+/-12% for the LCX. By pacing, the lactate extraction ratio of the drained blood from the ischemic myocardium was +10+/-3% in the GCV blood, whereas it was -10+/-4% (p<0.01) in the CS blood. Thus, this new thermodilution catheter with a balloon was found to be useful for measuring regional coronary hemodynamics and also for evaluating the regional myocardial metabolism.

Different mechanisms of isoproterenol-induced and nitroglycerin-induced syncope during head-up tilt in patients with unexplained syncope: important role of epinephrine in nitroglycerin-induced syncope.

INTRODUCTION: A reduction in left ventricular volume and an increase in epinephrine levels have been reported in tilt-induced neurally mediated syncope. To compare the mechanisms of isoproterenol-induced and nitroglycerin-induced syncope during head-up tilt and to investigate the role of catecholamines, the temporal changes in plasma levels of norepinephrine and epinephrine and in left ventricular volume were measured. METHODS AND RESULTS: The first study population consisted of 90 patients with syncope of unknown etiology and 12 control subjects. The second study population consisted of 43 patients with unexplained syncope. In the first study, head-up tilt (80 degree angle) was conducted for 40 minutes, and norepinephrine and epinephrine levels were measured. In the second study, all patients were randomly allocated to either isoproterenol test (20 patients) or nitroglycerin test (23 patients) for 20-minute head-up tilt. Isoproterenol infusion was given at a rate of 1 to 3 microg/min. Intravenous infusion of nitroglycerin was started at 250 microg/hour with increasing dosages up to 1,500 microg/hour. Norepinephrine and epinephrine were measured in peripheral venous blood. Left ventricular volumes were measured by echocardiography with patients in the supine position and during head-up tilt every 1 minute. End-diastolic volume and end-systolic volume were calculated. In the first study, 61 patients demonstrated a positive response and 29 patients demonstrated a negative response. Plasma norepinephrine changes during head-up tilt were not significantly different, whereas epinephrine levels were significantly higher in the positive patients than in the negative and control subjects (148 +/- 118 pg/mL vs 66 +/- 31 pg/mL and 55 +/- 27 pg/mL). Thirteen of the 20 patients given isoproterenol and 15 of the 23 patients given nitroglycerin showed a positive head-up tilt (65.0% vs 65.2%; P = NS). During isoproterenol and nitroglycerin infusion head-up tilt, epinephrine in the positive group determined by the nitroglycerin test was significantly higher than that in the other three groups (103 +/- 38 pg/mL vs 60 +/- 33 pg/mL, 31 +/- 21 pg/mL, and 50 +/- 52 pg/mL). In contrast, end-systolic volume was significantly smaller in the positive group than in the other three groups based on findings of the isoproterenol test. CONCLUSION: The findings suggest that nitroglycerin triggers head-up tilt-induced syncope by increasing epinephrine levels, whereas isoproterenol induces syncope by decreasing left ventricular volume.

Bone formation by transplanted human osteoblasts cultured within collagen sponge with dexamethasone in vitro.

To apply osteoblasts to bone reconstruction, we proved that transplanted osteoblasts possessed the differentiated osteoblastic function and formed bonelike tissue in vivo after transplantation. First, we confirmed that dexamethasone (Dex) promoted the expression of osteoblastic phenotype in human osteoblast culture using reverse-transcription-polymerase chain reaction (RT-PCR). These osteoblasts were cultured for 10 days within collagen sponge, which consists of denatured type I collagen, in the presence or absence of 10(-7) M Dex. The osteoblasts along with collagen sponge were transplanted into the trapezius muscles of 8-week-old severe combined immunodeficiency (SCID) mice, and the transplants were harvested at 2, 4, 6, and 8 weeks. At 2 weeks, Dex-treated osteoblasts formed bonelike tissue, the quantity of which increased in a time-dependent manner to 8 weeks. This bonelike tissue was composed of mineralized collagen matrix newly synthesized by the transplanted osteoblasts. This mineralized matrix was separated from the osteoblasts by nonmineralized matrixlike osteoid. Furthermore, many osteocytic cells were observed in this mineralized matrix. A high expression of alkaline phosphatase (ALPase) and osteocalcin was detected in the transplanted cells surrounding the bonelike tissue. In situ hybridization for human-specific alu sequence indicated that newly formed bone was of donor origin. The transplants of nontreated cells failed to form bonelike tissue. The transplants of collagen sponge alone formed no bonelike tissue. These studies indicate that Dex-treated human osteoblasts possess the differentiated osteoblastic function and are able to form bone tissue in vivo. These new findings are of use in facilitating the application of osteoblasts to bone reconstruction.

Establishment and characterization of chondrocyte cell lines from the costal cartilage of SV40 large T antigen transgenic mice.

Complete understanding of the physiology and pathology of the cartilage is essential to establish treatments for a variety of cartilage disorders and defects such as rheumatoid arthritis, congenital malformations, and tumors of cartilage. Although synthetic materials have been used in many cases, they possess inherent problems including wear of the materials and low mechanical strength. Autograft has been considered very effective to overcome these problems. However, the limitation of the transplant volume is a major problem in autograft to be overcome. The costal cartilage is the most serious candidate for donor site transplantation, since it is the largest permanent hyaline cartilage in the body. To investigate the possibility using the costal cartilage as a transplant source, we have established and characterized three mouse chondrocyte cell lines (MCC-2, MCC-5, and MCC-35) derived from the costal cartilage of 8-week-old male SV40 large T-antigen transgenic mice. At confluence, all the cell lines formed nodules that could be positively stained with alcian blue (pH 2.5). The size of nodules gradually increased during culturing time. After 2 and 6 weeks of culture, RT-PCR analysis demonstrated that all three cell lines expressed mRNA from the cartilage-specific genes for type II collagen, type XI collagen, aggrecan, and link protein. Furthermore, type X collagen expression was detected in MCC-5 and MCC-35 but not in MCC-2. Any phenotypic changes were not observed over 31 cell divisions. Immunocytochemistry showed further that MCC-2, MCC-5, and MCC-35 produced cartilage-specific proteins type II collagen and type XI collagen, while in addition MCC-5 and MCC-35 produced type X collagen. Treatment with 1alpha, 25-dihydroxyvitamin D(3) inhibited cell proliferation and differentiation of the three cell lines in a dose-dependent manner. These phenotypic characteristics have been found consistent with chondrocyte cell lines established from cartilage tissues other than costal cartilage. In conclusion, costal cartilage shows phenotypic similarities to other cartilages, i.e., articular cartilage and embryonic limbs, suggesting that costal cartilage may be very useful as the donor transplantation site for the treatment of cartilage disorders. Furthermore, the cell lines established in this study are also beneficial in basic research of cartilage physiology and pathology.

Circulating skeletal stem cells.

We report the isolation of adherent, clonogenic, fibroblast-like cells with osteogenic and adipogenic potential from the blood of four mammalian species. These cells phenotypically resemble but are distinguishable from skeletal stem cells found in bone marrow (stromal stem cells, "mesenchymal stem cells"). The osteogenic potential of the blood-borne cells was proven by an in vivo transplantation assay in which either polyclonal or single colony-derived strains were transplanted into the subcutis of immunocompromised mice, and the donor origin of the fully differentiated bone cells was proven using species-specific probes. This is the first definitive proof of the existence of circulating skeletal stem cells in mammals.

Endothelial dysfunction and decreased exercise tolerance in interferon-alpha therapy in chronic hepatitis C: relation between exercise hyperemia and endothelial function.

BACKGROUND: We previously reported that reversible endothelial dysfunction is caused by interferon-alpha therapy (IFN) in patients with chronic hepatitis C. In experimental studies, limb blood flow during exercise is reported to be dependent on endothelium-derived nitric oxide. HYPOTHESIS: The purpose of this study was to confirm the effect of IFN on endothelial function and to investigate whether exercise hyperemia is dependent on endothelial function in humans. METHODS: We performed symptom-limited exercise treadmill testing and measured flow-mediated vasodilation (FMD, endothelium-dependent vasodilation) and sublingual glyceryl-trinitrate-induced dilation (GTN-D, 0.3 mg, endothelium-independent vasodilation) in the brachial artery by using high-resolution ultrasound in 10 patients with chronic active hepatitis C (age 53 +/- 11 years, 2 men, 8 women) before and immediately after administration of recombinant interferon 2b (10 million U/day) for 4 weeks. RESULTS: There were no significant abnormal findings in any patients in routine studies of 24-h ambulatory electrocardiogram monitoring, two-dimensional echocardiography, and exercise treadmill testing both before and after treatment. Leg fatigue and exhaustion were the reasons for termination of exercise treadmill testing in each patient. Pressure rate product was calculated at rest and peak exercise. Interferon-alpha therapy significantly (p<0.05) decreased FMD (6.8 +/- 3.1 vs. 1.9 +/- 2.6%), exercise treadmill testing tolerance time (437 +/- 89 vs. 395 +/- 62 s) and peak pressure rate product (283 +/- 41 vs. 241 +/- 47 mmHg x beats/min x 10(-2)), but not GTN-D (13.4 +/- 5.4 vs. 17.0 +/- 5.5%). The change of FMD due to IFN significantly and highly correlated with exercise treadmill testing tolerance time (r = 0.86, p<0.001), but not with change of peak pressure rate product, suggesting that FMD is more closely related to the condition of the peripheral circulation than is cardiac performance. CONCLUSION: These results suggest that IFN in patients with chronic hepatitis C impairs endothelial function and exercise tolerance, and that endothelial function might be at least partly involved in exercise hyperemia in humans.

Pedicled bone flap formation using transplanted bone marrow stromal cells.

HYPOTHESIS: Transplanted osteoprogenitor cells derived from cultured bone marrow stromal cells (BMSCs) can be used to fabricate pedicled bone flaps. DESIGN: Prospective, randomized experimental trials. SETTING: Basic science research laboratory. MATERIALS: Immunodeficient female NIH-Bg-Nu-Xid mice, aged 3 months. INTERVENTION: The BMSCs were harvested from the long bones of C57Bl/6 transgenic mice carrying the type Ialpha1 collagen-chloramphenicol acetyl transferase reporter gene construct; their numbers were expanded in tissue culture. Treated mice received BMSC transplantations around the common carotid artery and internal jugular vein, the aorta and its venae comitantes, or the saphenous artery and vein; control mice received a sham transplant in comparable recipient sites. MAIN OUTCOME MEASURES: Mice underwent harvesting from 4 weeks to 2 years after transplantation. Transplants were evaluated via histological, immunohistochemical, and angiographic analyses. RESULTS: Compared with the controls, which formed no bone, 32 of 37 BMSC-containing transplants formed a vascularized bone island that was perfused specifically and solely by its common carotid artery vascular source. Mature transplants consisted of well-developed lamellar, corticocancellous bone whose osteocytes were derived from the grafted BMSCs; hematopoietic tissue derived from the recipient mouse. Transplants formed as early as 4 weeks and remained stable in size as late as 108 weeks. CONCLUSIONS: Bone marrow stromal cells can be used to create vascularized bone flaps in mice; these bone constructs are vascularized by their pedicle and therefore can potentially be transferred to a recipient site using microsurgical techniques. These findings provide proof of principle of an additional clinical application of BMSC transplantation techniques.

Angioplasty decreases prolonged QT dispersion in patients with angina pectoris but not in patients with prior myocardial infarction.

BACKGROUND AND HYPOTHESIS: Prolonged QT dispersion (QTd) is shortened by successful percutaneous transluminal coronary angioplasty (PTCA) in patients with ischemic heart disease. Particularly, QTd plays an important role in the prognostication in patients with prior myocardial infarction (MI). However, whether the effect of PTCA on QTd differs in patients with and without prior MI is not clear, and this study sought to clarify this question. METHODS: In 41 consecutive patients with ischemic heart disease, we measured QTd from a routine 12-lead electrocardiogram taken at 72 h before and after successful PTCA. Patients were divided into two groups based on the presence or absence of prior MI: Group 1 consisted of 24 patients with angina (61 +/- 11 years old) without prior MI and Group 2 was comprised of 17 patients (69 +/- 10 years old) with prior MI. QTd was calculated as the difference between the maximum and minimum QT and QT corrected for heart rate (QTc), using Bazett's formula for calculating QTcd. All measurements were obtained manually and blindly. RESULTS: In Group 1, 15 of 24 patients (63%) demonstrated multivessel disease and 16 of 24 (67%) patients had high QTd > 60 ms. Percutaneous transluminal coronary angioplasty decreased QTd and QTcd in Group 1 (QTd, from 83 +/- 35 to 57 +/- 19 ms, p < 0.05 ; QTcd, from 89 +/- 37 to 63 +/- 33 ms, p < 0.05), whereas no changes were observed in Group 2 (QTd, from 73 +/- 25 to 69 +/- 22 ms, NS; QTcd, from 80 +/- 30 to 79 +/- 28 ms, NS). QTd is more sensitive to decrease by successful PTCA in patients with angina than in patients with prior MI. CONCLUSIONS: The effect of successful PTCA on inhomogeneity of ventricular repolarization reflected by QTd in patients with prior MI is different from that in patients without prior MI.

Significance of circulatory epinephrine levels in exercise-induced neurally mediated syncope.

BACKGROUND AND HYPOTHESIS: Previous research has failed to document temporal changes in epinephrine levels in patients with neurally mediated syncope associated with exercise. The purpose of this study was to investigate the role of circulatory catecholamines in exercise-induced neurally mediated syncope, specifically focusing on epinephrine levels. METHODS: The present study deals with temporal changes of circulatory catecholamine levels during head-up tilt tests (40 min, 80 degree tilt) in 62 patients with syncope of unknown origin, 7 of whom had syncope associated with exercise (exercise-induced group, 19+/-3 years). Data were compared with 10 control subjects (control group, 45+/-23 years). Of the 55 patients with syncope not associated with exercise, 32 tested positive for the head-up tilt tests (positive group, 31+/-16 years) and 23 patients tested negative (negative group, 46+/-19 years). Blood samples for circulatory catecholamine assay were obtained from the antecubital vein in the baseline supine position 2 min after the tilt started, every 10 min during tilt, and at the time of the onset of symptoms or the end of tilt. Levels of norepinephrine and epinephrine were determined using the high-pressure liquid chromatography (HPLC) method (pg/ml). RESULTS: Plasma norepinephrine levels among the four groups were similar at the supine position and during tilt testing. In contrast, patients in the exercise-induced group had significantly higher maximum epinephrine levels during head-up tilt testing than the other three groups (288+/-191 vs. 148+/-117, 66+/-31, and 54+/-27 pg/ml, respectively, p < 0.05). Patients in the positive group had higher maximum epinephrine levels than those in the negative group (p <0.05). Also, patients in the exercise-induced group and those in the positive group had a significantly shorter tilt-testing time than patients in the negative and control groups. CONCLUSIONS: A marked increase of epinephrine was observed during head-up tilt testing in patients with neurally mediated syncope associated with exercise. The present findings further accelerate the identification of the role of epinephrine in the mechanisms behind neurally mediated syncope associated with exercise.

Expression of telomerase components in oral keratinocytes and squamous cell carcinomas.

Telomerase activity was measured using a telomeric repeat amplification protocol (TRAP), and expressions of the telomerase components, telomerase associated protein 1 (hTEP1), human telomerase RNA component (hTR), and human telomerase reverse transcriptase (hTERT) were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) in cultured normal oral keratinocytes and oral squamous cell carcinoma (SCC) cells. Telomerase localization was analyzed by in situ hybridization (ISH) in normal, precancerous and cancerous oral tissues. There was a strong correlation of telomerase activity with the expression levels of hTERT but not with hTEP1 or hTR mRNA in the cultured cells. Not only hTEP1 and hTR but also hTERT expression were detected in the basal cells of normal oral mucosa, and the cells expressing these mRNAs were also seen in the upper layer of leukoplakia of gingiva, and a heterogeneous pattern of expression was observed in the oral SCC tissues. These results indicate that there are at least two steps in the increase of telomerase activity during carcinogenesis in oral squamous cells; a change in distribution of cells expressing these telomerase components and the over-expression of hTERT gene in individual cells.

Expression of matrix metalloproteinase 3 in experimental atherosclerotic plaques.

In atherosclerotic lesions, matrix metalloproteinases produced by foam cells (macrophages) are thought to increase plaque instability, promote plaque rupture, by degradating extracellular matrix. To investigate the relationship between the expression of these proteinases and the histologic appearance of atheromas, immunohistochemical analysis of matrix metalloproteinase 3 and cell-type markers was performed in atherosclerotic plaques induced in rabbit abdominal aortas by high-cholesterol diets and mechanical injury. In addition to an antibody against matrix metalloproteinase 3, RAM-11 and HHF-35 were used to detect macrophages and smooth muscle cells, respectively. Matrix metalloproteinase 3 was expressed diffusely within the plaques with a fibrofatty histologic pattern. In plaques with foam cell accumulation, matrix metalloproteinase 3 was seen in areas rich in foam cells and the smooth muscle cells near the lumen. In the plaques with fewer macrophages, the proteinase was expressed only in such smooth muscle cells. Matrix metalloproteinase 3 was expressed in the smooth muscle cells in plaques of all histologic types, and macrophages also expressed the metalloproteinase when present in significant numbers. These findings suggest that macrophage accumulation plays an important pathophysiologic role in causing the instability of atherosclerotic lesions by increasing the levels of matrix metalloproteinase 3.