Validating the Automatic Independent Component Analysis of DSA.

Time-density curve analysis of DSA provides useful blood flow information. However, manually selecting the ROI is time-consuming. We developed an automatic technique to provide arterial, capillary, and venous vasculatures with corresponding time-density curves. This study retrospectively analyzed the data of 36 patients with unilateral carotid stenosis. We found that the full width at half maximum of the time-density curve for the automatically segmented capillary vasculature is a suitable representation of the cerebral circulation time.

A comparison of left and right atrial fibroblasts reveals different collagen production activity and stress-induced mitogen-activated protein kinase signalling in rats.

AIM: Atrial fibrosis plays a pivotal role in the pathophysiology of heart failure (HF). The left atrium (LA) experiences greater fibrosis than the right atrium (RA) during HF. It is not clear whether LA cardiac fibroblasts contain distinctive activities that predispose LA to fibrosis. METHODS: LA and RA fibrosis were evaluated in healthy and isoproterenol-induced HF Sprague Dawley rats. Rat LA and RA primary isolated fibroblasts were subjected to proliferation assay, oxidative stress assay, cell migration analysis, collagen measurement, cytokine array and Western blot. RESULTS: Healthy rat LA and RA had a similar extent of collagen deposition. HF significantly increased fibrosis to a greater severity in LA than in RA. Compared to isolated RA fibroblasts, the in vitro experiments showed that isolated LA fibroblasts had higher oxidative stress and exhibited higher collagen, transforming growth factor-ß1, connective tissue growth factor production and less vascular endothelial growth factor (VEGF) production, but had similar migration, myofibroblast differentiation and proliferation activities. VEGF significantly increased the collagen production ability of LA fibroblasts, but not RA fibroblasts. LA fibroblasts had more phosphorylated ERK1/2 and P38 expression. ERK inhibitor (PD98059, 50 µmol L-1 ) significantly attenuated collagen production and increased VEGF production in RA fibroblasts but not in LA fibroblasts. P38 inhibitor (SB203580, 30 µmol L-1 ) significantly attenuated collagen production in LA fibroblasts but not in RA fibroblasts. P38 inhibitor also significantly increased VEGF production in RA and LA fibroblasts. CONCLUSIONS: Differences in profibrotic activity between LA and RA fibroblasts may be caused by different responses to mitogen-activated protein kinase signalling.

Peritherapeutic Hemodynamic Changes of Carotid Stenting Evaluated with Quantitative DSA in Patients with Carotid Stenosis.

BACKGROUND AND PURPOSE: Quantitative data from DSA have become important tools for understanding hemodynamic changes of intracranial lesions. In this study, we evaluated 8 hemodynamic parameters in patients before and after carotid artery angioplasty. MATERIALS AND METHODS: DSA images of 34 patients with carotid stenosis who underwent angioplasty and stent placement were retrospectively analyzed. Eleven ROIs (M1, M2, A1, A2, the parietal vein, superior sagittal sinus, internal jugular vein, and 4 in the ICA) were selected on color-coded DSA. Eight hemodynamic parameters (bolus arrival time, TTP, relative TTP, full width at half maximum, wash-in slope, washout slope, maximum enhancement, and area under the curve) were measured from the time-concentration curves of these ROIs. The dependent t test for paired samples was applied to these parameters before and after stent placement. RESULTS: We found that the treatment significantly reduced TTP, relative TTP, bolus arrival time, and washout slope at all arterial ROIs and full width at half maximum and area under the curve at some arterial ROIs. Bolus arrival time was significantly reduced after treatment for all arterial ROIs, the parietal vein, and the superior sagittal sinus. The maximum enhancement and wash-in slope did not show significant changes after treatment. After treatment, the relative TTP from the ICA to M1, M2, and the parietal vein returned to normal values. CONCLUSIONS: In addition to TTP and relative TTP, other parameters can be used to evaluate peritherapeutic cerebral hemodynamic changes. Bolus arrival time has the potential to evaluate brain circulation at arterial and venous sites, especially when TTP cannot be measured because of an incomplete time-concentration curve.

Impact of lumbar instrumented circumferential fusion on the development of adjacent vertebral compression fracture.

We evaluated the impact of lumbar instrumented circumferential fusion on the development of adjacent level vertebral compression fractures (VCFs). Instrumented posterior lumbar interbody fusion (PLIF) has become a popular procedure for degenerative lumbar spine disease. The immediate rigidity produced by PLIF may cause more stress and lead to greater risk of adjacent VCFs. However, few studies have investigated the relationship between PLIF and the development of subsequent adjacent level VCFs. Between January 2005 and December 2009, a total of 1936 patients were enrolled. Of these 224 patients had a new VCF and the incidence was statistically analysed with other covariants. In total 150 (11.1%) of 1348 patients developed new VCFs with PLIF, with 108 (72%) cases at adjacent segment. Of 588 patients, 74 (12.5%) developed new subsequent VCFs with conventional posterolateral fusion (PLF), with 37 (50%) patients at an adjacent level. Short-segment fusion, female and age older than 65 years also increased the development of new adjacent VCFs in patients undergoing PLIF. In the osteoporotic patient, more rigid fusion and a higher stress gradient after PLIF will cause a higher adjacent VCF rate.

Anti-inflammatory effects of platelet biomaterials in a macrophage cellular model.

BACKGROUND AND OBJECTIVES: Recent clinical data suggested that platelet materials used in regenerative medicine exert anti-inflammatory effects. One must understand whether functionality varies among platelet preparations and also the role of the various protein compartments. MATERIALS AND METHODS: Platelet-poor-plasma (PPP), platelet lysate with cell debris (PL) or cell-free (CFPL), platelet gel releasate (PGR) and solvent/detergent-treated PL (SDPL) were prepared from four apheresis platelet donations. Protein profile was examined by SDS-PAGE, and growth factors and cytokines by ELISA, multiplexed Luminex assay and cytokine array. Anti-inflammatory activity was evaluated in RAW 264.7 mouse macrophages treated for 24 h with the blood fractions followed by 24 h of stimulation with 500 ng/ml lipopolysaccharides (LPS). Inflammatory marker nitric oxide (NO) was determined by colorimetry, tumour necrosis factor (TNF)-α by ELISA and inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 by Western blotting. RESULTS: Proteins, growth factors and cytokines composition differed among preparations. Blood fractions alone did not stimulate inflammatory markers expression. Following LPS stimulus, NO and iNOS expressions were significantly inhibited (P < 0.001) by all blood fractions, but inhibition was more pronounced with SDPL. In addition, only SDPL inhibited TNF-α (P < 0.001) and COX-2 expressions. CONCLUSIONS: All the plasma and platelet fractions evaluated in this study exert an anti-inflammatory effect on macrophages, suggesting that both the plasma and platelet proteomes contribute to anti-inflammation. However, the extent and nature of the anti-inflammatory action vary among products. Further studies are needed to better understand the functionality of platelet biomaterials and optimize their clinical use.

The dipeptidyl peptidase-4 inhibitor-sitagliptin modulates calcium dysregulation, inflammation, and PPARs in hypertensive cardiomyocytes.

BACKGROUND: Hypertension induces cardiac dysfunction, calcium (Ca(2+)) dysregulation, and arrhythmogenesis. Dipeptidyl peptidase (DPP)-4 inhibitors, an antidiabetic agent with anti-inflammation and anti-hypertension potential, may regulate peroxisome proliferator-activated receptors (PPARs)-α, -γ, and -δ and Ca(2+) homeostasis. OBJECTIVE: The purpose of this study was to investigate whether DPP-4 inhibitor, sitagliptin, can modulate PPARs and Ca(2+) handling proteins in hypertensive hearts. METHODS: A Western blot analysis was used to evaluate protein expressions of myocardial PPAR isoforms, tumor necrosis factor (TNF)-α, interleukin (IL)-6, sarcoplasmic reticulum ATPase (SERCA2a), Na(+)-Ca(2+) exchanger (NCX), ryanodine receptor (RyR), voltage-dependent Ca(2+) (CaV1.2), slow-voltage potassium currents (Kvs), angiotensin II type 1 receptor (AT1R), and receptor of advanced glycated end-products (RAGE) from Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHR), and SHR treated with sitagliptin (10mg/kg for 4weeks). Conventional microelectrodes were used to record action potentials (APs) in the ventricular myocytes from each group. RESULTS: Compared to the control group, SHR had lower cardiac PPAR-α and PPAR-δ protein expressions, but had greater cardiac PPAR-γ levels, and TNF-α, IL-6, RAGE, and AT1R protein expressions, which were ameliorated in the sitagliptin-treated SHR. SHR had prolonged QT interval and AP duration with less SERCA2a and RyR, and greater CaV1.2 expressions, which were also attenuated in sitagliptin-treated SHR. CONCLUSIONS: Sitagliptin significantly changed the cardiac electrophysiological characteristics and Ca(2+) regulation, which may have been caused by its effects on cardiac PPARs, proinflammatory cytokines, and AT1R.

Peroxisome proliferator-activated receptors modulate cardiac dysfunction in diabetic cardiomyopathy.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality among patients with diabetes mellitus (DM). Chronic inflammation and derangement of myocardial energy and lipid homeostasis are common features of DM. The transcription factors of peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily, which are important in regulating energy and lipid homeostasis. There are three PPAR isoforms, α, γ, and δ, and their roles have been increasingly recognized to be important in CVD. These three isoforms are expressed in the heart and play pivotal roles in myocardial lipid metabolism, as well as glucose and energy homeostasis, and contribute to extra metabolic roles with effects on inflammation and oxidative stress. Moreover, regulation of PPARs may have significant effects on cardiac electrical activity and arrhythmogenesis. This review describes the roles of PPARs and their agonists in DM cardiomyopathy, inflammation, and cardiac electrophysiology.

Cordyceps militaris and mycelial fermentation induced apoptosis and autophagy of human glioblastoma cells.

This study is the first report that investigated the apoptosis-inducing effects of Cordyceps militaris (CM) and its mycelial fermentation in human glioblastoma cells. Both fractions arrested the GBM8401 cells in the G0/G1 phase, whereas the U-87MG cells were arrested at the G2/M transitional stage. Western blot data suggested that upregulation of p53 and p21 might be involved in the disruption of cell cycle progression. Induction of chromosomal condensation and the appearance of a sub-G1 hypodipoid population further supported the proapoptogenicity, possibly through the activation of caspase-3 and caspase-8, and the downregulation of antiapoptotic Bcl-2 and the upregulation of proapoptotic Bax protein expression. Downregulation of mammalian target of rapamycin and upregulation of Atg5 and LC3 II levels in GBM8401 cells implicated the involvement of autophagy. The signaling profiles with mycelial fermentation treatment indicated that mycelial fermentation triggered rapid phosphorylation of Akt, p38 MAPK, and JNK, but suppressed constitutively high levels of ERK1/2 in GBM8401 cells. Mycelial fermentation treatment only significantly increased p38 MAPK phosphorylation, but decreased constitutively high levels of Akt, ERK1/2, and JNK phosphorylation in U-87MG cells. Pretreatment with PI3K inhibitor wortmannin and MEK1 inhibitor PD98059 prevented the mycelial fermentation-induced cytotoxicity in GBM8401 and U-87MG cells, suggesting the involvement of PI3K/Akt and MEK1 pathways in mycelial fermentation-driven glioblastoma cell apoptosis and autophagy.

Steroidogenic Factor 1 (NR5A1) resides in centrosomes and maintains genomic stability by controlling centrosome homeostasis.

SF-1 (Steroidogenic Factor 1, NR5A1) is a tissue-specific transcription factor critical for the growth, development and differentiation of steroidogenic and a few other endocrine tissues. But how SF-1 regulates cell growth is not entirely clear. Here we found that SF-1 was localized to the centrosome in addition to the nucleus, and SF-1 depletion by shRNA caused centrosome over-duplication, aberrant mitosis and genomic instability, leading to a reduction of cell number. Centrosome amplification defect was rescued by both wild-type SF-1 and transcription-defective SF-1-G35E, suggesting a non-genomic activity of SF-1 involved in centrosome homeostasis. In addition, we identified in SF-1 a centrosome localization signal, whose overexpression led to reduced localization of both SF-1 and γ-tubulin to the centrosome. Our results uncover a novel role of SF-1 in the control of centrosome homeostasis and genomic stability.

Management of long-standing mandibular dislocation.

Long-standing dislocation of the temporomandibular joint (TMJ) is rare. The management of this disorder is still controversial. This paper presents the authors' experience of managing long-standing dislocation of the TMJ, and their attempt to develop guidelines for the management of this problem. They also show magnetic resonance images of two patients with long-standing dislocation of the TMJ.

Proinflammatory cytokine and ligands modulate cardiac peroxisome proliferator-activated receptors.

BACKGROUND: Peroxisome proliferator-activated receptors (PPAR) mediate inflammatory processes and alter cardiac function. However, it is not clear whether inflammatory cytokines or PPAR ligands regulate PPARs in the cardiomyocytes to modulate cardiac functions. We investigated the effects of tumour necrosis factor-alpha (TNF-alpha) and PPAR ligands on the expression of PPARs in HL-1 cardiomyocytes. MATERIALS AND METHODS: HL-1 cardiomyocytes were incubated with and without TNF-alpha (1, 10, 25 and 50 ng mL(-1)) or PPAR ligands (rosiglitazone, pioglitazone and fenofibrate) at concentrations of 0.1, 1 and 10 microM for 24 h. The cells also received SN-50 (NF-kappaB inhibitor, 50 microg mL(-1)), ascorbic acid (100 microM) and coenzyme Q10 (10 microM) alone or combined with TNF-alpha. RESULTS: Using reverse transcriptase-polymerase chain reaction and Western blot, we found that incubation of TNF-alpha (50 ng mL(-1)) for 24 h decreased PPAR-alpha, but increased PPAR-gamma without altering PPAR-delta. These effects were not changed by co-administration of SN-50. However, co-administration of ascorbic acid prevented the effect of TNF-alpha both on PPAR-alpha and PPAR-gamma. Coenzyme Q10 partially attenuated the effect of TNF-alpha on PPAR-gamma but did not alter its effect on PPAR-alpha. The administration of rosiglitazone (10 microM) and pioglitazone (10 microM) for 24 h increased PPAR-gamma mRNA, but did not alter PPAR-alpha or PPAR-delta. Moreover, fenofibrate (0.1, 1 and 10 microM) increased PPAR-gamma without any effects on PPAR-alpha or PPAR-delta. CONCLUSIONS: Oxidative stress causes the regulations of PPAR-alpha and PPAR-gamma in the TNF-alpha-treated cardiomyocytes. The up-regulation of PPAR-gamma by PPAR ligands may contribute to their anti-inflammation effects.

High-mobility group box 1 protein activates hepatic stellate cells in vitro.

OBJECTIVES: Our previous study noticed remarkably elevated titers of anti-high-mobility group box 1 (HMGB1) antibodies in sera during the tolerance induction phase of a rat tolerogenic orthotopic liver transplantation (OLT) as well as in sera of clinically drug-free patients. We hypothesized that the release of nonhistone nuclear protein HMGB1 during rejection may play a pathogenic role in deteriorating post-OLT graft functions, such as inducing liver fibrosis. This study sought to investigate whether HMGB1 can directly activate hepatic stellate cells (HSCs) and drive them toward fibrogenesis. METHODS: The cultured HSCs were treated with recombinant HMGB1. RT-PCR and Western blotting analysis were used to measure alpha-smooth muscle actin (alpha-SMA) expression. Conditioned media were collected for gelatin zymography to monitor the activities of collagen-degrading matrix metalloproteinases (MMPs). RESULTS: HMGB1 at concentrations > 1 ng/mL significantly stimulated HSC growth as revealed by proliferation and BrdU assays. alpha-SMA gene and protein expression were significantly up-regulated by HMGB1, whereas the MMP-2, but not MMP-9, activity was suppressed by HMGB1 treatment. CONCLUSION: Our data suggested that HMGB1 protein, once released during the rejection phase of OLT, activated HSCs and exhibited profibrogenic effects on liver grafts either by increasing the HSC population and extracellular matrix content in liver grafts, or by transforming HSCs into myofibroblasts. Neutralization with anti-HMGB1 antibody was suggested to be a therapeutic modality applicable to prevent fibrogenesis in post-OLT liver grafts.

Histone H1 vaccine therapy for overcoming acute rejection in experimental organ transplantation.

OBJECTIVE: In a rat tolerogenic orthotopic liver transplantation (OLT) model, the recipient serum (post-OLT serum) shows strong immunosuppressive activity. In our previous reports, we suggested that autoreactive antibody (Ab) against histone H1 is a major immunosuppressive factor in this serum. The present study sought to determine whether up-regulation of anti-histone H1 Ab by histone H1 vaccination led to tolerance. MATERIALS AND METHODS: Using mixed lymphocyte reactions (MLR) and heterotopic heart transplantations (HHT), the alloreactive T-cell responses and allograft survivals of histone H1-immunized rats were compared with those of control rats. Cytokine and cellular profiles were determined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. RESULTS: The alloreactive T-cell response of histone H1-immunized rats was significantly lower than that of control rats, although there was no difference in nonspecific T-cell activation between the 2 groups. The allograft survival of histone H1-immunized rats was significantly prolonged after HHT. The major histocompatibility complex (MHC) class II and CD25 molecules of histone H1-immunized rats were significantly down-regulated compared with those of control rats. Moreover, the serum cytokine profile was modified by the immunization with histone H1. CONCLUSIONS: These results suggest that histone H1 vaccination of transplant recipients leads to the production of immunosuppressive factors and the modification of cytokine/cellular profiles.

Role of antinuclear antibodies in experimental and clinical liver transplantation.

OBJECTIVE: We recently reported that autoreactive antibodies (Abs) against nuclear histone H1 was transiently induced at an early phase after orthotopic liver transplantation (OLT) in a tolerogenic rat OLT model and possessed immunosuppressive activity. It was also reported that nuclear antigen, high-mobility group box 1 (HMGB1) protein was one of the initiators of the immune reaction. The present study sought to evaluate the role of antinuclear Abs in experimental and clinical liver transplantation. MATERIALS AND METHODS: We prepared 3 animal models: natural tolerance model (DA liver into PVG); acute rejection model (DA liver into LEW); and drug-induced tolerance model (acute rejection model + cyclosporine [CsA]). In addition, we examined clinical samples, including 1 drug-free patient, to measure the antihistone H1/HMGB1 titers at various times after OLT. RESULTS: In a natural tolerance model, antihistone H1 and HMGB1 Ab was induced during the rejection and the tolerance induction phases, respectively. Those Ab responses were also confirmed in a drug-induced tolerance model, whereas no such responses were shown in an acute rejection model. In our clinical drug-free patient, antihistone H1/HMGB1 titer was significantly higher after cessation of CsA than that in healthy volunteers. CONCLUSIONS: Antinuclear Ab is actively expressed in accordance with overcoming rejection episodes with subsequent tolerance induction in both a natural tolerance model and a drug-induced tolerance model. We also observed a similar tendency in our clinical drug-free patient. These results suggested that antinuclear Abs may be useful markers to determine the timing to withdraw immunosuppressants.

FK506 independently upregulates transforming growth factor beta and downregulates inducible nitric oxide synthase in cultured human keratinocytes: possible mechanisms of how tacrolimus ointment interacts with atopic skin.

BACKGROUND: Tacrolimus ointment (FK506) has been used in recent years for the treatment of atopic dermatitis (AD), with favourable results. Most of the therapeutic efficacy of FK506 in AD has been attributed to its immunomodulatory effects on different immune cell types, but its effects on keratinocytes (KCs) have rarely been discussed. Studies have shown that low expression of transforming growth factor (TGF)-beta and high expression of nitric oxide synthase (NOS) are implicated in the pathogenesis of AD. OBJECTIVES: To investigate the direct effects of FK506 on KCs in terms of TGF-beta and inducible NOS (iNOS), and to explore the interactions between TGF-beta and iNOS in the KC system. METHODS: Cultured human KCs treated with different concentrations of FK506 were used for investigation. The changes in the KC system induced by FK506 were documented in terms of TGF-beta and iNOS using enzyme-linked immunosorbent assay and Western blotting techniques, respectively. The gene expression of both TGF-beta and iNOS was also determined. A certain amount of tumour necrosis factor (TNF)-alpha was introduced to mimic atopic skin in vivo. RESULTS: Our results showed that the release of TGF-beta was upregulated in FK506-treated KCs, particularly in the presence of TNF-alpha, while the expression of iNOS was downregulated. The gene expression of iNOS was also downregulated, as shown by reverse transcriptase-polymerase chain reaction analysis. However, the addition of TNF-alpha did not further downregulate the expression of iNOS protein, suggesting that FK506 may regulate TGF-beta and iNOS through different pathways. CONCLUSIONS: Our findings indicate that the direct effects of FK506 on KCs probably contribute to its therapeutic efficacy in the treatment of AD.