However, urbanization maintains exposure to the crowd infections that lack immunoregulatory roles, while accelerating loss of exposure to the natural environment. This effect is

most pronounced in individuals of low socioeconomic status (SES) MLN0128 order who lack rural second homes and rural holidays. Interestingly, large epidemiological studies indicate that the health benefits of living close to green spaces are most pronounced for individuals of low SES. Here we discuss the immunoregulatory role of the natural environment, and how this may interact with, and modulate, the proinflammatory effects of psychosocial stressors in low SES individuals. “
“Since their discovery as a distinct T helper (Th) cell lineage, Th17 cells have been extensively investigated both in mice and in humans. These studies have identified factors involved in their

differentiation and effector functions and have also revealed a high degree of flexibility that seems to be a characteristic of the Th17-cell lineage. In this review, we discuss recent studies addressing the heterogeneity of human Th17 cells, their differentiation requirements, their migratory capacities, and their role in defense against fungi and extracellular bacteria. Human T cells producing IL-17 were described as early as the late 1990s in the context of chronic inflammatory conditions Dabrafenib order such as rheumatoid arthritis and airway inflammation [1, 2], but it was only in 2005 that they were recognized as a
age of effector T cells [3]. Three lines of evidence obtained in the mouse system supported this notion. First, pathogenic inflammatory T cells produced high levels of IL-17A, IL-17F, and TNF and were dependent on

IL-23 rather than IL-12 for their expansion [3]. Second, naïve CD4+ T cells acquired IL-17-, but not IFN-γ- or IL-4-producing capacity, when activated in vitro in the presence of TGF-β and IL-6 or IL-23 [4-6]. Third, overexpression of the orphan nuclear receptor RORγt was sufficient to else induce differentiation of Th17 cells, while deficiency of RORγt in T cells attenuated autoimmune disease due to lack of tissue-infiltrating Th17 cells [7]. From these groundbreaking studies, the field has progressed at an astonishing pace, taking advantage of new and powerful technologies that have become accessible in recent years. As in many other areas of immunology, discoveries from studies performed in both experimental animal models and in human systems have contributed to our current understanding of the Th17 system and the role these cells play in physiology and pathology. Here, we will review, in particular, studies that address the heterogeneity of human Th17 cells, their differentiation requirements, their migratory capacities, and their role in defense against pathogens. To perform their function, effector, and memory T cells have to migrate to specific tissue sites, which are marked by the presence of constitutive or inflammatory chemokines [8].

infantum[14], have been reported previously to down-regulate CD1a expression. L. donovani was also shown to prevent activation of CD1-restricted T cells by DCs, which may represent a survival strategy by avoiding parasite glycolipid recognition [12]. L. amazonensis click here was able to alter DC differentiation by inducing a significant decrease in CD1a and CD80 expression and a significant increase in CD86 expression, causing down-regulation of the Th1 adaptive immune response [16]. We did not observe significant down-regulation of CD80 or increase of CD86. This could

be attributed to differences in the biology of Lm and L. amazonensis. In the last part of our work we showed that, despite their intracellular location, Lm clones did not stimulate IL-12p70, TNF-α or IL-10

production by DCs. In agreement with our results, others have reported that the uptake of the parasites alone by immature DCs provided an insufficient stimulus for cytokine production [6,11–13,25]. However, in the presence of an appropriate co-stimulation, and depending on the life stage and species involved, Leishmania parasites were shown to be able to modulate cytokine production by human DCs. We showed that, independently of their virulence, Lm clones were able to induce a decrease of IL-12p70 secretion during LPS-induced maturation of DC. Interestingly, although the LV Lm clone find more was not internalized by DCs, it was able to down-regulate IL-12p70 production during DC maturation similarly to the high virulent clone. It has been suggested that Leishmania-induced maturation does not require infection of DC and that direct recognition of parasites by DCs could be sufficient [28]. In agreement with our data, altered DC responsiveness to exogenous stimuli in the presence of Leishmania parasites and antigens has been reported by others [12,16,25]. L. donovani parasites for and

excreted–secreted antigens from L. donovani and Lm inhibited strongly IL-12p70 secretion by mature DCs [25]. Leishmania phosphoglycans family of virulence-associated antigens were able to inhibit DC maturation [29]. Conversely, it was reported that Lm was able to prime DC for CD40L-dependent IL-12p70 production [6,11,30] in a life stage and species- and strain-dependent manner [11]. This variability of Leishmania parasites ability to modulate a human DCs cytokine response could be explained not only by intrinsic differences between Leishmania species or strains or infective stage, but also by differences in the specific culture conditions such as the nature of priming and triggering signals used to induce maturation.

Significant production of interleukin-12 in the human PBMCs was observed after oral administration of Lactobacillus casei spp. casei and L. casei Shirota (Ogawa et al., 2006). The augmentation of phagocytosis activity and the percentage of phagocytotic cells after the probiotic intake compared with the other see more time points demonstrated efficient enhancement of innate immunity in an elderly population after 4 weeks of probiotic cheese consumption. Additionally, the increase in phagocytosis activity related to the consumption of control cheese indicates that the starter strains also have immune stimulation properties at least for the

phagocytosis. The increase in phagocytosis activity might play a role in the observed enhancement of NK cytotoxicity as it has been reported that the phagocytosis of bacteria by monocytes provides an additional signal on accessory cells inducing NK cell activation (Haller et al., 2002). NK cells’ activity is known to be important for immune surveillance against cancer cells and pathogenic infection. The incidence of cancer and the rate of mortality were reported to be higher in populations with a low NK activity compared with those with higher NK activities (Morales & Ottenhof, 1983; Imai et al., 2000; Ogata et al., 2001). Moreover, phagocytosis measurement is a useful tool in the assessment of macrophage function in LY294002 purchase immunotoxicological and immunopharmacological evaluations (Musclow et al., 1991). However, with the

present findings, further studies are needed to investigate whether there is an association of this size effect of immune modulation with clinical benefits. The general health parameters for the subjects were within

the physiological ranges throughout the course of the study. Although the mean values for erythrocytes, hemoglobin, hematocrit, and % HDL cholesterol were slightly lower after the probiotic intake, they were all within the normal ranges and were not significantly different from the baseline or the wash-out values. The two individuals with high CRP values (43.2 and 50.9 mg L−1) were suffering from urinary tract and respiratory infection, respectively. The values influenced the mean after the consumption of Clomifene the control cheese so that a significant difference was observed between the baseline and the run-in. A recent study (Hostmark et al., 2009) reported that cheese intake was negatively associated with triacylglycerols and HDL cholesterol. The amount of cheese consumed in this study was constant throughout the study and no correlation could be found between the amount of cheese consumption and the serum lipids. Considering that there were no significant changes in the total cholesterol or the HDL cholesterol level during the study, and the values were in the normal ranges, there seems to be no risk associated with the amount of cheese consumed. However, these values are worthwhile monitoring in future studies when cheese is used as a probiotic carrier.

abortus rough strain RB51 and smooth strain 2308 to stimulate murine bone marrow-derived DC (BMDC) activation and function based on the cell surface expression of costimulatory molecule and cytokine production. This study assessed simultaneously, for the first time, the differential ability of live, HK and IR rough and smooth strains of B. abortus https://www.selleckchem.com/products/PF-2341066.html at the same doses to stimulate DC activation and function. Female 6–8-week-old BALB/c mice were obtained from Charles River Laboratories Inc. (Wilmington, MA). Mice were used under animal care protocols approved by Institutional Animal Care and Use Committee at Virginia Tech. BMDCs were generated,

as described previously (Inaba et al., 1992). Briefly, tibias and fibulas of 7–8-week-old BALB/c mice were incised and bone marrow (BM) cells were removed. Following red blood cell lysis and filtration, the cells were resuspended and plated in RPMI 1640 complete media with 10% non-heat-inactivated fetal bovine serum and 20 ng mL−1 rGM-CSF (recombinant Granulocyte colony stimulating factor; Invitrogen, Carlsbad, CA). The cells were incubated at 37 °C in 5% CO2. Fresh media containing rGM-CSF was added at days 2, 4 and 5 and harvested on day 6. The cells harvested on day 6 were typically 70% CD11c+ and displayed low levels of major

histocompatibility complex (MHC) class II, CD40 and CD86 expression, consistent with immature DCs. Flow cytometry was performed to confirm DC activation status (Inaba et al., 1992). Stock cultures of live-attenuated rough B. abortus vaccine strain RB51 and virulent smooth Wnt mutation strain 2308 from our culture collection (Schurig et al., 1991; Vemulapalli et al., 2000) were stored at −80 °C. An aliquot each of strain RB51 and strain

2308 were subjected to γ-irradiation using a 60Co source irradiator with a radiation output of 2200 rads min−1 (Model 109-68R by J.L. Shepherd and Associates, San Fernando, CA) for 3 h (396 krads Erastin datasheet of γ-radiation). Aliquots of strain RB51 and strain 2308 were subjected to heat killing by incubating in an 80 °C water bath for 60 min. IR and HK bacterial preparations were confirmed to be nonviable by plating aliquots on TSA plates and confirming lack of growth following 4 days of incubation. All experiments with Brucella were performed in our CDC-approved (C2003 1120-0016) Biosafety Level-3 facility. On day 6, DCs were harvested and plated at 5 × 105 cells per well in 24-well plates and stimulated with live, IR or HK strain RB51 or strain 2308 at 1 : 10 (DC : Brucella) or 1 : 100 CFUs per well (i.e. 5 × 106 or 5 × 107 CFU equivalents per well of IR or HK B. abortus). Stimulation was enhanced by a short spin at 400 g for 5 min at room temperature. The stimulated cells were incubated for 4 h at 37 °C in 5% CO2. Then cells were washed with media containing gentamicin (Sigma, St. Louis, MO) 30 μg mL−1. The stimulated cells were incubated for an additional 20 h in complete media with 10 ng mL−1 rGM-CSF and 30 μg mL−1 gentamicin.

Efficacy of AGP in both endotoxemia and CLP support the potential utility of this novel, natural colloidal resuscitation fluid. The ability of AGP to maintain liver perfusion and decrease leukocyte adherence to the liver microvasculature could arise from numerous previously suggested Sorafenib purchase potential mechanisms, ranging from altering the ratio

of pro-inflammatory to anti-inflammatory cytokines and signals in hepatic inflammation, to restoring glycocalyx/barrier functions of the liver microcirculation, to directly binding and sequestering LPS. Of these possibilities, we selected the last one for further investigation, given that it was amenable to testing using methodologies already employed in this study. When AGP was combined with LPS and then injected intraperitoneally, it attenuated the pro-inflammatory effects of LPS on the hepatic microcirculation, at least with respect to leukocyte adhesion to PSV and sinusoidal perfusion. AGP has been shown to bind to LPS in two in vitro studies [25, 16]. If AGP bound LPS in the peritoneal space, it may have prevented the endotoxin from reaching the circulation and exerting systemic

effects, given the slow uptake of AGP from the peritoneal space into the circulation detected in our clearance experiments with radiolabeled AGP. Alternatively, LPS and AGP may not have interacted in the peritoneal space, but instead both reached Temozolomide chemical structure the circulation, where AGP exerted the anti-inflammatory effects we previously observed. To discriminate more fully between these

possibilities, we amended our experimental endotoxemia protocol to permit administration of both AGP and LPS intravenously, by reducing the LPS dose to 0.08 mg/kg, avoiding the mortality likely to ensue from an intravascular 5 mg/kg LPS dose. While administration of AGP just prior to LPS injection into the vasculature resulted in a non-significant trend toward decreased inflammation, pre-incubation of AGP with LPS significantly improved liver perfusion and reduced leukocyte adherence in both the post-sinusoidal venules and the sinusoids. Although in hindsight the latter experiment was likely underpowered, taken together our results support the concept that AGP is an LPS-binding mafosfamide protein and demonstrate this binding can have consequences in vivo. The anti-inflammatory effects of AGP manifested in the hepatic microcirculation are consistent with previous reports that infusion of pharmacological quantities of AGP purified from healthy cattle or humans limited mortality in disease models of uncontrolled inflammation [15, 20, 26]. However, they differ from two reports suggesting that AGP mediates a failure of leukocyte migration to the site of infection, both in normal and diabetic mice subjected to the CLP procedure. Mestriner et al. found that human AGP administered at the remarkably low dose of 4 μg/rat (approximately 0.

Two-way repeated measures ANOVA was used to determine the effects of group, treatment, and group–treatment interactions on measured variables (Sigmastat, Chicago, IL, USA). For all ANOVA procedures, Student–Newman–Keuls post hoc analysis was used to identify pair-wise differences among specific groups. Significance was assessed at p < 0.05. PVNS was performed on arcade bridge arterioles to determine responsiveness to sympathetic nerve stimulation [24]. These arterioles, originating from the thoracodorsal and 11th intercostal arteries, are the site of the majority of vascular resistance in the spinotrapezius muscle and hence of major importance in regulation

of blood flow in the muscle [5]. A beveled micropipette was filled with 0.9% saline, attached to a Grass Stimulator (Model Transmembrane Transporters activator S9; Grass Instruments, Quincy, MA, USA), and the tip was brought to gently rest in the arteriolar adventitia. The CX-4945 in vitro perivascular nerves were

stimulated between 20 and 60 seconds to develop stable constriction at a random frequency of 2–16 Hz. The observation site was distal to the stimulation site by 2–5 mm in the direction of flow. Microvascular reactivity was assessed first under normal superfusate conditions, then in the presence of phentolamine (1 μm). Arterioles were allowed to recover greater than two minutes between stimulations to return to baseline diameter. AH was induced in a separate set of rats through the stimulation of muscular contraction to determine the impact of PMMTM exposure on metabolically induced vasodilation as previously described [24]. Briefly, electrodes were attached to the rostral and caudal ends of the muscle and attached to a Grass Stimulator. Muscular contraction was induced at a frequency of 2–12 Hz randomly for one minute. The l-NMMA was added following normal superfusate responses. Arterioles were allowed to recover greater than two minutes between stimulations to return to baseline diameter.

Intraluminal infusion was performed on a separate set of rats as previously described [35]. Briefly, a micropipette was positioned in line with the stream of blood within an arcade bridge arteriole. Following an acclimation period, ejection of A23187 was performed for three minutes at 10–40 PSI via a Picospritzer II (World Precision Instruments Inc., Sarasota, FL, USA). Isolated mesenteric Rucaparib research buy or coronary arteries were equilibrated until the vessels achieved spontaneous tone, then myogenic responsiveness was determined from 0–90 mmHg (coronary) and 0–105 mmHg (mesenteric) in 15 mmHg increments as previously described [26, 27]. Endothelium-dependent arteriolar dilation was assessed with ACh and A23187. NO sensitivity was assessed with the NO donor Spermine NONOate. Adrenergic sensitivity was assessed with PE. Following the addition of each agent, a washout period was performed to allow the vessel to return to basal tone prior to the addition of the next vasoactive agent.

5 Four of these had clinical and biochemical improvement, with sustained graft function. In Nachman et al.’s series, the majority of patients received Cyclophosphamide

(12/16) as treatment, with 11/16 attaining a complete remission.4 The duration of Cyclophosphamide treatment was not stated. The use of plasma exchange is well documented in AAV-affecting native kidneys and while its use in the transplant recurrence setting lacks prospective data it is likely that many clinicians are using it particularly as for native AAV when there is pulmonary involvement or high ANCA titres. The monoclonal anti-CD20 antibody, Rituximab, is widely used as an alternative to Cyclophosphamide in inducing remission in AAV-affecting native kidney disease and its use in treating recurrent Aurora Kinase inhibitor vasculitis in the transplant setting is emerging as an alternative to Cyclophosphamide. The ideal time to transplant patients who have ESRD from AAV is not yet clear, although there is general consensus that there should be clinical remission at the time of transplantation. Little et al.’s series from European vasculitis group EUVAS showed that the strongest predictor of death was transplantation <1 year post-vasculitis remission.9 ANCA positivity at the time of transplantation did not increase the risk of relapse or graft loss, which is in concordance with the series of Nachman et al.4 We report a case of recurrent AAV in the renal allograft,

successfully treated with Cyclophosphamide, plasma exchange and increased-dose Prednisolone. Kidney transplantation is a safe and viable option for those with ESRD secondary MK-2206 order to AAV. Overall, graft survival is excellent, and comparable with transplantation for other causes of ESRD. Relapse rates vary, but are perhaps lower

with modern immunosuppression and while there are several emerging potential treatment options for relapse at this stage, including the use of plasma exchange and Rituximab, Cyclophosphamide remains the cornerstone of therapy. None. “
“Metabolic syndrome (MS) is associated with higher mortality and morbidity in the general population. However, the effect of MS and its individual components on clinical Methocarbamol outcomes in non-diabetic peritoneal dialysis (PD) patients has not been widely studied in India. Our aim was to study the prevalence of MS in non-diabetic PD patients who were on PD for at least 3 months and to analyze the influence of MS and its individual components on clinical outcomes of these patients on subsequent follow up. We prospectively included 163 non-diabetic PD patients (mean age 45.1 ± 16.2 years, 104 male). MS was defined using the modified National Cholesterol Education Programme (ATP III) criteria. Outcomes of patients with and without MS were compared. Of the 163 non-diabetic PD patients, 84 (51.5%) patients had MS. The mean follow up duration was 24.0 ± 14.0 patient months. Patients with MS had significantly greater body mass index (P = 0.007), Systolic BP (P = 0.

The placental vascular dysfunction does extend to other fetal vascular beds including endothelial cells from umbilical vessels, where there are reports of elevated basal iNOS activity and altered sensitivity to insulin. There is emerging evidence of epigenetic modulation of fetal endothelial HM781-36B supplier genes in diabetes and long-term vascular consequences

of this. Thus, placental vascular dysfunction in diabetes may be contributing to and describing disturbances in the fetal vasculature, which may produce an overt pathological response in later life if challenged with additional cardiovascular stresses. “
“Please cite this paper as: Arkill KP, Neal CR, Mantell JM, Michel CC, Qvortrup K, Rostgaard J, Bates DO, Knupp C, Squire JM. 3D reconstruction of the glycocalyx structure in mammalian capillaries using electron tomography. Microcirculation 19: 343–351, 2012. Objective:  Visualising the molecular strands making up the glycocalyx in the lumen of small blood vessels has proved to be difficult using conventional transmission electron microscopy techniques. Images obtained from tissue stained in a variety of ways have revealed a regularity in the organisation of the proteoglycan components of the glycocalyx layer (fundamental spacing about 20 nm), but require a large sample number. Attempts to visualise the glycocalyx face-on (i.e. in a direction perpendicular to the endothelial cell layer in the Cisplatin purchase lumen and directly

applicable for permeability modelling) has had limited success (e.g. freeze fracture). A new approach is therefore needed. Methods:  Here we demonstrate the effectiveness of using the relatively novel electron microscopy technique of 3D electron tomography on two differently stained glycocalyx preparations. A tannic acid staining

method and a novel staining technique using Lanthanum Dysprosium Glycosamino Glycan adhesion (the LaDy GAGa method). Results:  3D electron tomography reveals details of the architecture of the glycocalyx just above the endothelial cell layer. The LaDy GAGa method visually appears to show more complete coverage and more depth than the Tannic Acid staining method. Conclusion:  The tomographic reconstructions show a potentially significant improvement in determining much glycocalyx structure over standard transmission electron microscopy. “
“Please cite this paper as: Ella, Yang, Clifford, Gulia, Dora, Meininger, Davis and Hill (2010). Development of an Image-Based System for Measurement of Membrane Potential, Intracellular Ca2+ and Contraction in Arteriolar Smooth Muscle Cells. Microcirculation17(8), 629–640. Objective:  Changes in smooth muscle cell (SMC) membrane potential (Em) are critical to vasomotor responses. As a fluorescent indicator approach would lessen limitations of glass electrodes in contracting preparations, we aimed to develop a Forster (or fluorescence) resonance energy transfer (FRET)-based measurement for Em.

Fig. S3. Insulin autoantibody titres in unmated female non-obese diabetic (NOD) mice (group A1) and in NOD dams mated with haploidentical males (group C1) before breeding at age 10 weeks, and after weaning at age 16 weeks. Insulin autoantibody titres are expressed

as delta counts per minute (cpm). The horizontal lines indicate the median insulin autoantibody titre per treatment group. There are no significant differences between groups. “
“Department of Immunogy, School of Basic Medical Sciences, Xiang Ya School of Medicine, Central South University, Hunan, P. R. China The concept of DC-based tumour vaccine has been tested both clinically and experimentally for the past two decades. Even though only limited success has been achieved to date, DC vaccination remains a promising immunological approach EX 527 against tumours and deserves further exploration. It aims to elicit and establish specific immunity to destroy tumours. By such an approach, find more DC are used not only as a vector to deliver tumour antigens, but also as a “natural adjuvant” to boost vaccine efficacy. Tumours are however of mutated “self”, to which the host immune system is essentially tolerated in the absence of external perturbation otherwise. Such a live cell-based approach

is unfortunately extremely sensitive to, hence its efficacy inevitably limited by, the tumour microenvironment. Certain immunosuppressive mechanisms triggered by the tumour cells are therefore major obstacles against successful DC vaccination. Attempts have since been made in order to overcome these hurdles. This brief review summarises some of the earlier and current findings, and compares the effectiveness of various approaches used in these studies. It focuses particularly on strategies aimed at enhancing DC immunogenicity, through molecular modifications and functional

conditioning of the cell vectors, targeting Interleukin-3 receptor both the positive and negative regulators of DC functions. By dissecting the roles of DC in immunity versus tolerance induction, and the very mechanisms underlying autoimmunity, we examine further and try to explain how the suppressed or “misguided” immunity may be alternatively switched-on and more effectively redirected for cancer therapy. The immune system, in particular the adaptive arm, plays evidently important roles in restricting tumour growth and development 1. T lymphocytes are known to be essential in mediating the anti-tumour immune responses 2–4. Tumours are, however, clones of mutated cells that have arisen from the body’s own tissues. To prevent autoimmunity, it is believed that the immune system needs to be “educated” early in life (thymic selection) 5, 6, and continuously through adulthood (peripheral tolerance mechanisms) 7, during which T cells with potential self-reactivities are largely removed or immunologically “silenced”.

We report a progressive lower extremity lymphedema (LEL) case successfully treated with a ladder-shaped LVA. A 67-year-old female with secondary LEL refractory to conservative treatments underwent LVA. A ladder-shaped

LVA was performed at the left ankle. In the ABT-263 mouse ladder-shaped LVA, 3 lymphatic vessels and 1 vein were anastomosed in a side-to-side fashion; 2 lymphatic vessels next to the vein were anastomosed to the vein, and the other lymphatic vessel was anastomosed to the nearby lymphatic vessel. Using ladder-shaped LVA, 6 lymph flows of 3 lymphatic vessels could be bypassed into a vein. Six months after the LVA operation, her left LEL index decreased from 212 to 195, indicating edematous volume reduction. Ladder-shaped CHIR 99021 LVA may be a useful option when there are 3 lymphatic vessels and 1 vein in a surgical field. © 2013 Wiley Periodicals, Inc. Microsurgery 34:404–408, 2014. “
“This study evaluated the results of repair of the radius defect with a vascularized tissue

engineered bone graft composed by implanting mesenchymal stem cells (MSCs) and a vascular bundle into the xenogeneic deproteinized cancellous bone (XDCB) scaffold in a rabbit model. Sixty-four rabbits were used in the study. Among them, four rabbits were used as the MSCs donor. Other 57 rabbits were divided into five groups. In group one (n = 9), a 1.5 cm bone defect was created with no repair. In group two (n = 12), the bone defect was repaired by a XDCB graft alone. In group three (n = 12), the defect was repaired by a XDCB graft that included a vascular bundle. In group four (n = 12), the defect was repaired by a XDCB graft seeded with MSCs. In group Idoxuridine five (n = 12), the defect was repaired by a XDCB graft including a vascular bundle and MSCs implantation. The rest three rabbits were used as the normal control for the biomechanical test. The results of X-ray and histology at postoperative intervals (4, 8, and 12 weeks) and biomechanical examinations at 12 weeks showed that combining MSCs and a vascular bundle implantation

resulted in promoting vascularization and osteogenesis in the XDCB graft, and improving new bone formation and mechanical property in repair of radius defect with this tissue engineered bone graft. These findings suggested that the vascularized tissue engineered bone graft may be a valuable alternative for repair of large bone defect and deserves further investigations. © 2011 Wiley-Liss, Inc. Microsurgery, 2011. “
“The comparisons of two different methods of donor nephrectomy were performed in this study. Fisher inbred rats were used as donors and recipients of kidneys. In method A (the conventional technique), meticulous blunt dissection of the abdominal aorta, inferior vena cava, and renal arteries/veins, was followed by ligating and cutting the superior mesenteric artery and small vessels entering the above vessels. Both donor kidneys were irrigated after the suprarenal aorta and inferior vena cava were cross-clamped (n = 10).