Effect of exercise training on insulin sensitivity, mitochondria and computed tomography muscle attenuation in overweight women with and without polycystic ovary syndrome.

AIMS/HYPOTHESIS: Polycystic ovary syndrome (PCOS) is an insulin resistant (IR) state. Increased skeletal muscle lipid content and impaired mitochondrial biogenesis have been implicated in the pathogenesis of IR. We investigated whether differences in these variables explain the IR of women affected by PCOS and whether improvements in IR with exercise are reflected by changes in these variables. METHODS: Sixteen PCOS and 13 non-PCOS overweight women were assessed, and eight PCOS and seven non-PCOS women were reassessed after 12 weeks of moderate and vigorous exercise training. Outcomes included insulin sensitivity (glucose infusion rate [GIR]), skeletal muscle gene expression and protein abundance, enzyme activity of selected mitochondrial components, and computed tomography (CT) attenuation-estimated muscle lipid. RESULTS: GIR was lower in women with PCOS versus those without (p = 0.01) and increased with exercise in both groups. Baseline CT muscle attenuation suggested a trend to less muscle lipid in PCOS, which increased with exercise training, with a difference in the change in muscle lipid (p = 0.01, age-corrected), compared with non-PCOS women. GIR correlated with PGC1A gene expression across the whole group; skeletal muscle expression of mitochondrial biogenesis markers was not different between groups at baseline, or after training. Neither lipid changes nor mitochondrial changes correlated with changes in GIR. CONCLUSIONS/INTERPRETATION: Differences in IR in women with and without PCOS were not explained by differences in skeletal muscle lipid or mitochondrial parameters. Improvements in IR with exercise were dissociated from mitochondrial parameters. CT muscle attenuation suggested a differential capacity of PCOS muscle to store lipid compared with non-PCOS. TRIAL REGISTRATION: Clinicaltrials.gov ISRCTN84763265. FUNDING: National Health & Medical Research Council (Grant number 606553), Monash University and The Jean Hailes Foundation.

Psychological distress and unmet supportive care needs in cancer patients and carers who contact cancer helplines.

Cancer information services are a highly accessible source of support for people affected by cancer. To date the nature and extent of distress experienced by such callers and their unmet support needs have not been well described. A cross-sectional survey of 354 cancer patients and 336 carers who reported elevated distress on contact with a cancer information service assessed socio-demographic variables; anxiety, depression and somatization; unmet supportive care needs; cancer-specific distress; presenting problems; post-traumatic growth. Adjustment to cancer was most commonly reported; followed by anxiety. In all, 53.4% of patients and 45.2% of carers reached caseness in anxiety, depression or somatization. Carers had higher distress ratings and intrusive thinking compared to patients; whereas patients had higher somatization. For patients, most unmet supportive care needs were psychological; for carers unmet needs were related to health care services and information related to the person diagnosed with cancer. Being single, unemployed, in treatment, having higher initial distress scores, higher intrusion and avoidance predicted poorer outcomes. Information service frameworks should include distress screening and clear triage and referral processes for psychological care.

Exercise decreases anti-müllerian hormone in anovulatory overweight women with polycystic ovary syndrome: a pilot study.

Polycystic ovary syndrome (PCOS) is a common condition in women associated with menstrual irregularity and anovulation. While obesity worsens and weight loss or exercise improves reproduction function in PCOS, the mechanism for this is unclear. The aim of this study was to examine the effect of exercise on ovarian hormones [anti-Müllerian hormone (AMH)] and menstrual and ovulatory function in women with and without PCOS. Overweight women with (n=7) and without (n=8) PCOS of comparable age, weight and BMI undertook a 12-week intensified endurance exercise training program (1 h 3 times/week) with no structured energy restriction. Primary outcomes were AMH, ovulation (weekly urinary pregnanediol) and menstrual regularity. Secondary outcomes were insulin resistance (euglycemic hyperinsulinemic clamp) and body composition (computed tomography and dual X-ray absorptiometry). Exercise decreased BMI, total and android fat mass and improved insulin sensitivity for all women. AMH was significantly higher in women with PCOS compared to controls before (p<0.001) and after exercise (p=0.001). There was a significant interaction between AMH changes with exercise and PCOS status (p=0.007) such that women without PCOS had no change in AMH (+1.4±5.2 pmol/l, p=0.48) while women with PCOS had a decrease in AMH (- 13.2±11.7 pmol/l, p=0.025). Exercise is associated with improvements in ovarian hormones in women with abnormal ovarian function. This suggests that mechanisms associated with ovarian dysfunction can be improved by exercise in PCOS.

Analysis of canine myeloid-derived suppressor cells (MDSCs) utilizing fluorescence-activated cell sorting, RNA protection mediums to yield quality RNA for single-cell RNA sequencing.

Fluorescence-activated cell sorting (FACS) is a branch of flow cytometry that allows for the isolation of specific cell populations that can then be further analyzed by single-cell RNA sequencing (scRNA-seq). When utilizing FACS for population isolation prior to sequencing, it is essential to consider the protection of RNA from RNase activity, environmental conditions, and the sorting efficiency to ensure optimum sample quality. This study aimed to optimize a previously published MDSC flow cytometry strategy to FACS sort canine Myeloid-Derived Suppressor Cells (MDSC) with various permutations of RNAlater ™ and RiboLock™ before and after FACS sorting. Concentrations of RNAlater™ greater than 2 % applied before flow analysis affected cell survival and fluorescence, whereas concentrations ≤ 2 % and time ≤ 4 h had little to no effect on cells. To shorten the procedural time and to enhance the sorting of rare populations, we used a primary PE-conjugated CD11b antibody and magnetic column. The combination of RiboLock™ pre- and post-sorting for FACS provided the best quality RNA as determined by the RNA integrity number (RIN ≥ 7) for scRNA-seq in a normal and dog and a dog with untreated oral melanoma dog. As proof of principle, we sequenced two samples, one from a normal dog another from a dog with untreated oral melanoma. Applying scRNA-Seq analysis using the 10X Genomic platform, we identified 6 clusters in the Seurat paired analysis of MDSC sorted samples. Two clusters, with the majority of the cells coming from the melanoma sample, had genes that were upregulated (> log2); these included MMP9, MMP1, HPGD, CPA3, and GATA3 and CYBB, CSTB, COX2, ATP6, and COX 17 for cluster 5 and 6 respectively. All genes have known associations with MDSCs. Further characterization using pathway analysis tools was not attempted due to the lower number of cells sequenced in the normal sample. The benefit deriving from the results of the study helped to gain data consistency when working with cells prone to RNase activity, and the scRNA-seq provided data showing transcriptional heterogeneity in MDSC populations and potentially identifying previously unreported or rare cell populations.

Novel inflammatory markers in overweight women with and without polycystic ovary syndrome and following pharmacological intervention.

BACKGROUND: Polycystic ovary syndrome (PCOS) is associated with reproductive and metabolic abnormalities. AIM: The aim of this study was to assess novel inflammatory markers [adipokines leptin, adiponectin, and leptinadiponectin ratio (L/A)] in overweight women with and without PCOS and to examine alterations in these markers [aldosterone, leptin, adiponectin, and L/A] with pharmacological interventions modulating insulin resistance (IR) in PCOS. MATERIALS/SUBJECTS AND METHODS: Overweight age, and body mass index (BMI)-matched women with (no.=80) or without PCOS (no.=27) were assessed cross-sectionally. Subjects with PCOS were then randomised to 6 months metformin (1 g b.d, no.=26) or oral contraceptive pill (OCP) (35 g ethinyl estradiol/2 mg cytoproterone acetate, no.=30). Outcome measures were leptin, adiponectin, L/A, aldosterone, highly sensitive C-reactive protein, lipid profile, IR, and androgen levels. RESULTS: Leptin levels were lower (156.4+/-85.9 vs 208.5+/-105.2 ng/ml, p=0.015) while adiponectin and L/A were not different between women with and without PCOS. Following intervention, IR increased for the OCP and decreased for metformin, however leptin and aldosterone decreased equivalently with the OCP and metformin with no difference between each treatment (p=0.583 and p=0.801, respectively). There was no change in adiponectin or L/A with the OCP or metformin. On multiple regression, the only baseline predictor of leptin was BMI (r(2)=0.485, p<0.001) and the strongest predictor of change in leptin was change in weight (r(2)=0.402, p<0.001). CONCLUSIONS: Alterations in leptin between women with and without PCOS and following pharmacological interventions are primarily related to adiposity and not IR. Aldosterone was reduced equivalently with metformin and the OCP despite differential effects on IR.

Exercise and insulin resistance in PCOS: muscle insulin signalling and fibrosis.

OBJECTIVE: Mechanisms of insulin resistance in polycystic ovary syndrome (PCOS) remain ill defined, contributing to sub-optimal therapies. Recognising skeletal muscle plays a key role in glucose homeostasis we investigated early insulin signalling, its association with aberrant transforming growth factor ß (TGFß)-regulated tissue fibrosis. We also explored the impact of aerobic exercise on these molecular pathways. METHODS: A secondary analysis from a cross-sectional study was undertaken in women with (n = 30) or without (n = 29) PCOS across lean and overweight BMIs. A subset of participants with (n = 8) or without (n = 8) PCOS who were overweight completed 12 weeks of aerobic exercise training. Muscle was sampled before and 30 min into a euglycaemic-hyperinsulinaemic clamp pre and post training. RESULTS: We found reduced signalling in PCOS of mechanistic target of rapamycin (mTOR). Exercise training augmented but did not completely rescue this signalling defect in women with PCOS. Genes in the TGFß signalling network were upregulated in skeletal muscle in the overweight women with PCOS but were unresponsive to exercise training except for genes encoding LOX, collagen 1 and 3. CONCLUSIONS: We provide new insights into defects in early insulin signalling, tissue fibrosis, and hyperandrogenism in PCOS-specific insulin resistance in lean and overweight women. PCOS-specific insulin signalling defects were isolated to mTOR, while gene expression implicated TGFß ligand regulating a fibrosis in the PCOS-obesity synergy in insulin resistance and altered responses to exercise. Interestingly, there was little evidence for hyperandrogenism as a mechanism for insulin resistance.

An investigation of the impact of the location and timing of antigen-specific T cell division on airways inflammation.

It is widely accepted that allergic asthma is orchestrated by T helper type 2 lymphocytes specific for inhaled allergen. However, it remains unclear where and when T cell activation and division occurs after allergen challenge, and whether these factors have a significant impact on airways inflammation. We therefore employed a CD4-T cell receptor transgenic adoptive transfer model in conjunction with laser scanning cytometry to characterize the location and timing of T cell division in asthma in vivo. Thus, for the first time we have directly assessed the division of antigen-specific T cells in situ. We found that accumulation of divided antigen-specific T cells in the lungs appeared to occur in two waves. The first very early wave was apparent before dividing T cells could be detected in the lymph node (LN) and coincided with neutrophil influx. The second wave of divided T cells accumulating in lung followed the appearance of these cells in LN and coincided with peak eosinophilia. Furthermore, accumulation of antigen-specific T cells in the draining LN and lung tissue, together with accompanying pathology, was reduced by intervention with the sphingosine 1-phosphate receptor agonist FTY720 2 days after challenge. These findings provide greater insight into the timing and location of antigen-specific T cell division in airways inflammation, indicate that distinct phases and locations of antigen presentation may be associated with different aspects of pathology and that therapeutics targeted against leukocyte migration may be useful in these conditions.

Dissecting the contribution of innate and antigen-specific pathways to the breach of self-tolerance observed in a murine model of arthritis.

BACKGROUND: The relative roles of innate immunity and antigen-specific T cells in rheumatoid arthritis remain controversial. Previous studies demonstrated that T-helper type 1 cells of irrelevant antigen specificity (ovalbumin) induced a transient arthritis in BALB/c mice, which recapitulates many of the pre-articular and articular features of human disease and is associated with the emergence of autoreactive T and B-cell responses to joint-specific antigens. However, the mechanisms underlying this phenomenon were unclear. OBJECTIVES: The aim of this study was to dissect the relative contribution of innate and heterologous antigen-specific pathways to the breach of self-tolerance and pathology observed in this model and how this may result from modified T and B-cell interactions. METHODS: To address this issue, experimental arthritis was elicited either by a non-specific inflammatory stimulus alone, by activation of T cells of an irrelevant specificity or a combination of both. RESULTS: The non-specific inflammatory response generated by lipopolysaccharide led to articular inflammation and cartilage erosion, but did not break tolerance to joint-specific antigens. In contrast, local activation of T cells of an irrelevant specificity produced a similar pathological picture but, in addition, induced T-cell responses to unrelated joint-specific antigens with associated activation of autoreactive B cells. These effects could be further potentiated by the addition of lipopolysaccharide. CONCLUSION: These data demonstrate that non-specific inflammation alone is insufficient to breach self-tolerance. In contrast, T cells of an irrelevant specificity, when triggered locally in an antigen-specific manner, can breach self-tolerance leading to arthritis and autoantibody production, which can then be amplified in a non-specific manner.

Imaging T-cell movement in the brain during experimental cerebral malaria.

T-cells are known to play a role in the pathology associated with experimental cerebral malaria, although it has not previously been possible to examine their behaviour in brain. Using multiphoton laser scanning microscopy, we have examined the migration and movement of these cells in brain tissue. We believe that this approach will help define host-parasite interactions and examine how intervening in these relationships affects the development of cerebral pathology.

Assessment of iron status and the role for iron-replacement therapy in anaemic cancer patients under the care of a specialist palliative care unit.

Anaemia is common in advanced cancer, may develop for several reasons, and is not always symptomatic. Our observations of the seemingly indiscriminate prescription of iron-replacement therapy (IRT) for anaemic palliative care patients, and our practice of discontinuing IRT in patients with normal red-cell indices, prompted a study to determine (1) the prevalence of anaemia in our patients, (2) what proportion had iron deficiency, (3) the prevalence and benefits of IRT and (4) the prevalence of side effects attributable to IRT. The prevalence of anaemia was 65%. We found a 9% prevalence of iron deficiency, and suggestive but inconclusive evidence of iron deficiency in a further 41%, but only three (27%) of these patients had typical iron deficiency red-cell indices. Only two patients within the study population were taking IRT. Haemoglobin increased significantly in one, but fell in the other, and both experienced side effects attributable to iron. IRT should neither be indiscriminately prescribed nor withheld for anaemic palliative care patients, and the decision should not be based on red-cell indices alone. When symptomatic anaemia is found in patients whose general condition indicates that IRT would be acceptable, iron status should be fully assessed. A therapeutic trial of IRT may be justified where ferritin is elevated, but other parameters suggest iron deficiency.

Characterization of myeloid-derived suppressor cells and cytokines GM-CSF, IL-10 and MCP-1 in dogs with malignant melanoma receiving a GD3-based immunotherapy.

Melanoma in humans and canines is an aggressive and highly metastatic cancer. The mucosal forms in both species share genetic and histopathologic features, making dogs a valuable spontaneous disease animal model. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells of myeloid origin with immunosuppressive capabilities, which are increased in many human cancers and contribute to tumor immune evasion. They are a possible target to improve immunotherapy outcomes. Current information regarding MDSCs in canines is minimal, limiting their use as translational model for the study of MDSCs. The objective of this study was to characterize major MDSCs subsets (monocytic and polymorphonuclear) and the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 10 (IL-10) and monocyte chemoattractant protein-1 (MCP-1) in canines with malignant melanoma and to evaluate changes in MDSCs and the cytokines over time in response to a GD3-based active immunotherapy. Whole blood and serum collected from 30 healthy controls and 33 patients enrolled in the University of Florida melanoma vaccine trial were analyzed by flow cytometry with canine specific CD11b, MHCII and anti-human CD14 antibodies to assess ostensibly polymorphonuclear-MDSC (CD11b+ MHCII- CD14-) and monocytic-MDSC (CD11b+ MHCII- CD14+) subsets. IL-10, MCP-1 and both MDSCs subsets were significantly elevated in melanoma dogs versus controls. Both MDSCs subsets decreased significantly following GD3-based immunotherapy administration but no significant changes in cytokines were seen over time. To our knowledge, this is the first report documenting increased monocytic-MDSCs in canine melanoma. This is consistent with human malignant melanoma data, supporting dogs as a valuable model for therapeutic intervention studies.

Tumour necrosis factor-alpha blockade suppresses murine allergic airways inflammation.

Asthma is a heterogeneous disease that has been increasing in incidence throughout western societies and cytokines, including proinflammatory tumour necrosis factor alpha (TNF-alpha), have been implicated in the pathogenesis of asthma. Anti-TNF-alpha therapies have been established successfully in the clinic for diseases such as rheumatoid arthritis and Crohn's disease. TNF-alpha-blocking strategies are now being trialled in asthma; however, their mode of action is poorly understood. Based on the observation that TNF-alpha induces lymph node hypertrophy we have attempted to investigate this as a mechanism of action of TNF-alpha in airway inflammation by employing two models of murine airway inflammation, that we have termed short and long models, representing severe and mild/moderate asthma, respectively. The models differ by their immunization schedules. In the short model, characterized by eosinophilic and neutrophilic airway inflammation the effect of TNF-alpha blockade was a reduction in draining lymph node (DLN) hypertrophy, eosinophilia, interleukin (IL)-5 production and immunoglobulin E (IgE) production. In the long model, characterized by eosinophilic inflammation, TNF-alpha blockade produced a reduction in DLN hypertrophy and IL-5 production but had limited effects on eosinophilia and IgE production. These results indicate that anti-TNF-alpha can suppress DLN hypertrophy and decrease airway inflammation. Further investigations showed that anti-TNF-alpha-induced inhibition of DLN hypertrophy cannot be explained by preventing l-selectin-dependent capture of lymphocytes into the DLN. Given that overall TNF blockade was able to suppress the short model (severe) more effectively than the long model (mild/moderate), the results suggest that TNF-alpha blocking therapies may be more effective in the treatment of severe asthma.

In-utero and neonatal exposure to secondhand smoke causes vascular dysfunction in newborn rats.

OBJECTIVES: We sought to determine the effects of secondhand smoke (SHS) exposure on vascular reactivity in newborn and infant rats. BACKGROUND: Secondhand smoke exposure increases cardiovascular risk. Secondhand smoke-induced endothelial dysfunction has been demonstrated in older teenagers and young adults. We have previously shown in adult rabbits that SHS induces atherogenesis and endothelial dysfunction. The effects of SHS on vascular function in the offspring of SHS-exposed mothers and in infants are unknown. METHODS: In this study the effects of in-utero (21 days) and neonatal (28 days) exposure to SHS were examined in 80 rats, 4 weeks of age, in a 2-by-2 design study. Rats were exposed to sidestream smoke in smoking chambers. Aortic rings were excised and isometric force responses to phenylephrine, acetylcholine, A23187 and nitroglycerin were studied in organ baths. RESULTS: Neonatal SHS exposure reduced animal weight (p=0.009). In-utero exposure increased the sensitivity (decreased the EC50) of aortic rings to phenylephrine (p < 0.0005), as did neonatal exposure (p=0.01). Maximal contraction to phenylephrine was reduced by in-utero exposure (p=0.04). In-utero SHS exposure reduced maximal endothelium-dependent relaxation to acetylcholine (p=0.04) and increased the EC50 (p=0.05), suggesting impaired sensitivity to acetylcholine. In-utero exposure decreased the sensitivity (increased the EC50) to the endothelium-independent vasodilator nitroglycerin (p=0.003). CONCLUSIONS: Secondhand smoke has detrimental effects on vascular smooth muscle function in the newborn.

Testosterone worsens endothelial dysfunction associated with hypercholesterolemia and environmental tobacco smoke exposure in male rabbit aorta.

OBJECTIVES: To assess the effects of interaction of sex hormones, hypercholesterolemia (HC) and environmental tobacco smoke (ETS) exposure on endothelium-dependent relaxation, we examined vascular reactivity in vitro in an animal model of atherogenesis. BACKGROUND: Animal and human studies indicate the presence of interactions between classic coronary artery disease risk factors and endothelium-dependent relaxation. Sex hormones have also been shown to influence release of endothelium-derived relaxing factor. METHODS: New Zealand White rabbits were randomized to receive either an HC diet (n = 8) or ETS exposure plus HC diet (n = 8). Eight rabbits receiving a normal diet, without exposure to ETS, served as the control group. The HC diet consisted of 3% soybean oil and 0.3% cholesterol by weight over 13 weeks. The source of ETS was sidestream smoke of 4 cigarettes/15 min, 6 h/day, 5 days/week over 10 weeks in a smoking chamber. Rabbits were killed, and fresh aortic rings were harvested and maintained in oxygenated Krebs solution in an organ bath at 37 degrees C. Rings were precontracted with norepinephrine and exposed to acetylcholine in increasing doses, and isometric tension was recorded. Rings were also exposed to physiologic concentrations (1 nmol/liter) of either 17-beta-estradiol, testosterone or progesterone before pre-contraction with norepinephrine and relaxation with acetylcholine. Endothelium-independent relaxation was studied using nitroglycerin. The surface area of the ring covered by lipids was measured by Sudan IV staining. RESULTS: HC and ETS significantly reduced endothelium-dependent relaxation (p = 0.01 and p < 0.0005, respectively) and caused atherogenesis (p < 0.0005 and p = 0.047, respectively) but did not affect endothelium-independent relaxation. Incubation with estradiol and estradiol plus progesterone did not influence endothelium-dependent relaxation. Testosterone reduced endothelium-dependent relaxation (p = 0.049) and augmented the endothelial dysfunction associated with ETS exposure and HC (p = 0.03). CONCLUSIONS: Both HC and ETS are atherogenic and impair endothelial function but do not affect endothelium-independent relaxation. Physiologic levels of estradiol and estradiol plus progesterone do not affect endothelium-dependent relaxation. Physiologic levels of testosterone impair relaxation and augment the endothelial dysfunction associated with ETS exposure and HC.

Effects of second-hand smoke and gender on infarct size of young rats exposed in utero and in the neonatal to adolescent period.

OBJECTIVES: We sought to assess the effects of second-hand smoke (SHS) and gender on infarct size in young rats exposed in utero or in the neonatal to adolescent period, or both. BACKGROUND: We previously demonstrated that exposure to SHS increases infarct size in a rat model of ischemia and reperfusion, with a dose-response relation. These results are consistent with epidemiologic studies demonstrating that SHS increases risk of death from heart disease. METHODS: Thirty-one pregnant female rats were randomly divided into two groups: those exposed to SHS and a control group (non-SHS). After 3 weeks, each rat had given birth to 10 to 12 rats. One hundred one neonatal rats were divided into four groups according to exposure to SHS in utero (SHSu) and randomized to SHS exposure in the neonatal to adolescent period (SHSna). After 12 weeks, all rats were subjected to 17 min of left coronary artery occlusion and 2 h of reperfusion. RESULTS: Birth mortality was higher in the SHSu group than in the non-SHSu group (11.9% vs. 2.8%, p < 0.001). Body weight of neonatal rats at 3 and 4 weeks in the two SHSu groups was lower than that of rats in the two non-SHSu groups (p < 0.001). Exposure to SHSna increased endothelin-1 levels in plasma (p = 0.001). In all 70 young rats who survived the neonatal period, infarct size (Infarct mass/Risk area x 100%) was greater in the SHSna groups than in the non-SHSna groups (p = 0.005) and in the male groups than in the female groups (p < 0.001). CONCLUSIONS: Exposure to SHS in the neonatal to adolescent period and male gender increased myocardial infarct size in a young rat model of ischemia and reperfusion. These results are consistent with epidemiologic studies demonstrating that SHS increases the health risk to neonates and adolescents.

Effect of physiological mechanical perturbations on intact human myocardial repolarization.

OBJECTIVE: The objective of this study was to investigate the relationship between acute decreases in right ventricular volume during Valsalva strain (with resultant changes in autonomic neural tone) and measures of local endocardial repolarization time independent of heart rate and autonomic neural tone. METHODS: Patients implanted with a stimulus to T wave (Stim-T) sensing pacemaker specially adapted to output a validate measure of beat to beat local repolarization (n = 9) performed Valsalva manoeuvers (40 mmHg for 15 s) while paced at a cycle length of 500 ms. Stim-T intervals were measured before and after autonomic blockade (Block: 0.03 mg/kg i.v. atropine +/- 0.15 mg/kg propranolol). Right ventricular end diastolic volume was estimated by simultaneous 2D-echocardiography. RESULTS: Without autonomic blockade, compared to baseline, repolarization significantly prolonged during Valsalva strain (1.1 +/- 0.7%) and shortened during release (-1.4 +/- 1.0%). After block, strain related repolarization prolongation was also observed (1.0 +/- 0.6%), with significantly less release related repolarization shortening (-0.8 +/- 0.8%) compared to pre-block (P < 0.05). Right ventricular end diastolic volume decreased during strain by 11 +/- 10 and 9 +/- 16% from baseline, pre- and post-block respectively (P < 0.05). CONCLUSION: In a chronically instrumented human model, an acute physiologic volume reduction modestly prolongs right ventricular repolarization independent of changes in rate or autonomic tone.

The greater glycan content of recombinant human thyroid peroxidase of mammalian than of insect cell origin facilitates purification to homogeneity of enzymatically protein remaining soluble at high concentration.

Structural studies on thyroid peroxidase (TPO), a major thyroid autoantigen, require milligram amounts of pure protein. We found that the human TPO ectodomain (amino acid residues 1-848) generated in insect cells did not remain in solution at high concentrations after affinity purification. In contrast, the TPO ectodomain secreted by mammalian (Chinese hamster ovary) cells, although generated to a lesser extent (1 vs. 8 mg/liter), remained in solution at high concentration (10 mg/ml) after purification to homogeneity. This purified material was well recognized by TPO autoantibodies, but lacked enzymatic activity. We attempted to restore activity by culturing the Chinese hamster ovary cells in the presence of added heme. TPO enzymatic activity was clearly detected in conditioned medium from cells cultured in hematin and hemin, but not in protoporphyrin IX (all at 1 mg/liter). Heme prosthetic group incorporation into affinity-purified TPO was highest for hematin and hemin, but unchanged for protoporphyrin IX (OD 410/280 nm ratios of 0.25, 0.23, and 0.14, respectively). Enzymatic activity was now evident with hemin (mean +/- SE, 27.2 +/- 2.6; n = 3; guaiacol units/mg protein), hematin (24.1 +/- 1.6), and, to a lesser extent, protoporphyrin IX (3.6 +/- 0.2). Culturing cells in 20 mg/liter hematin, the maximum concentration tolerated, increased enzymatic activity even further (45.6 +/- 0.6 guaiacol units/mg protein). All purified TPO preparations were homogeneous on PAGE and of similar size (105 kDa). Enzymatic deglycosylation showed a complex carbohydrate contribution of 13 kDa (unlike the 2.3 kDa in insect cell TPO). In conclusion, this is the first report on the purification to homogeneity of recombinant human TPO of mammalian cell origin. Unlike TPO generated in insect cells, mammalian TPO remains soluble at high concentration, possibly because of its greater carbohydrate content. This enzymatically active, recombinant human TPO may be useful for future structural studies.

Effects of L-arginine on atherogenesis and endothelial dysfunction due to secondhand smoke.

Secondhand smoke (SHS) and hypercholesterolemia increase cardiovascular risk. We hypothesized that L-arginine, the precursor of nitric oxide (NO), might protect against atherogenesis and endothelial dysfunction caused by SHS. The effects of L-arginine supplementation (2.25% solution ad libitum) and SHS (smoking chambers for 10 weeks) were examined in 32 hypercholesterolemic rabbits. Eight normal rabbits served as controls. Acetylcholine- and nitroglycerin-induced vasorelaxation was assessed in aortic rings precontracted with norepinephrine. Hypercholesterolemia increased intimal lesion area (P=0.012), reduced endothelium-dependent relaxation (P=0.009), and reduced basal (P=0.005) and stimulated (P<0.0005) production of NOs. SHS increased intimal lesion area (P=0. 01) norepinephrine-induced contraction (P=0.001) and reduced endothelium-dependent relaxation (P=0.02). SHS-induced increase in norepinephrine contraction was abolished by the inhibition of NO synthase and removal of endothelium. L-Arginine improved endothelium-dependent relaxation (P=0.001) and attenuated SHS-induced endothelial dysfunction (P=0.007) and atherogenesis (P=0. 001). Basal production of nitrogen oxides correlated inversely with intimal lesion area (r=-0.66; P<0.0005) and stimulated production of NOs correlated with endothelium-dependent relaxation (r=-0.66; P<0. 001). SHS causes endothelial dysfunction and increased adrenergic responsiveness and atherogenesis in hypercholesterolemic rabbits. Chronic dietary supplementation with the NO precursor L-arginine mitigates these effects. The adverse vascular consequences of SHS appear to be mediated via deleterious effects on endothelial function.

Chronic dietary L-arginine prevents endothelial dysfunction secondary to environmental tobacco smoke in normocholesterolemic rabbits.

Our goal was to determine whether environmental tobacco smoke causes endothelial dysfunction in the absence of hypercholesterolemia and whether such an effect can be prevented by supplementation with L-arginine. Environmental tobacco smoke exposure is associated with an increase in coronary artery disease events and mortality. We have previously demonstrated that environmental tobacco smoke causes endothelial dysfunction and atherosclerosis in rabbits with diet-induced hypercholesterolemia and atherosclerosis and that chronic dietary L-arginine supplementation prevents this. The effects of L-arginine supplementation (2.25% solution ad libitum) and environmental tobacco smoke (smoking chambers for 10 weeks) were examined with a 2 x 2 design in 32 rabbits fed a normal diet. Acetylcholine, calcium ionophore A23187, and nitroglycerin-induced vasorelaxation were assessed in aortic rings precontracted with phenylephrine. Endothelial L-arginine levels were measured by chromatography. Chronic L-arginine supplementation increased serum (P < .001) and endothelial (P = .003) L-arginine levels. Environmental tobacco smoke reduced endothelium-dependent acetylcholine-induced relaxation, and L-arginine blocked this adverse effect (P = .04). Environmental tobacco smoke tended to increase phenylephrine-induced contraction (P = .06). Neither environmental tobacco smoke nor L-arginine influenced A23187-induced relaxation nor endothelium-independent nitroglycerin-induced relaxation. Endothelial dysfunction secondary to environmental tobacco smoke may occur in the absence of diet-induced hypercholesterolemia and atherosclerosis. Chronic dietary supplementation with a nitric oxide donor such as L-arginine offsets the endothelial dysfunction associated with environmental tobacco smoke in normocholesterolemic rabbits, possibly through substrate loading of the nitric oxide pathway.

Free radical pathology in chronic arterial disease.

The generation of toxic oxygen metabolites is more usually associated with inflammation. However, pathological free radical reactions can cause tissue damage by adversely affecting prostacyclin (PGI2) synthesis allowing initiation of coagulation. We have assessed changes in the red cell defence to toxic oxygen metabolite generation, viz measurement of glutathione concentration (GSH) and superoxide dismutase activity (SOD). GSH and SOD were measured in 20 patients with peripheral arterial disease, 22 patients with vasculitis, and 11 patients with angina, and compared to 17 matched controls. The 53 subjects with arterial disease had significantly lower SOD levels: in contrast GSH levels were significantly higher. Extracellularly plasma thiol levels (PSH) were low and caeruloplasmin (Cp) levels were high. We suggest that free radical pathology exists not only in inflammatory vascular disease but also in atherosclerosis.