The role of self-reliance and denial in the help-seeking process for eating disorders among university students.

OBJECTIVE: This study investigated how self-reliance and denial influence the relationship between help-seeking attitudes and behaviour. METHOD: Australian university students (N = 406) completed an online survey and females with elevated eating disorder (ED) concerns and high impairment were included in the study (N = 137). Participants completed measures of help-seeking attitudes, perceived barriers, and actual help-seeking behaviour. Via moderated logistic regression, we examined self-reliance and denial as perceived barriers to help-seeking. RESULTS: Of the total sample, over 33.7% of university students reported substantial ED concerns and impairment of whom 65.0% believed they needed help. While a majority reported that help-seeking would be useful (85.4%), only a minority of participants had sought professional help for their concerns (38.7%). Self-reliance and denial were frequently endorsed barriers and moderated the relationship between help-seeking attitudes and behaviours. CONCLUSION: ED concerns are common among university students and perceived barriers play a moderating role between attitudes and help-seeking. Future prevention and early intervention programs should address students' denial, while the importance of reaching out for professional help (rather than relying on themselves) could be highlighted with peer support.

An investigation of the dual continua model of mental health in the context of eating disorder symptomatology using latent profile analysis.

OBJECTIVE: The dual continua model of mental health suggests that mental well-being and mental illness are two distinct continua that are interrelated in their contributions to overall mental health. This study investigated the dual continua model in individuals with eating disorder symptoms. METHOD: Female university students (N = 346) completed measures of mental well-being, eating disorder symptomatology, clinical impairment and psychological distress. Confirmatory factor and latent profile analyses were used to derive underlying mental health profiles. RESULTS: Results revealed two oblique factors representing the mental well-being and eating disorder symptomatology constructs and four unique mental health profiles that were partially consistent with the dual continua model emerged: 'flourishing', 'vulnerable', 'partially symptomatic and content', and 'languishing'. The derived mental health profiles had unique characteristics described by psychological distress and clinical impairment. CONCLUSIONS: The results did not provide conclusive evidence for the dual continua model as they could also be interpreted in a manner consistent with the unidimensional approach. While the dual continua model is a promising avenue to understand mental health in a way that looks beyond symptoms, these results caution against its rapid adoption and encourage future research to understand how eating disorder symptomatology and positive mental health assets contribute to overall mental health.

From Screening to Targeted Degradation: Strategies for the Discovery and Optimization of Small Molecule Ligands for PCSK9.

Proprotein convertase substilisin-like/kexin type 9 (PCSK9) is a serine protease involved in a protein-protein interaction with the low-density lipoprotein (LDL) receptor that has both human genetic and clinical validation. Blocking this protein-protein interaction prevents LDL receptor degradation and thereby decreases LDL cholesterol levels. Our pursuit of small-molecule direct binders for this difficult to drug PPI target utilized affinity selection/mass spectrometry, which identified one confirmed hit compound. An X-ray crystal structure revealed that this compound was binding in an unprecedented allosteric pocket located between the catalytic and C-terminal domain. Optimization of this initial hit, using two distinct strategies, led to compounds with high binding affinity to PCSK9. Direct target engagement was demonstrated in the cell lysate with a cellular thermal shift assay. Finally, ligand-induced protein degradation was shown with a proteasome recruiting tag attached to the high-affinity allosteric ligand for PCSK9.

Advising women with diabetes in pregnancy to express breastmilk in late pregnancy (Diabetes and Antenatal Milk Expressing [DAME]): a multicentre, unblinded, randomised controlled trial.

BACKGROUND: Infants of women with diabetes in pregnancy are at increased risk of hypoglycaemia, admission to a neonatal intensive care unit (NICU), and not being exclusively breastfed. Many clinicians encourage women with diabetes in pregnancy to express and store breastmilk in late pregnancy, yet no evidence exists for this practice. We aimed to determine the safety and efficacy of antenatal expressing in women with diabetes in pregnancy. METHODS: We did a multicentre, two-group, unblinded, randomised controlled trial in six hospitals in Victoria, Australia. We recruited women with pre-existing or gestational diabetes in a singleton pregnancy from 34 to 37 weeks' gestation and randomly assigned them (1:1) to either expressing breastmilk twice per day from 36 weeks' gestation (antenatal expressing) or standard care (usual midwifery and obstetric care, supplemented by support from a diabetes educator). Randomisation was done with a computerised random number generator in blocks of size two and four, and was stratified by site, parity, and diabetes type. Investigators were masked to block size but masking of caregivers was not possible. The primary outcome was the proportion of infants admitted to the NICU. We did the analyses by intention to treat; the data were obtained and analysed masked to group allocation. This trial is registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12611000217909. FINDINGS: Between June 6, 2011, and Oct 29, 2015, we recruited and randomly assigned 635 women: 319 to antenatal expressing and 316 to standard care. Three were not included in the primary analysis (one withdrawal from the standard care group, and one post-randomisation exclusion and one withdrawal from the antenatal expressing group). The proportion of infants admitted to the NICU did not differ between groups (46 [15%] of 317 assigned to antenatal expressing vs 44 [14%] of 315 assigned to standard care; adjusted relative risk 1·06, 95% CI 0·66 to 1·46). In the antenatal expressing group, the most common serious adverse event for infants was admission to the NICU for respiratory support (for three [<1%] of 317. In the standard care group, the most common serious adverse event for infants was moderate to severe encephalopathy with or without seizures (for three [<1%] of 315). INTERPRETATION: There is no harm in advising women with diabetes in pregnancy at low risk of complications to express breastmilk from 36 weeks' gestation. FUNDING: Australian National Health and Medical Research Council.

Insights into activity and inhibition from the crystal structure of human O-GlcNAcase.

O-GlcNAc hydrolase (OGA) catalyzes removal of ßα-linked N-acetyl-D-glucosamine from serine and threonine residues. We report crystal structures of Homo sapiens OGA catalytic domain in apo and inhibited states, revealing a flexible dimer that displays three unique conformations and is characterized by subdomain α-helix swapping. These results identify new structural features of the substrate-binding groove adjacent to the catalytic site and open new opportunities for structural, mechanistic and drug discovery activities.

Integration of Affinity Selection-Mass Spectrometry and Functional Cell-Based Assays to Rapidly Triage Druggable Target Space within the NF-κB Pathway.

The primary objective of early drug discovery is to associate druggable target space with a desired phenotype. The inability to efficiently associate these often leads to failure early in the drug discovery process. In this proof-of-concept study, the most tractable starting points for drug discovery within the NF-κB pathway model system were identified by integrating affinity selection-mass spectrometry (AS-MS) with functional cellular assays. The AS-MS platform Automated Ligand Identification System (ALIS) was used to rapidly screen 15 NF-κB proteins in parallel against large-compound libraries. ALIS identified 382 target-selective compounds binding to 14 of the 15 proteins. Without any chemical optimization, 22 of the 382 target-selective compounds exhibited a cellular phenotype consistent with the respective target associated in ALIS. Further studies on structurally related compounds distinguished two chemical series that exhibited a preliminary structure-activity relationship and confirmed target-driven cellular activity to NF-κB1/p105 and TRAF5, respectively. These two series represent new drug discovery opportunities for chemical optimization. The results described herein demonstrate the power of combining ALIS with cell functional assays in a high-throughput, target-based approach to determine the most tractable drug discovery opportunities within a pathway.

Structure and Function of the Hypertension Variant A486V of G Protein-coupled Receptor Kinase 4.

G-protein-coupled receptor (GPCR) kinases (GRKs) bind to and phosphorylate GPCRs, initiating the process of GPCR desensitization and internalization. GRK4 is implicated in the regulation of blood pressure, and three GRK4 polymorphisms (R65L, A142V, and A486V) are associated with hypertension. Here, we describe the 2.6 Å structure of human GRK4α A486V crystallized in the presence of 5'-adenylyl ß,γ-imidodiphosphate. The structure of GRK4α is similar to other GRKs, although slight differences exist within the RGS homology (RH) bundle subdomain, substrate-binding site, and kinase C-tail. The RH bundle subdomain and kinase C-terminal lobe form a strikingly acidic surface, whereas the kinase N-terminal lobe and RH terminal subdomain surfaces are much more basic. In this respect, GRK4α is more similar to GRK2 than GRK6. A fully ordered kinase C-tail reveals interactions linking the C-tail with important determinants of kinase activity, including the αB helix, αD helix, and the P-loop. Autophosphorylation of wild-type GRK4α is required for full kinase activity, as indicated by a lag in phosphorylation of a peptide from the dopamine D1 receptor without ATP preincubation. In contrast, this lag is not observed in GRK4α A486V. Phosphopeptide mapping by mass spectrometry indicates an increased rate of autophosphorylation of a number of residues in GRK4α A486V relative to wild-type GRK4α, including Ser-485 in the kinase C-tail.

Safety and efficacy of antenatal milk expressing for women with diabetes in pregnancy: protocol for a randomised controlled trial.

INTRODUCTION: Many maternity providers recommend that women with diabetes in pregnancy express and store breast milk in late pregnancy so breast milk is available after birth, given (1) infants of these women are at increased risk of hypoglycaemia in the first 24 h of life; and (2) the delay in lactogenesis II compared with women without diabetes that increases their infant's risk of receiving infant formula. The Diabetes and Antenatal Milk Expressing (DAME) trial will establish whether advising women with diabetes in pregnancy (pre-existing or gestational) to express breast milk from 36 weeks gestation increases the proportion of infants who require admission to special or neonatal intensive care units (SCN/NICU) compared with infants of women receiving standard care. Secondary outcomes include birth gestation, breastfeeding outcomes and economic impact. METHODS AND ANALYSIS: Women will be recruited from 34 weeks gestation to a multicentre, two arm, unblinded randomised controlled trial. The intervention starts at 36 weeks. Randomisation will be stratified by site, parity and diabetes type. Women allocated to the intervention will be taught expressing and encouraged to hand express twice daily for 10 min and keep an expressing diary. The sample size of 658 (329 per group) will detect a 10% difference in proportion of babies admitted to SCN/NICU (85% power, α 0.05). Data are collected at recruitment (structured questionnaire), after birth (abstracted from medical record blinded to group), and 2 and 12 weeks postpartum (telephone interview). DATA ANALYSIS: the intervention group will be compared with the standard care group by intention to treat analysis, and the primary outcome compared using χ(2) and ORs. ETHICS AND DISSEMINATION: Research ethics approval will be obtained from participating sites. Results will be published in peer-reviewed journals and presented to clinicians, policymakers and study participants. TRIAL REGISTRATION NUMBER: Australian Controlled Trials Register ACTRN12611000217909.

Structural basis for selective small molecule kinase inhibition of activated c-Met.

The receptor tyrosine kinase c-Met is implicated in oncogenesis and is the target for several small molecule and biologic agents in clinical trials for the treatment of cancer. Binding of the hepatocyte growth factor to the cell surface receptor of c-Met induces activation via autophosphorylation of the kinase domain. Here we describe the structural basis of c-Met activation upon autophosphorylation and the selective small molecule inhibiton of autophosphorylated c-Met. MK-2461 is a potent c-Met inhibitor that is selective for the phosphorylated state of the enzyme. Compound 1 is an MK-2461 analog with a 20-fold enthalpy-driven preference for the autophosphorylated over unphosphorylated c-Met kinase domain. The crystal structure of the unbound kinase domain phosphorylated at Tyr-1234 and Tyr-1235 shows that activation loop phosphorylation leads to the ejection and disorder of the activation loop and rearrangement of helix αC and the G loop to generate a viable active site. Helix αC adopts a orientation different from that seen in activation loop mutants. The crystal structure of the complex formed by the autophosphorylated c-Met kinase domain and compound 1 reveals a significant induced fit conformational change of the G loop and ordering of the activation loop, explaining the selectivity of compound 1 for the autophosphorylated state. The results highlight the role of structural plasticity within the kinase domain in imparting the specificity of ligand binding and provide the framework for structure-guided design of activated c-Met inhibitors.

Addressing the midwifery workforce crisis: evaluating an employment model for undergraduate midwifery students at a tertiary maternity hospital in Melbourne, Australia.

BACKGROUND AND AIMS: In Victoria, maternity services are under significant strain due to increased numbers of women giving birth and critical workforce shortages. Hospitals have experienced challenges in adequately staffing maternity units, particularly on postnatal wards. In 2008, a tertiary maternity hospital in Melbourne introduced a model where undergraduate midwifery students were employed as Division 2 nurses (SMW_Div2) (enrolled nurses), to work in the postnatal area only. This study explored the pilot employment model from the perspective of the SMW_Div2 and hospital midwives. METHODS: A web-based survey was administered to hospital midwives and the SMW_Div2s in the employment model in January 2010. The survey explored the views of midwives and SMW_Div2s regarding the perceived impact of the model on workforce readiness, recruitment and retention, and clinical competence and confidence. FINDINGS: Forty-seven of 158 midwives (30%) and five of nine SMW_Div2s employed in the model responded to the survey. Both groups considered the model to have benefits for the organisation, including increased: student workforce readiness; clinical confidence and competence; and organisational loyalty. Both groups also considered that the model would facilitate: workforce recruitment; a teaching and learning culture within the organisation; and enhanced partnerships between students, hospitals and universities. Caution was expressed regarding workload and the need for ongoing support for SMW_Div2s working in the model. DISCUSSION AND CONCLUSION: SMW_Div2s and midwives were positive about the introduction of the paid employment model at the Women's. The findings are consistent with evaluations of similar programs in the nursing setting. The employment model has potential short and long term individual and organisational advantages, which is important in the context of increasing births and workforce shortages. Progression of such models will be contingent on the collaboration and cooperation of the various stakeholders involved in maternity workforce and education.

Diabetes and antenatal milk expressing: a pilot project to inform the development of a randomised controlled trial.

OBJECTIVE: infants of women with diabetes in pregnancy are at increased risk of hypoglycaemia. If the infant's blood glucose is low and the mother is unable to breast feed/provide sufficient expressed breast milk, infants are often given formula. Some hospitals encourage women with diabetes to express breast milk before birth. However, there is limited evidence for this practice, including its impact on labour and birth, e.g. causing premature birth may be a concern. A pilot study was undertaken to establish the feasibility of conducting an adequately powered randomised controlled trial to evaluate this practice. DESIGN: consecutive eligible women with pre-existing or gestational diabetes (requiring insulin), planning to breast feed and attending the study hospital were offered participation. INCLUSION CRITERIA: 34-36 weeks of gestation; singleton pregnancy; cephalic presentation; and able to speak, read and write in English. EXCLUSION CRITERIA: history of spontaneous preterm birth, antepartum haemorrhage, placenta praevia and suspected fetal compromise. Women were encouraged to express colostrum twice a day from 36 weeks of gestation, and advised how to store the colostrum, which was frozen for their infant's use after birth. They were asked to keep a diary documenting their expressing. DATA: demographic questionnaire, telephone interview at six and 12 weeks postpartum and medical record data. SETTING: a public, tertiary, women's hospital in Melbourne, Australia. PARTICIPANTS: 43 women with diabetes in pregnancy (requiring insulin). FINDINGS: cardiotocographs were undertaken after the first expressing episode and none of the infants showed any sign of fetal compromise. Forty per cent of infants received formula in the 24 hours postpartum. The proportion of infants receiving any breast milk at six weeks was 90%, and this decreased to 75% at 12 weeks. No women showed evidence of hypoglycaemia post expressing. The intervention was positively received by most women; 95% said that they would express antenatally again if the practice proved to be beneficial. The amount of colostrum varied according to the number of expressions, the length of time in the study and the time spent expressing, with a median of 14 days expressing and 39.6 ml of colostrum obtained. KEY CONCLUSIONS: the small number of women in this pilot was not an adequate number to examine safety or efficacy, but this study does provide evidence that it would be feasible and desirable to conduct a randomised controlled trial of antenatal milk expressing for women with diabetes requiring insulin in pregnancy. IMPLICATIONS FOR PRACTICE: it is important that this widespread practice undergoes rigorous evaluation to assess both efficacy and safety. Until such evidence is available, the authors suggest that the routine encouragement of antenatal milk expressing in women with diabetes in pregnancy should cease.

Structural definition and substrate specificity of the S28 protease family: the crystal structure of human prolylcarboxypeptidase.

BACKGROUND: The unique S28 family of proteases is comprised of the carboxypeptidase PRCP and the aminopeptidase DPP7. The structural basis of the different substrate specificities of the two enzymes is not understood nor has the structure of the S28 fold been described. RESULTS: The experimentally phased 2.8 A crystal structure is presented for human PRCP. PRCP contains an alpha/beta hydrolase domain harboring the catalytic Asp-His-Ser triad and a novel helical structural domain that caps the active site. Structural comparisons with prolylendopeptidase and DPP4 identify the S1 proline binding site of PRCP. A structure-based alignment with the previously undescribed structure of DPP7 illuminates the mechanism of orthogonal substrate specificity of PRCP and DPP7. PRCP has an extended active-site cleft that can accommodate proline substrates with multiple N-terminal residues. In contrast, the substrate binding groove of DPP7 is occluded by a short amino-acid insertion unique to DPP7 that creates a truncated active site selective for dipeptidyl proteolysis of N-terminal substrates. CONCLUSION: The results define the structure of the S28 family of proteases, provide the structural basis of PRCP and DPP7 substrate specificity and enable the rational design of selective PRCP modulators.

Expression, purification and crystallization of human prolylcarboxypeptidase.

Prolylcarboxypeptidase (PrCP) is a lysosomal serine carboxypeptidase that cleaves a variety of C-terminal amino acids adjacent to proline and has been implicated in diseases such as hypertension and obesity. Here, the robust production, purification and crystallization of glycosylated human PrCP from stably transformed CHO cells is described. Purified PrCP yielded crystals belonging to space group R32, with unit-cell parameters a = b = 181.14, c = 240.13 A, that diffracted to better than 2.8 A resolution.

Cardioprotective and antiapoptotic effects of heme oxygenase-1 in the failing heart.

BACKGROUND: Heme oxygenase-1 (HO-1) is an inducible stress-response protein that imparts antioxidant and antiapoptotic effects. However, its pathophysiological role in cardiac remodeling and chronic heart failure (HF) is unknown. We hypothesized that induction of HO-1 in HF alleviates pathological remodeling. METHODS AND RESULTS: Adult male nontransgenic and myocyte-restricted HO-1 transgenic mice underwent either sham operation or coronary ligation to induce HF. Four weeks after ligation, nontransgenic HF mice exhibited postinfarction left ventricular (LV) remodeling and dysfunction, hypertrophy, fibrosis, oxidative stress, apoptosis, and reduced capillary density, associated with a 2-fold increase in HO-1 expression in noninfarcted myocardium. Compared with nontransgenic mice, HO-1 transgenic HF mice exhibited significantly (P<0.05) improved postinfarction survival (94% versus 57%) and less LV dilatation (end-diastolic volume, 46+/-8 versus 85+/-32 microL), mechanical dysfunction (ejection fraction, 65+/-9% versus 49+/-16%), hypertrophy (LV/tibia length 4.4+/-0.4 versus 5.2+/-0.6 mg/mm), interstitial fibrosis (11.2+/-3.1% versus 18.5+/-3.5%), and oxidative stress (3-fold reduction in tissue malondialdehyde). Moreover, myocyte-specific HO-1 overexpression in HF promoted tissue neovascularization and ameliorated myocardial p53 expression (2-fold reduction) and apoptosis. In isolated mitochondria, mitochondrial permeability transition was inhibited by HO-1 in a carbon monoxide (CO)-dependent manner and was recapitulated by the CO donor tricarbonylchloro(glycinato)ruthenium(II) (CORM-3). HO-1-derived CO also prevented H2O2-induced cardiomyocyte apoptosis and cell death. Finally, in vivo treatment with CORM-3 alleviated postinfarction LV remodeling, p53 expression, and apoptosis. CONCLUSIONS: HO-1 induction in the failing heart is an important cardioprotective adaptation that opposes pathological LV remodeling, and this effect is mediated, at least in part, by CO-dependent inhibition of mitochondrial permeability transition and apoptosis. Augmentation of HO-1 or its product, CO, may represent a novel therapeutic strategy for ameliorating HF.

Aprotinin modifies left ventricular contractility and cytokine release after ischemia-reperfusion in a dose-dependent manner in a murine model.

BACKGROUND: Periods of ischemia-reperfusion (I/R) during cardiac surgery are associated with transient left ventricular (LV) dysfunction and an inflammatory response. In this study, we examined the potential dose-dependent effects of aprotinin (APRO) on LV contractility and cytokine release in the setting of I/R. METHODS: An index of LV contractility, LV maximal elastance (E(max)), was measured at baseline, 30 min of ischemia, and 60 min of reperfusion by microtransducer volumetry. Mice were randomized as follows: (a) APRO 20,000 kallikrein-inhibiting units (KIU)/kg (n = 11); (b) APRO 4 x 10(4) KIU/kg (n = 10); (c) APRO 8 x 10(4) KIU/kg (n = 10); and (d) vehicle (saline; n = 10). APRO doses were calculated to reflect half, full, and twice the clinical Hammersmith dosing schedule. After I/R, plasma was collected for cytokine measurements. RESULTS: After I/R, E(max) decreased from the baseline value by more than 40% in the vehicle group as well as in the APRO 4 x 10(4) KIU/kg and APRO 8 x 10(4) KIU/kg groups (P < 0.05). However, E(max) returned to near baseline values in the APRO 2 x 10(4) KIU/kg group. Tumor necrosis factor (TNF) increased 10-fold after I/R, but it was reduced with higher APRO doses. CONCLUSIONS: This study demonstrated that a low dose of APRO provided protective effects on LV contractility, whereas higher doses suppressed TNF release. These unique findings suggest that there are distinct and independent mechanisms of action of APRO in the context of I/R.

Endothelin-A receptor inhibition after cardiopulmonary bypass: cytokines and receptor activation.

BACKGROUND: Basic studies have suggested that cross-talk exists between the endothelin-A receptor (ET-AR) and tumor necrosis factor signaling pathway. This study tested the hypothesis that administration of an ET-AR antagonist at the separation from cardiopulmonary bypass would alter the tumor necrosis factor activation in the early postoperative period. METHODS: Patients (n = 44) were randomly allocated to receive bolus infusion of vehicle, 0.1, 0.5, 1, or 2 mg/kg of the ET-AR antagonist (sitaxsentan), at the separation from cardiopulmonary bypass (n = 9, 9, 9, 9, and 8, respectively). Plasma levels of tumor necrosis factor-alpha and soluble tumor necrosis factor receptor 1 and 2 were measured. RESULTS: Compared with the vehicle group at 24 hours, plasma levels of tumor necrosis factor-alpha and tumor necrosis factor receptor 2 (indicative of receptor activation) were reduced in the 1 mg/kg ET-AR antagonist group (by approximately 13 pg/mL and approximately 0.5 ng/mL, respectively; p < 0.05). Plasma tumor necrosis factor receptor I levels also decreased (by approximately 1 ng/mL) after infusion of the higher doses of the ET-AR antagonist and remained lower (by approximately 3 ng/mL) at 24 hours after infusion (p < 0.05). In addition, a dose effect was observed between the ET-AR antagonist and these indices of tumor necrosis factor activation (p < 0.01). CONCLUSIONS: This study demonstrated a mechanistic relationship between the ET-AR and tumor necrosis factor receptor activation in the post-cardiac surgery period. Thus, in addition to the potential cardiovascular effects, a selective ET-AR antagonist can modify other biological processes relevant to the post-cardiac surgery setting.

Aprotinin exacerbates left ventricular dysfunction after ischemia/reperfusion in mice lacking tumor necrosis factor receptor I.

Aprotinin is a serine protease inhibitor with diverse biological effects; until recently, it was utilized in the context of ischemia/reperfusion (I/R). It has been hypothesized that a signaling pathway modulated by aprotinin in the context of I/R is the tumor necrosis factor-alpha receptor (TNFR) pathway. An intact mouse model of I/R (30 min ischemia and 60 min reperfusion) was used and left ventricular (LV) peak + maximal rate of left ventricular (LV) peak pressure (dP/dt) was measured in wild-type mice (WT, C57BL/6; n = 10), WT mice with aprotinin (4 mL/kg; n = 10), transgenic mice devoid of the TNFRI (TNFRI-null; n = 10), and TNFRI-null with aprotinin (n=10). Following I/R, LV peak + dP/dt decreased in both WT groups, but remained similar to baseline values in the TNFRI-null group. In contrast, aprotinin caused a marked reduction in LV peak + dP/dt in the TNFRI-null group following I/R. Soluble plasma TNF levels increased in the WT and TNFRI-null mice with I/R and was reduced with aprotinin. Soluble TNFRI and TNFRII levels, indicative of TNF activation, increased in the WT mice following I/R and remained elevated with aprotinin. Soluble TNFRII levels were increased in the TNFRI-null mice following I/R and remained elevated with aprotinin. The new and unique findings of this study were twofold. First, aprotinin failed to improve LV function after I/R despite a reduction in circulating TNF levels. Second, genetic ablation of TNFRI uncovered a negative inotropic effect of aprotinin. These findings demonstrate that complex biological pathways and interactions are affected with broad spectrum serine protease inhibition, which are relevant to myocardial function in the context of I/R.

Aprotinin exerts differential and dose-dependent effects on myocardial contractility, oxidative stress, and cytokine release after ischemia-reperfusion.

BACKGROUND: Cardiac surgery can result in left ventricular ischemia and reperfusion (I/R), the release of cytokines such as tumor necrosis factor, and oxidative stress with release of myeloperoxidase. Although aprotinin has been used in cardiac surgery, the likely multiple effects of this serine protease inhibitor limit clinical utility. This study tested the hypothesis that different aprotinin doses cause divergent effects on left ventricular contractility, cytokine release, and oxidative stress in the context of I/R. METHODS: Left ventricular I/R (30 minutes I, 60 minutes R) was induced in mice, and left ventricular contractility (maximal end-systolic elastance) determined. Mice were randomly allocated to 2 x 10(4) kallikrein inhibitory units (KIU)/kg aprotinin (n = 11), 4 x 10(4) KIU/kg aprotinin (n = 10), and vehicle (saline, n = 10). Based upon a fluorogenic assay, aprotinin doses of 2 and 4 x 10(4) KIU/kg resulted in plasma concentrations similar to those of the half and full Hammersmith doses, respectively. RESULTS: After I/R, maximal end-systolic elastance fell by more than 40% from baseline (p < 0.05), and this effect was attenuated by 2 x 10(4) KIU/kg but not 4 x 10(4) KIU/kg aprotinin. Tumor necrosis factor increased by more than 60% from control (p < 0.05) with I/R, but was reduced with 4 x 10(4) KIU/kg aprotinin. Myeloperoxidase increased with I/R, and was reduced to the greatest degree by 2 x 10(4) KIU/kg aprotinin. CONCLUSIONS: Aprotinin influences left ventricular contractility, cytokine release, and oxidative stress, which are dose dependent. These results provide mechanistic evidence that multiple pathways are differentially affected by aprotinin in a context relevant to cardiac surgery.