Comparison of Fecal Calprotectin and Myeloperoxidase in Predicting Outcomes in Inflammatory Bowel Disease.

BACKGROUND: Biomarkers have been proposed as surrogate treatment targets for the management of inflammatory bowel disease (IBD); however, their relationship with IBD-related complications remains unclear. This study investigated the utility of neutrophil biomarkers fecal calprotectin (fCal) and fecal myeloperoxidase (fMPO) in predicting a complicated IBD course. METHODS: Participants with IBD were followed for 24 months to assess for a complicated IBD course (incident corticosteroid use, medication escalation for clinical disease relapse, IBD-related hospitalizations/surgeries). Clinically active IBD was defined as Harvey-Bradshaw index >4 for Crohn's disease (CD) and simple clinical colitis activity index >5 for ulcerative colitis (UC). Area under the receiver-operating-characteristics curves (AUROC) and multivariable logistic regression assessed the performance of baseline symptom indices, fCal, and fMPO in predicting a complicated disease IBD course at 24 months. RESULTS: One hundred and seventy-one participants were included (CD, n = 99; female, n = 90; median disease duration 13 years [interquartile range, 5-22]). Baseline fCal (250 µg/g; AUROC = 0.77; 95% confidence interval [CI], 0.69-0.84) and fMPO (12 µg/g; AUROC = 0.77; 95% CI, 0.70-0.84) predicted a complicated IBD course. Fecal calprotectin (adjusted OR = 7.85; 95% CI, 3.38-18.26) and fMPO (adjusted OR = 4.43; 95% CI, 2.03-9.64) were associated with this end point after adjustment for other baseline variables including clinical disease activity. C-reactive protein (CRP) was inferior to fecal biomarkers and clinical symptoms (pdifference < .05) at predicting a complicated IBD course. A combination of baseline CRP, fCal/fMPO, and clinical symptoms provided the greatest precision at identifying a complicated IBD course. CONCLUSIONS: Fecal biomarkers are independent predictors of IBD-related outcomes and are useful adjuncts to routine clinical care.

Changes to insulin sensitivity in glucose clearance systems and redox following dietary supplementation with a novel cysteine-rich protein: A pilot randomized controlled trial in humans with type-2 diabetes.

We recently developed a novel keratin-derived protein (KDP) rich in cysteine, glycine, and arginine, with the potential to alter tissue redox status and insulin sensitivity. The KDP was tested in 35 human adults with type-2 diabetes mellitus (T2DM) in a 14-wk randomised controlled pilot trial comprising three 2×20 g supplemental protein/day arms: KDP-whey (KDPWHE), whey (WHEY), non-protein isocaloric control (CON), with standardised exercise. Outcomes were measured morning fasted and following insulin-stimulation (80 mU/m2/min hyperinsulinaemic-isoglycaemic clamp). With KDPWHE supplementation there was good and very-good evidence for moderate-sized increases in insulin-stimulated glucose clearance rate (GCR; 26%; 90% confidence limits, CL 2%, 49%) and skeletal-muscle microvascular blood flow (46%; 16%, 83%), respectively, and good evidence for increased insulin-stimulated sarcoplasmic GLUT4 translocation (18%; 0%, 39%) vs CON. In contrast, WHEY did not effect GCR (-2%; -25%, 21%) and attenuated HbA1c lowering (14%; 5%, 24%) vs CON. KDPWHE effects on basal glutathione in erythrocytes and skeletal muscle were unclear, but in muscle there was very-good evidence for large increases in oxidised peroxiredoxin isoform 2 (oxiPRX2) (19%; 2.2%, 35%) and good evidence for lower GPx1 concentrations (-40%; -4.3%, -63%) vs CON; insulin stimulation, however, attenuated the basal oxiPRX2 response (4%; -16%, 24%), and increased GPx1 (39%; -5%, 101%) and SOD1 (26%; -3%, 60%) protein expression. Effects of KDPWHE on oxiPRX3 and NRF2 content, phosphorylation of capillary eNOS and insulin-signalling proteins upstream of GLUT4 translocation AktSer437 and AS160Thr642 were inconclusive, but there was good evidence for increased IRSSer312 (41%; 3%, 95%), insulin-stimulated NFκB-DNA binding (46%; 3.4%, 105%), and basal PAK-1Thr423/2Thr402 phosphorylation (143%; 66%, 257%) vs WHEY. Our findings provide good evidence to suggest that dietary supplementation with a novel edible keratin protein in humans with T2DM may increase glucose clearance and modify skeletal-muscle tissue redox and insulin sensitivity within systems involving peroxiredoxins, antioxidant expression, and glucose uptake.

The role of oxidants and vitamin C on neutrophil apoptosis and clearance.

We have investigated the role of neutrophil oxidants in the surface changes that result in recognition and uptake of neutrophils by macrophages. We have shown that H2O2 produced by stimulated neutrophils is essential for the surface expression of phosphatidylserine. This does not occur in neutrophils from mice with chronic granulomatous disease and may explain the formation of granuloma in this condition. We have also investigated the role of intracellular vitamin C on neutrophil apoptosis. Cells from vitamin C-deficient mice were found to be less likely to undergo both spontaneous and oxidant-induced apoptosis, with eventual necrosis being the most probable outcome.

Regulation of apoptosis by vitamin C. Specific protection of the apoptotic machinery against exposure to chlorinated oxidants.

We have investigated the ability of intracellular vitamin C to protect human umbilical vein endothelial cells from exposure to hypochlorous acid (HOCl) and a range of derived chloramines. Ascorbate provided minimal protection against the cytotoxicity induced by these oxidants, as measured by propidium iodide uptake. In contrast, there was a marked effect on apoptosis, monitored by caspase-3 activation and phosphatidylserine exposure. Extended incubation of the cells with glycine chloramine or histamine chloramine completely blocked apoptosis initiated in the cells by serum withdrawal. This effect was significantly abrogated by ascorbate. Inhibition of apoptosis required the oxidant to be present for an extended period after serum withdrawal and occurred prior to caspase-3 activation. General protection of thiols by ascorbate was not responsible for the protection of apoptosis, because intracellular oxidation by HOCl or chloramines was not prevented in supplemented cells. The results suggest a new role for vitamin C in the regulation of apoptosis. We propose that, by protection of an oxidant-sensitive step in the initiation phase, ascorbate allows apoptosis to proceed in endothelial cells under sustained oxidative stress.

Hypochlorous acid causes caspase activation and apoptosis or growth arrest in human endothelial cells.

We have investigated the effect of hypochlorous acid (HOCl) on cultured human umbilical-vein endothelial cells and shown that, whereas higher concentrations cause rapid necrosis, smaller amounts of this oxidant induce apoptosis or growth arrest. Exposure to 20-40 nmol of HOCl per 1.2x10(5) cells initiated apoptosis that was determined morphologically, by the identification of apoptotic nuclei with Hoechst 33342, and by detection of phosphatidylserine on the outer membrane. Degraded DNA was detected by flow cytometry. HOCl induced caspase activity; specific inhibition of caspases was shown to prevent apoptosis. No caspase activation could be detected with 50 nmol of HOCl per 1.2x10(5) cells, a dose that caused more extensive necrosis. Lower doses of HOCl, which did not cause cell death, resulted in a transient growth arrest that was apparent with as little as 5 nmol of HOCl per 1.2x10(5) cells. These results show that HOCl can modify cellular responses that are dependent on signal transduction pathways in a manner similar to that of other oxidants.

Physician assistants and nurse practitioners: rural-urban settings and reimbursement for services.

Evidence based on productivity measures, salaries and costs of medical education indicates that physician assistants and nurse practitioners are cost-effective. Managed care suggests that health maintenance organizations (HMOs) would seek to utilize these professionals. Moreover, underserved rural areas would utilize physician assistants and nurse practitioners to provide access. This study examined the role of payment sources in the utilization of physician assistants and nurse practitioners using the 1994 National Hospital Ambulatory Medical Care Survey (NHAMCS) conducted by the National Center for Health Statistics, U.S. Centers for Disease Control and Prevention. Rural vs. urban results were compared. The study found that significant rural-urban differences exist in the relationships between payment sources and the utilization of physician assistants and nurse practitioners. The study also found that payment source affects varied for physicians, physician assistants and nurse practitioners who saw outpatients in hospital settings. Surprisingly, prepaid and HMO types of reimbursements are shown to have no relationship with physician assistant and nurse practitioner utilization, and this finding is the same for both rural and urban patient visits. After controlling for other influences, the study shows that physicians, physician assistants and nurse practitioners are each as likely as the other to be present at a rural managed care visit. However, physicians are much more likely than physician assistants and nurse practitioners to be present at an urban managed care visit.

Caspase involvement in the induction of apoptosis by the environmental toxicants tributyltin and triphenyltin.

Organotin compounds such as tributyltin (TBT) and triphenyltin (TPT) can kill target cells by triggering apoptosis. The mechanism by which these environmental toxicants activate the apoptotic program is currently unclear. We have studied the effect of TBT and TPT in the human Hut-78 and Jurkat T-lymphocyte cell lines. Within 1 h there was a 30-fold increase in caspase activity, as measured by the cleavage of the fluorescent peptide DEVD-AMC. Morphological changes characteristic of apoptosis, such as membrane blebbing and nuclear fragmentation, were readily detectable. Blocking caspase activity with the peptide inhibitor z-VAD-fmk prevented all subsequent apoptotic changes. The optimal concentration range for induction of apoptosis was 0.5 to 5 microM TBT. TPT was also able to trigger caspase activity in the lymphocyte cell lines, but it took over 2 h to detect and occurred at a lower concentration range of 0.01 to 1 microM. Higher concentrations of TBT and TPT caused cell necrosis, and we showed that these concentrations were able to inhibit caspase activity in apoptotic cells. TBT and TPT were able interact with a vicinal thiol compound, similar to the known caspase inhibitor phenylarsine oxide, providing a potential mechanism for caspase inhibition. We propose that vicinal thiol proteins may be a general biological target of these organotin compounds, leading to the induction of caspase activity and apoptosis at low concentrations, and more extensive cell damage and necrotic cell death at higher concentrations.

Methods for quantifying phagocytosis and bacterial killing by human neutrophils.

This paper reviews a variety of methods available for quantifying phagocytosis and bacterial killing by neutrophils. We outline the advantages and disadvantages of each technique, with the selection of a technique for research or analytical purposes being dependent on the information required and the resources available. A detailed protocol is provided for a comprehensive microbiological technique that measures both phagocytosis and killing in a single assay, along with a kinetic analysis for measuring and calculating separate rate constants for the two events. The kinetic analysis can be easily adapted to other methods to give the same quantitative information.

Involvement of caspases in neutrophil apoptosis: regulation by reactive oxygen species.

Human neutrophils have a short half-life and are believed to die by apoptosis or programmed cell death both in vivo and in vitro. We found that caspases are activated in a time-dependent manner in neutrophils undergoing spontaneous apoptosis, concomitant with other characteristic features of apoptotic cell death such as morphologic changes, phosphatidylserine (PS) exposure, and DNA fragmentation. The treatment of neutrophils with agonistic anti-Fas monoclonal antibodies (MoAbs) significantly accelerated this process. However, in cells treated with the potent neutrophil activator phorbol 12-myristate 13-acetate (PMA), caspase activity was only evident after pharmacologic inhibition of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Similarily, inhibition of the NADPH oxidase in constitutive and Fas/APO-1-triggered apoptosis resulted in increased rather than suppressed levels of caspase activity, suggesting that reactive oxygen species may prevent caspases from functioning optimally in these cells. Moreover, oxidants generated via the NADPH oxidase were essential for PS exposure during PMA-induced cell death, but not for neutrophils undergoing spontaneous apoptosis. We conclude that caspases are an important component of constitutive and Fas/APO-1-triggered neutrophil apoptosis. However, these redox sensitive enzymes are suppressed in activated neutrophils, and an alternate oxidant-dependent pathway is used to mediate PS exposure and neutrophil clearance under these conditions.

Redox regulation of apoptotic cell death.

Apoptosis is a special form of cell death, which can be triggered by a variety of signals and pathophysiological conditions, including oxidative stress. Activation of members of the caspase family of cysteine proteases is a crucial event in the apoptotic death program. Being cysteine proteases, the caspases are sensitive to the redox status of the cell, and their activity is blocked by excessive oxidative stress. Thus, alterations of intracellular redox status may either trigger or block the apoptotic death program, depending on the severity of the oxidative stress.

Activation of NF-kappaB in human neutrophils during phagocytosis of bacteria independently of oxidant generation.

We have exposed human neutrophils to opsonized Staphylococcus aureus and used an electrophoretic mobility shift assay to show activation of the transcription factor NF-kappaB above basal levels. Activation was evident within 10 min and was increased with higher bacteria:neutrophil ratios. The neutrophil NADPH oxidase inhibitor diphenylene iodonium, catalase, and other oxidant scavengers did not inhibit NF-kappaB activation, and no activation was seen with added hydrogen peroxide. Oxidants produced during phagocytosis, therefore, are not involved in the activation mechanism.

Cytochrome c release and caspase activation in hydrogen peroxide- and tributyltin-induced apoptosis.

The ability of H2O2 and tributyltin (TBT) to trigger pro-caspase activation via export of cytochrome c from mitochondria to the cytoplasm was investigated. Treatment of Jurkat T lymphocytes with H2O2 resulted in the appearance of cytochrome c in the cytosol within 2 h. This was at least 1 h before caspase activation was observed. TBT caused cytochrome c release already after 5 min, followed by caspase activation within 1 h. Measurement of mitochondrial membrane potential (delta psi(m)) showed that both H2O2 and TBT dissipated delta psi(m), but with different time courses. TBT caused a concomitant loss of delta psi(m) and release of cytochrome c, whereas cytochrome c release and caspase activation preceded any apparent delta psi(m) loss in H2O2-treated cells. Thus, our results suggest that different mechanisms are involved in triggering cytochrome c release with these apoptosis-inducing agents.

Importance of the redox state of cytochrome c during caspase activation in cytosolic extracts.

The export of cytochrome c from mitochondria to the cytoplasm has been detected during apoptosis. Addition of cytochrome c to cytosolic extracts can activate the caspases, suggesting that this export could be an important intracellular signal for initiating the apoptotic programme. We have investigated the mechanism of caspase activation by cytochrome c. Mitochondrial cytochrome c normally shuttles electrons between complexes III and IV of the electron transport chain. Interaction with these complexes is dependent on electrostatic interactions via a polylysine binding pocket. Cytosolic caspase activation was only observed with intact holocytochrome c, and increasing the ionic composition of the extracts prevented activation, suggesting that stringent allosteric interactions between cytochrome c and other cytoplasmic factors are necessary. Cytochrome c was fully reduced within 5 min of addition to the cytosolic extracts. Potassium ferricyanide could maintain cytochrome c in an oxidized state, but care was taken to use ferricyanide at concentrations where its polyanion effect did not cause interference. The oxidized form of cytochrome c was able to activate the caspases. We conclude that reduced cytochrome c will function in the cytoplasm; however, its reduction is not a critical step, and electron transfer from cytochrome c to its cytoplasmic-binding partner(s) is not necessary in the pathway leading to apoptosis.

Redox regulation of apoptotic cell death in the immune system.

Apoptosis is a special form of cell death, which can be triggered by a variety of signals and pathophysiological conditions, including oxidative stress. Activation of members of the caspase family of cysteine proteases is a crucial event in the apoptotic death program. Being cysteine proteases, the caspases are sensitive to the redox status of the cell, and their activity is blocked by excessive oxidative stress. Thus, alterations of intracellular redox status may either trigger or block the apoptotic death program, depending on the severity of the oxidative stress.

Redox regulation of the caspases during apoptosis.

Apoptosis is now widely recognized as being a distinct process of importance both in normal physiology and pathology. In the current paradigm for apoptotic cell death, the activity of a family of proteases, caspases, related to interleukin-1 beta-converting enzyme (ICE) orchestrates the multiple downstream events, such as cell shrinkage, membrane blebbing, glutathione (GSH) efflux, and chromatin degradation that constitute apoptosis. Recent studies suggest that mitochondria could be the principle sensor and that the release of mitochondrial factors, such as cytochrome c, is the critical event governing the fate of the cell.--One of the most reproducible inducers of apoptosis is mild oxidative stress, although it is unclear how an oxidative stimulus can activate the caspase cascade. Oxidative modification of proteins and lipids has also been observed in cells undergoing apoptosis in response to nonoxidative stimuli, suggesting that intracellular oxidation may be a general feature of the effector phase of apoptosis. The caspases themselves are cysteine-dependent enzymes and, as such, appear to be redox sensitive. Indeed, our recent work on hydrogen peroxide-mediated apoptosis suggests that prolonged or excessive oxidative stress can actually prevent caspase activation. A physiological example of this is the NADPH oxidase-derived oxidants generated by stimulated neutrophils that prevent caspase activation in these cells. Pursuant to these findings, stimulated neutrophils appear to use a specialized caspase-independent pathway to initiate phosphatidylserine (PS) exposure and subsequent phagocytic clearance. The possible implications of these dual roles for reactive oxygen species in apoptosis, that is, induction and inhibition of caspases, are discussed in the present review.

The number of times attended as a factor in the success of a comprehensive hospital-based approach to health promotion.

This study assesses the relationship between a perceived change in a health-related behavior or attitude and the number of times an individual participates in a weekly hospital-based health promotion program. A survey was distributed to participants in April 1995. A univariate probit evaluation shows that the number of times attended and age have positive effects on the likelihood of change. As the number of times an individual attends increases, the more likely the person is to change, possibly revealing a reinforcement mechanism. It is important to learn the most effective way to raise attendance so the reinforcement process reaches more individuals.

Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis.

The induction of apoptosis in Jurkat T-lymphocytes with 50 microM hydrogen peroxide was associated with caspase activation. Caspase activity was first detected 3 h after treatment, and the morphological features of apoptosis were apparent by 6 h. At higher concentrations of hydrogen peroxide there was no detectable caspase activity, and the cells died by necrosis. Cells treated with hydrogen peroxide were impaired in their ability to undergo Fas-mediated apoptosis. This appeared to be the result of direct inhibition of the cysteine-dependent caspases. The cells were able to recover and undergo apoptosis at later times. Therefore, hydrogen peroxide has two distinct effects. It initially inhibits the caspases and delays apoptosis. Then, depending on the degree of the initial oxidative stress, the caspases are activated and the cells die by apoptosis, or they remain inactive and necrosis occurs. We discuss the physiological implications of cells having to maintain a reducing environment during apoptosis to allow the caspases to function.