Attenuated induction of epithelial and leukocyte serine antiproteases elafin and secretory leukocyte protease inhibitor in Crohn's disease

Elafin (or skin-derived antileukoprotease) and secretory leukocyte protease inhibitor (SLPI) are serine antiproteases antagonizing human neutrophil elastase (HNE), thereby preventing tissue injury from excessive release of proteolytic enzymes by inflammatory cells. Furthermore, elafin and SLPI are "defensin-like" molecules with broad antimicrobial activity. The balance between proteases and antagonists may critically determine inflammatory processes in Crohn's disease (CD) and ulcerative colitis (UC). Real-time PCR was performed to quantitate colonic, proinflammatory cytokine IL-8, protease (HNE), and antiprotease mRNA (elafin and SLPI) in a total of 340 biopsies from 117 patients (47 CD, 45 UC, 25 controls). Histological inflammation was scored, and HNE, elafin, and SLPI were localized and semiquantified by immunostaining in 51 colonic paraffin sections (23 CD, 11 UC, 17 controls). Proinflammatory IL-8, degree of histological inflammation, and granulocyte content were similar in UC and CD. Elafin stained predominantly in the epithelium and SLPI in mucosal inflammatory cells. HNE mRNA levels and immunostaining were increased equally in both forms of inflammatory bowel disease. Levels of mRNA and immunostaining of the antiproteases elafin and SLPI were enhanced strongly in inflamed versus noninflamed UC. It is surprising that comparing inflamed versus noninflamed CD, this increase was significantly less pronounced for elafin and even lacking for SLPI. Despite comparable degrees of inflammation and protease levels, the induction of both antiproteases was attenuated in CD. This could contribute to the transmural depth of tissue destruction in CD. Elafin and SLPI may be added to the list of defensin-like peptides with diminished induction in CD versus UC.     

Microbiome and chronic inflammatory bowel diseases

It is nowadays generally accepted that the microbiome is a central driver of chronic inflammatory bowel diseases based on observations from human patients as well as inflammatory rodent models. Many studies focussed on different aspects of microbiota and some scientists believe that a primary dis-balance results in a direct microbial induced inflammatory situation. It is also clear that the microbiome is influenced by environmental and genetic factors and is also tightly regulated by host defense molecules such as antimicrobial peptides (defensins et al.). Different lines of investigations showed different complex antimicrobial barrier defects in inflammatory bowel diseases which also influence the composition of the microbiome and generally impact on the microbial-mucosal interface. In this review, we aim to discuss the bigger picture of these different aspects and current views and conclude about therapeutic consequences for future concepts beyond anti-inflammatory treatment.     

ADP-stimulated contraction: A predictor of thin-filament activation in cardiac disease

Diastolic dysfunction is general to all idiopathic dilated (IDCM) and hypertrophic cardiomyopathy (HCM) patients. Relaxation deficits may result from increased actin–myosin formation during diastole due to altered tropomyosin position, which blocks myosin binding to actin in the absence of Ca²⁺. We investigated whether ADP-stimulated force development (without Ca²⁺) can be used to reveal changes in actin–myosin blockade in human cardiomyopathy cardiomyocytes. Cardiac samples from HCM patients, harboring thick-filament (MYH7_mut, MYBPC3_mut) and thin-filament (TNNT2_mut, TNNI3_mut) mutations, and IDCM were compared with sarcomere mutation-negative HCM (HCM_smn) and nonfailing donors. Myofilament ADP sensitivity was higher in IDCM and HCM compared with donors, whereas it was lower for MYBPC3. Increased ADP sensitivity in IDCM, HCM_smn, and MYH7_mut was caused by low phosphorylation of myofilament proteins, as it was normalized to donors by protein kinase A (PKA) treatment. Troponin exchange experiments in a TNNT2_mut sample corrected the abnormal actin–myosin blockade. In MYBPC3_trunc samples, ADP sensitivity highly correlated with cardiac myosin-binding protein-C (cMyBP-C) protein level. Incubation of cardiomyocytes with cMyBP-C antibody against the actin-binding N-terminal region reduced ADP sensitivity, indicative of cMyBP-C’s role in actin–myosin regulation. In the presence of Ca²⁺, ADP increased myofilament force development and sarcomere stiffness. Enhanced sarcomere stiffness in sarcomere mutation-positive HCM samples was irrespective of the phosphorylation background. In conclusion, ADP-stimulated contraction can be used as a tool to study how protein phosphorylation and mutant proteins alter accessibility of myosin binding on actin. In the presence of Ca²⁺, pathologic [ADP] and low PKA-phosphorylation, high actin–myosin formation could contribute to the impaired myocardial relaxation observed in cardiomyopathies.Heart failure (HF) is a syndrome clinically defined as the inability of the heart to sufficiently supply blood to organs and tissues (1). Systolic dysfunction is present in approximately one-half of the HF population, whereas diastolic dysfunction is a common feature in almost all HF patients (2). Moreover, in hypertrophic cardiomyopathy (HCM), which is caused by mutations in genes encoding thin- and thick-filament proteins, impaired diastolic function is frequently observed (3). Impaired relaxation of the heart may be caused by high myofilament Ca²⁺ sensitivity. This increased sensitivity for Ca²⁺ would result in residual myofilament activation at diastolic [Ca²⁺], which may delay the onset of ventricular relaxation and limit proper filling of the heart. High myofilament Ca²⁺ sensitivity has been observed in both acquired and genetic forms of cardiomyopathy (3, 4). In human idiopathic dilated cardiomyopathy (IDCM), high myofilament Ca²⁺ sensitivity has been associated with reduced β-adrenergic receptor-mediated phosphorylation by protein kinase A (PKA) (4). Reduced PKA phosphorylation of cardiac troponin I (cTnI) and cardiac myosin-binding protein C (cMyBP-C) increases myofilament Ca²⁺ sensitivity (5–8). Likewise, high myofilament Ca²⁺ sensitivity is a common characteristic of HCM and may be caused by the mutant protein or by reduced PKA-mediated protein phosphorylation secondary to HCM disease progression (3, 9).Contractile performance of the heart muscle may thus be perturbed by mutation-induced and phosphorylation-mediated protein changes that affect thin-filament transitions. Ca²⁺-induced cardiac muscle contraction is tightly modulated by the troponin–tropomyosin complex that regulates the interactions between the actin thin filament and myosin thick filament (i.e., cross-bridge formation). Accordingly, the myofilaments oscillate between three transitions termed the blocked (B-state), closed (C-state), and open (M-state) states of thin-filament regulation that represent the distinct position of tropomyosin on actin (10–12) (Fig. 1). In the absence of Ca²⁺ (B state), tropomyosin sterically blocks the myosin-binding sites on actin (Fig. 1A). Upon electrical activation of cardiomyocytes, the rise of cytosolic [Ca²⁺] alters the conformation of the troponin–tropomyosin complex, which moves tropomyosin on actin and exposes myosin-binding sites (C state). Weakly bound cross-bridges (myosin-ADP-Pi) populate the C state (10, 12) (Fig. 1B). Transition to the M state involves release of inorganic phosphate (Pi) from the cross-bridge and strong-binding cross-bridge formation (myosin-ADP) that induces additional movement of tropomyosin, resulting in myofilament contraction and sliding (Fig. 1C).Open in a separate windowFig. 1.Three-state model of thin-filament activation. Seven actin monomers (circles), spanned by one tropomyosin dimer (red strand), together with the troponin complex (not depicted) comprise one functional unit (A₇TmTn). Two functional units are depicted, and individual myosins are shown as triangles (weak, weak-binding cross-bridges; strong, strong-binding cross-bridges). (A) B state (blocked); when ATP is present and cytoplasmic [Ca²⁺] is low and is not bound to cardiac troponin C (cTnC), tropomyosin is sterically blocking the myosin-binding sites on actin. (B) C state (Ca²⁺-induced); upon rise in cytoplasmic [Ca²⁺], Ca²⁺ binds to cTnC, inducing conformational changes of the troponin complex, resulting in a ∼25° movement of tropomyosin on the thin filament, thereby exposing myosin-binding sites on actin. In the C state, the myofilament is not yet activated as non–tension-generating cross-bridges bind weakly to actin. (C) M state (myosin induced); the strong binding of tension-generating cross-bridges induces a ∼10° movement of tropomyosin on actin, resulting in myofilament activation and contraction.The three-state model of cross-bridge interaction implies that the main task of Ca²⁺ is to uncover myosin-binding sites on actin and that formation of myosin-ADP represents the main regulator of force development and contraction. Notably, solution (10) and cryo-electron microscopy (13) studies have shown that in the absence of Ca²⁺ the myofilaments are not entirely blocked, as ∼5% of the thin filaments have tropomyosin localized in the C-state position. This observation suggests that conditions that promote myosin-ADP formation can trigger myofilament contraction in Ca²⁺-free conditions and thereby impair relaxation. Indeed, in membrane-permeabilized rabbit skeletal muscle fibers (14), bovine myocardium (15, 16) and human cardiac muscle (17) millimolar levels of ADP stimulate force development in the absence of Ca²⁺.Because ADP-stimulated contraction is due to myosin-ADP binding to the nonblocked sites of the thin filament in the absence of Ca²⁺, it provides an experimental tool to assess changes in tropomyosin’s position in acquired and genetic cardiomyopathies in which altered protein phosphorylation and mutant proteins may alter myofilament activation. In addition, it could represent a pathomechanism underlying the diastolic dysfunction seen in both disease states. Solution studies with mutant troponin proteins, which are known to cause HCM, showed a reduction in the B state at low-Ca²⁺ conditions compared with wild-type troponin proteins (18, 19). Mutation-induced irregularities in troponin–tropomyosin interactions disrupt the B state and shift the thin filament to the C state, increasing the available myosin-binding sites on actin.In addition to Ca²⁺-induced changes of the thin filament, tropomyosin location may also be altered by the thick-filament protein cMyBP-C. Recent evidence supports that the N-terminal extension of cMyBP-C binds the low-Ca²⁺–state (B-state) position of tropomyosin on actin and interferes with tropomyosin–actin interactions, dislocating tropomyosin into the C-state position (i.e., the presence of cMyBP-C sensitizes the thin filament to Ca²⁺) (20, 21). Because it was previously shown that in Ca²⁺-free conditions (B state) ∼5% of the thin filaments (lacking cMyBP-C) have tropomyosin localized in the C-state position (10), more myofilaments may be in the C state in the presence of cMyBP-C. We (22) and others (23) have shown that cMyBP-C mutations, which are a major cause of HCM, have a reduced level of healthy cMyBP-C protein compared with nonfailing hearts (i.e., haploinsufficiency), which may alter tropomyosin position on the thin filament.To verify whether ADP-stimulated contraction provides an experimental tool to assess mutation-induced and phosphorylation-mediated changes in thin-filament transitions, which precede Ca²⁺ activation of myofilaments, we tested the following hypotheses: (i) that IDCM and HCM samples with thin-filament mutations are more sensitive to ADP, as a result of a higher accessibility of myosin-binding sites on actin, whereas (ii) cMyBP-C haploinsufficient HCM myocardium has a reduced ADP sensitivity (i.e., less cMyBP-C causes reduced displacement of tropomyosin from the B state) compared with cells from nonfailing hearts. To answer our hypotheses, we activated membrane-permeabilized human cardiomyocytes in ADP containing Ca²⁺-free solutions. Cells were isolated from HCM patients with mutations in genes encoding thick-filament (MYH7, MYBPC3) and thin-filament (TNNT2, TNNI3) proteins and patients with IDCM and compared with cells from sarcomere mutation-negative HCM (HCM_smn) and nonfailing donors. Finally, we investigated whether the ADP level as observed in diseased hearts, in the presence of Ca²⁺, increases myofilament force development in cardiomyocytes from human cardiomyopathy hearts.We conclude that, in HCM with thin-filament mutations, tropomyosin’s ability to block myosin-binding sites on actin is reduced. This effect is exacerbated in HCM samples by the low PKA phosphorylation of myofilament proteins, which is also observed in human IDCM. In contrast, cMyBP-C HCM-causing mutations reduce accessibility of myosin for actin. The findings in this study provide evidence that ADP-mediated activation can be used as an experimental tool to reveal mutation- and phosphorylation-mediated changes in tropomyosin location on the thin filament.     

Contralateral hip fractures and other osteoporosis-related fractures in hip fracture patients: incidence and risk factors. An observational cohort study of 1,229 patients

Purpose

To report risk factors, 1-year and overall risk for a contralateral hip and other osteoporosis-related fractures in a hip fracture population.

Methods

An observational study on 1,229 consecutive patients of 50?years and older, who sustained a hip fracture between January 2005 and June 2009. Fractures were scored retrospectively for 2005–2008 and prospectively for 2008–2009. Rates of a contralateral hip and other osteoporosis-related fractures were compared between patients with and without a history of a fracture. Previous fractures, gender, age and ASA classification were analysed as possible risk factors.

Results

The absolute risk for a contralateral hip fracture was 13.8?%, for one or more osteoporosis-related fracture(s) 28.6?%. First-, second- and third-year risk for a second hip fracture was 2, 1 and 0?%. Median (IQR) interval between both hip fractures was 18.5 (26.6) months. One-year incidence of other fractures was 6?%. Only age was a risk factor for a contralateral hip fracture, hazard ratio (HR) 1.02 (1.006–1.042, p?=?0.008). Patients with a history of a fracture (33.1?%) did not have a higher incidence of fractures during follow-up (16.7?%) than patients without fractures in their history (14?%). HR for a contralateral hip fracture for the fracture versus the non-fracture group was 1.29 (0.75–2.23, p?=?0.360).

Conclusion

The absolute risk of a contralateral hip fracture after a hip fracture is 13.8?%, the 1-year risk was 2?%, with a short interval between the 2 hip fractures. Age was a risk factor for sustaining a contralateral hip fracture; a fracture in history was not.     

Long-term outcome of delirium during intensive care unit stay in survivors of critical illness: a prospective cohort study

Introduction

Delirium is associated with impaired outcome, but it is unclear whether this relationship is limited to in-hospital outcomes and whether this relationship is independent of the severity of underlying conditions. The aim of this study was to investigate the association between delirium in the intensive care unit (ICU) and long-term mortality, self-reported health-related quality of life (HRQoL), and self-reported problems with cognitive functioning in survivors of critical illness, taking severity of illness at baseline and throughout ICU stay into account.

Methods

A prospective cohort study was conducted. We included patients who survived an ICU stay of at least a day; exclusions were neurocritical care patients and patients who sustained deep sedation during the entire ICU stay. Delirium was assessed twice daily with the Confusion Assessment Method for the ICU (CAM-ICU) and additionally, patients who received haloperidol were considered delirious. Twelve months after ICU admission, data on mortality were obtained and HRQoL and cognitive functioning were measured with the European Quality of Life – Six dimensions self-classifier (EQ-6D). Regression analyses were used to assess the associations between delirium and the outcome measures adjusted for gender, type of admission, the Acute Physiology And Chronic Health Evaluation IV (APACHE IV) score, and the cumulative Sequential Organ Failure Assessment (SOFA) score throughout ICU stay.

Results

Of 1101 survivors of critical illness, 412 persons (37%) had been delirious during ICU stay, and 198 (18%) died within twelve months. When correcting for confounders, no significant association between delirium and long-term mortality was found (hazard ratio: 1.26; 95% confidence interval (CI) 0.93 to 1.71). In multivariable analysis, delirium was not associated with HRQoL either (regression coefficient: -0.04; 95% CI -0.10 to 0.01). Yet, delirium remained associated with mild and severe problems with cognitive functioning in multivariable analysis (odds ratios: 2.41; 95% CI 1.57 to 3.69 and 3.10; 95% CI 1.10 to 8.74, respectively).

Conclusions

In this group of survivors of critical illness, delirium during ICU stay was not associated with long-term mortality or HRQoL after adjusting for confounding, including severity of illness throughout ICU stay. In contrast, delirium appears to be an independent risk factor for long-term self-reported problems with cognitive functioning.     

Detection of ‘best’ positive end-expiratory pressure derived from electrical impedance tomography parameters during a decremental positive end-expiratory pressure trial

Introduction

This study compares different parameters derived from electrical impedance tomography (EIT) data to define ‘best’ positive end-expiratory pressure (PEEP) during a decremental PEEP trial in mechanically-ventilated patients. ‘Best’ PEEP is regarded as minimal lung collapse and overdistention in order to prevent ventilator-induced lung injury.

Methods

A decremental PEEP trial (from 15 to 0 cm H₂O PEEP in 4 steps) was performed in 12 post-cardiac surgery patients on the ICU. At each PEEP step, EIT measurements were performed and from this data the following were calculated: tidal impedance variation (TIV), regional compliance, ventilation surface area (VSA), center of ventilation (COV), regional ventilation delay (RVD index), global inhomogeneity (GI index), and intratidal gas distribution. From the latter parameter we developed the ITV index as a new homogeneity parameter. The EIT parameters were compared with dynamic compliance and the PaO₂/FiO₂ ratio.

Results

Dynamic compliance and the PaO₂/FiO₂ ratio had the highest value at 10 and 15 cm H₂O PEEP, respectively. TIV, regional compliance and VSA had a maximum value at 5 cm H₂O PEEP for the non-dependent lung region and a maximal value at 15 cm H₂O PEEP for the dependent lung region. GI index showed the lowest value at 10 cm H₂O PEEP, whereas for COV and the RVD index this was at 15 cm H₂O PEEP. The intratidal gas distribution showed an equal contribution of both lung regions at a specific PEEP level in each patient.

Conclusion

In post-cardiac surgery patients, the ITV index was comparable with dynamic compliance to indicate ‘best’ PEEP. The ITV index can visualize the PEEP level at which ventilation of the non-dependent region is diminished, indicating overdistention. Additional studies should test whether application of this specific PEEP level leads to better outcome and also confirm these results in patients with acute respiratory distress syndrome.     

Production of mouse monoclonal antibodies for the analysis of idiotypes in serum of patients with chronic lymphatic leukaemia

In this report a simple procedure for the production of murine monoclonal antibodies (MoAb) against the idiotype of malignant B cells is described. Mice were immunized with lymphoid cells from patients with B-cell chronic lymphocytic leukaemia (B-CLL). After fusion of the spleen cells, hybridoma supernatants were screened for anti-idiotypic MoAb in ELISA with immunoglobulins obtained from tumour-cell lysates, xenohybridomas and patients' sera. The anti-idiotypic MoAb were used to study tumour cells and serum immunoglobulins (Ig) from four different patients with B-CLL. It was found that the serum IgM and IgD in one patient shared the same idiotype. Evidence is presented that IgG-secreting cell populations are not restricted to lambda-Ig-light chain-expressing B-CLL cells. With the help of anti-idiotype MoAb accurate measurements of total and idiotype-positive serum immunoglobulin levels during chemotherapy were possible.