Liver frailty and all-cause mortality in the older participants of the Salus in Apulia Study

The liver contribution to the biological network underlying physical frailty in aging is underestimated. How best to measure this contribution magnitude and impact on health risk trajectories in frail individuals is not yet entirely clear. We analyzed the association of a novel liver frailty phenotype with the risk of death in older participants of the Salus in Apulia Study cohort. Clinical and physical examination, routine biomarkers, medical history, and anthropometry were analyzed in 1929 older adults (65?+). Physical frailty was classified by Cardiovascular Health Study criteria, and liver fibrosis risk by fibrosis-4 (FIB-4). The liver frailty phenotype was defined as physical frailty plus high-risk liver fibrosis (score?>?2.67). Physical frailty, high-risk liver fibrosis, and liver frailty subjects were compared to subjects without these conditions (non-frail). Proportional Cox regression tested the adjusted association between liver frailty and all-cause mortality for each category. The liver frailty prevalence was relatively low (3.8%), but higher in men (58.1%). Compared to non-frail older subjects, liver frailty subjects were significantly older (effect size (ES)???1.11, 95% confidence interval (CI)???1.35 to???0.87), with a lower education (ES 0.48, 95%CI 0.24 to 0.71) and higher multimorbidity (ES 15.81, 95%CI 4.20 to 27.41). Cox multivariate analyses showed a two-fold increased risk of overall mortality (hazard ratio 2.09, 95%CI 1.16–3.74) even after the adjustment for age, sex, education, and alcohol consumption. The liver frailty phenotype runs twice the risk of overall mortality compared with the non-frail population. This clinical tool, validated in a Southern Italian population, is based on simple sets of measures that can conveniently be assessed also in the primary care setting.

     

Mobilization of early hematopoietic progenitor cells with BB-10010: a genetically engineered variant of human macrophage inflammatory protein- 1 alpha

BB-10010 is a genetically engineered variant of human macrophage inflammatory protein-1 alpha with improved solution properties. We show here that it mobilizes stem cells into the peripheral blood. We investigated the mobilizing effects of BB-10010 on the numbers of circulating 8-day spleen colony-forming units (CFU-S8), CFU-S12, and progenitors with marrow repopulating ability (MRA). A single subcutaneous dose of BB-10010 caused a twofold increase in circulating numbers of CFU-S8, CFU-S12, and MRA 30 minutes after dosing. We also investigated the effects of granulocyte colony-stimulating factor (G- CSF) and the combination of G-CSF with BB-10010 on progenitor mobilization. Two days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA progenitors by 25.7-, 19.8-, and 27.7-fold. A single administration of BB-10010 after 2 days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA even further to 38-, 33-, and 100- fold. Splenectomy resulted in increased circulating progenitor numbers but did not change the pattern of mobilization. Two days of treatment with G-CSF then increased circulating CFU-S8, CFU-S12, and MRA by 64-, 69-, and 32-fold. A single BB-10010 administration after G-CSF treatment further increased them to 85-, 117-, and 140-fold, respectively, compared with control. We conclude that BB-10010 causes a rapid increase in the number of circulating hematopoietic progenitors and further enhances the numbers induced by pretreatment with G-CSF. BB- 10010 preferentially mobilized the more primitive progenitors with marrow repopulating activity, releasing four times the number achieved with G-CSF alone. Translated into a clinical setting, this improvement in progenitor cell mobilization may enhance the efficiency of harvest and the quality of grafts for peripheral blood stem cell transplantation.     

Time of catheter removal in candidemia and mortality

Background

The impact of central venous catheter (CVC) removal on the outcome of patients with candidemia is controversial, with studies reporting discrepant results depending on the time of CVC removal (early or any time during the course of candidemia).

Invasive fungal diseases in haematopoietic cell transplant recipients and in patients with acute myeloid leukaemia or myelodysplasia in Brazil

Invasive fungal disease (IFD) shows distinct regional incidence patterns and epidemiological features depending on the geographic region. We conducted a prospective survey in eight centres in Brazil from May 2007 to July 2009. All haematopoietic cell transplant (HCT) recipients and patients with acute myeloid leukaemia (AML) or myelodysplasia (MDS) were followed from admission until 1 year (HCT) or end of consolidation therapy (AML/MDS). The 12-month cumulative incidence (CI) of proven or probable IFD was calculated, and curves were compared using the Grey test. Among 237 AML/MDS patients and 700 HCT recipients (378 allogeneic, 322 autologous), the 1-year CI of IFD in AML/MDS, allogeneic HCT and autologous HCT was 18.7%, 11.3% and 1.9% (p <0.001), respectively. Fusariosis (23 episodes), aspergillosis (20 episodes) and candidiasis (11 episodes) were the most frequent IFD. The 1-year CI of aspergillosis and fusariosis in AML/MDS, allogeneic HCT and autologous HCT were 13.4%, 2.3% and 0% (p <0.001), and 5.2%, 3.8% and 0.6% (p 0.01), respectively. The 6-week probability of survival was 53%, and was lower in cases of fusariosis (41%). We observed a high burden of IFD and a high incidence and mortality for fusariosis in this first multicentre epidemiological study of IFD in haematological patients in Brazil.     

Role of cavities and hydration in the pressure unfolding of T4 lysozyme

It is well known that high hydrostatic pressures can induce the unfolding of proteins. The physical underpinnings of this phenomenon have been investigated extensively but remain controversial. Changes in solvation energetics have been commonly proposed as a driving force for pressure-induced unfolding. Recently, the elimination of void volumes in the native folded state has been argued to be the principal determinant. Here we use the cavity-containing L99A mutant of T₄ lysozyme to examine the pressure-induced destabilization of this multidomain protein by using solution NMR spectroscopy. The cavity-containing C-terminal domain completely unfolds at moderate pressures, whereas the N-terminal domain remains largely structured to pressures as high as 2.5 kbar. The sensitivity to pressure is suppressed by the binding of benzene to the hydrophobic cavity. These results contrast to the pseudo-WT protein, which has a residual cavity volume very similar to that of the L99A–benzene complex but shows extensive subglobal reorganizations with pressure. Encapsulation of the L99A mutant in the aqueous nanoscale core of a reverse micelle is used to examine the hydration of the hydrophobic cavity. The confined space effect of encapsulation suppresses the pressure-induced unfolding transition and allows observation of the filling of the cavity with water at elevated pressures. This indicates that hydration of the hydrophobic cavity is more energetically unfavorable than global unfolding. Overall, these observations point to a range of cooperativity and energetics within the T₄ lysozyme molecule and illuminate the fact that small changes in physical parameters can significantly alter the pressure sensitivity of proteins.The destabilization of proteins by pressure is a fundamental and highly informative probe of their structural free energy landscape but remains inadequately understood (1). The underlying determinants of pressure-induced unfolding have recently been a subject of several detailed investigations (2–10). Fundamentally, pressure-induced unfolding of proteins results from the population of nonnative conformations having a lower total system volume than the native structure seen at ambient pressure. Various mechanisms for pressure-induced unfolding have been proposed including changes in water structure that weaken the hydrophobic effect at high pressure (11, 12), increases in solvent density at the protein surface that contribute to a reduction in the total volume of the protein–water system (13, 14), and the elimination of cavities in the protein interior through exposure to solvent (3). With the development of high-pressure sample cells compatible with modern solution NMR probes (15), detailed measurements of proteins unfolding under pressure with atomic resolution have now become possible (5, 16–18). Recent studies of staphylococcal nuclease (SNase) compellingly argue that the filling of void volumes present in the native state is the primary determinant of pressure-induced unfolding (4–6). A critical aspect of a “destruction of voids” mechanism for pressure-induced unfolding of proteins is whether the voids or cavities are occupied with water in the folded state. Early investigations of buried hydrophobic pockets indicated that even large cavities are typically not hydrated, whereas hydrophilic cavities generally are occupied by water (19, 20). Many of the key studies impacting this question used the L99A single-point mutant of the model enzyme T₄ lysozyme (20).The L99A mutation creates an internal cavity with an estimated volume of ∼150–160 ų, large enough to accommodate three or four water molecules (21) (Fig. 1). Crystallographic investigation found no electron density within this pocket at ambient pressure (22, 23). In contrast, solution NMR and molecular-dynamics simulations suggest that the region of the protein around the hydrophobic pocket is highly dynamic, possibly to the extent that the pocket may be transiently accessible to solvent (22, 24–27). Crystallographic studies conducted at high pressure conversely suggested that the region around the pocket is rigid and exhibits increasing rigidity with increased pressure (23). Electron density also increased within the cavity as the hydrostatic pressure was increased (22), consistent with a pressure-induced filling of the hydrophobic cavity with water molecules. In contrast, fluorescence and small-angle X-ray scattering studies in bulk solution demonstrated that the protein is unfolded at these elevated pressures (2), suggesting that the crystal packing effects stabilize the protein. The hydrophobic cavity also provides a general, moderate-affinity binding site for small, relatively nonpolar ligands (28).Open in a separate windowFig. 1.Hydration of T₄ lysozyme L99A at ambient pressure (∼1 bar). A backbone ribbon representation of L99A [Protein Data Bank (PDB) ID code 1L90 (63)] is shown with the N-terminal domain (residues 13–65) illustrated in blue, and the C-terminal domain (residues 1–12 and 66–164) is colored green. The hydrophobic pocket created by the L99A mutation is shown as orange mesh, and the three tryptophan side chains are shown as stick representations. The helices are numbered as a reference for discussion in the text. Cyan spheres are shown at the positions of amide hydrogens where an NOE to the water resonance was detected. Yellow spheres indicate the positions of amide hydrogens within NOE distance (5 Å) of the interior of the hydrophobic pocket, but outside NOE distance to the protein surface. These are the sites where detection of NOEs to the water resonance would indicate hydration of the pocket. No NOE cross-peaks from these sites to the water resonance were observed, suggesting that the pocket is not hydrated at ambient pressure.T₄ lysozyme is one of the smallest known proteins to contain more than one cooperative folding unit. The folding of WT T₄ lysozyme has been examined in detail by using hydrogen–deuterium exchange approaches and has been shown to contain two domains that fold cooperatively and with distinct free energy profiles (29–32). The N-terminal domain is ∼6 kcal/mol less stable than the C-terminal domain. The cavity created by the L99A mutation is in the center of the C-terminal domain. The thermal stability of the L99A mutant is reduced compared with the WT protein by 16 °C (5 kcal/mol) (33), an effect that is partially abrogated by binding hydrophobic ligands to the cavity (28, 34).The L99A mutant of T₄ lysozyme provides a unique system to examine the hydration of internal pockets and the details of pressure-induced unfolding. In principle, protein–water interactions can be characterized by solution NMR methods (35), but severe artifacts often render the approach quite limited (36). Recently, it has been shown that various advantageous properties of proteins and water encapsulated within reverse micelles largely overcome these artifacts (37, 38). Here, we use this approach to directly measure the hydration of the internal cavity. High-pressure NMR is used to examine the pressure-induced response of the protein in bulk solution and under confinement by the reverse micelle. We demonstrate that the hydrophobic pocket appears to be essentially dehydrated at ambient pressure (∼1 bar) and that the pressure response of the protein is an unfolding of the C-terminal domain only, representing an inversion of the relative stability of the domains as a result of the cavity-creating mutation. This result is in contrast to the unfolding of the cysteine-free WT (WT*) protein, which shows only the earliest stages of pressure-induced subglobal unfolding. Furthermore, the L99A mutant with benzene occupying the cavity shows no evidence of pressure unfolding. Nanoscale confinement of the protein also suppresses the L99A pressure-induced unfolding transition (P_u) as a result of the restriction of conformational space imposed by the reverse micelle. In lieu of the pressure unfolding transition, the volume reduction imposed by increasing pressure is compensated for in the reverse micelle by progressively increasing incorporation of water into the cavity interior, essentially recapitulating the observations from high-pressure crystallography in a solution measurement. These findings have important implications with respect to the nature of pressure-induced unfolding, the roles of cavities in protein structural stability, and the effects of confinement, a critical parameter when considering the intracellular milieu, in which proteins must fold and carry out their functions.     

Risk Factors Associated With Early Invasive Pulmonary Aspergillosis in Kidney Transplant Recipients: Results From a Multinational Matched Case–Control Study

Risk factors for invasive pulmonary aspergillosis (IPA) after kidney transplantation have been poorly explored. We performed a multinational case–control study that included 51 kidney transplant (KT) recipients diagnosed with early (first 180 posttransplant days) IPA at 19 institutions between 2000 and 2013. Control recipients were matched (1:1 ratio) by center and date of transplantation. Overall mortality among cases was 60.8%, and 25.0% of living recipients experienced graft loss. Pretransplant diagnosis of chronic pulmonary obstructive disease (COPD; odds ratio [OR]: 9.96; 95% confidence interval [CI]: 1.09–90.58; p = 0.041) and delayed graft function (OR: 3.40; 95% CI: 1.08–10.73; p = 0.037) were identified as independent risk factors for IPA among those variables already available in the immediate peritransplant period. The development of bloodstream infection (OR: 18.76; 95% CI: 1.04–339.37; p = 0.047) and acute graft rejection (OR: 40.73, 95% CI: 3.63–456.98; p = 0.003) within the 3 mo prior to the diagnosis of IPA acted as risk factors during the subsequent period. In conclusion, pretransplant COPD, impaired graft function and the occurrence of serious posttransplant infections may be useful to identify KT recipients at the highest risk of early IPA. Future studies should explore the potential benefit of antimold prophylaxis in this group.     

Structural imaging of conjunctival filtering blebs in XEN gel implantation and trabeculectomy: a confocal and anterior segment optical coherence tomography study

Graefe's Archive for Clinical and Experimental Ophthalmology - To describe and compare the conjunctival filtering bleb features after XEN gel implantation and trabeculectomy using anterior...