Factors influencing heart rate variability in patients with severe aortic valve disease

Background and hypothesis: Heart rate variability (HRV) is an accepted tool for the assessment of cardiovascular autonomic tone. There are no sufficient data concerning its application to patients with severe aortic valve disease (AVD) requiring cardiac surgery. Methods: It was the aim of this study to examine HRV and its physiologic correlates in patients with severe aortic valve disease requiring cardiac surgery. The correlates of time domain indices of HRV obtained from 24-h Holter electrocardiographic recordings were analyzed in 36 consecutive patients (23 men and 13 women, mean age 62 ± 11 years) with AVD prior to cardiac surgery (aortic stenosis: 17 patients, aortic valve regurgitation: 3 patients, combined aortic valve disease: 16 patients). Results: Low values of HRV were found in the entire study group: SDNN 96.8 ± 30.9 ms, SDNNI 39.3 ± 14.4 ms, SDANN 86 ± 28.9 ms, and RMSSD 30 ± 18.1 ms. In a univariate analysis, there was no significant correlation between the time domain measures of HRV and age, gender, medication, left ventricular ejection fraction, peak aortic pressure gradient, fraction of aortic valve regurgitation, and left ventricular mass assessed by echocardiography. Patients in advanced functional classes of heart failure [New York Heart Association (NYHA) III or IV] had significantly lower values for SDNN (83.8 ± 33.6 vs. 107.3 ± 24.7 ms; p<0.05) and SDANN (72.7 ± 29.4 vs. 96.6 ± 24.3 ms; p<0.05) than patients in NYHA class I or II. Reassessment of HRV 1 week after aortic valve replacement was performed in 17 patients and showed a significant further decrease of SDNN (102.4 ± 29.7 vs. 61.5 ± 23.5 ms; p<0.001), SDNNI (40.7 ± 13.6 vs. 23.4 ± 12.4 ms; p<0.001) and SDANN (91.8 ±29.2 vs. 54.2 ± 22.8 ms;p<0.001). Conclusion: Patients with AVD requiring cardiac surgery reveal reduced time domain indices of HRV. This observation is pronounced in patients with a progressed clinical class of heart failure, whereas hemodynamic and echocardiographic parameters seem to have no significant influence on HRV parameters in this population. In addition, there is evidence of a further reduction of HRV time domain indices 1 week after uncomplicated aortic valve replacement.     

Azacitidine in 302 patients with WHO-defined acute myeloid leukemia: results from the Austrian Azacitidine Registry of the AGMT-Study Group

Data on efficacy and safety of azacitidine in acute myeloid leukemia (AML) with >30 % bone marrow (BM) blasts are limited, and the drug can only be used off-label in these patients. We previously reported on the efficacy and safety of azacitidine in 155 AML patients treated within the Austrian Azacitidine Registry (clinicaltrials.gov identifier NCT01595295). We herein update this report with a population almost twice as large (n?=?302). This cohort included 172 patients with >30 % BM blasts; 93 % would have been excluded from the pivotal AZA-001 trial (which led to European Medicines Agency (EMA) approval of azacitidine for high-risk myelodysplastic syndromes (MDS) and AML with 20–30 % BM blasts). Despite this much more unfavorable profile, results are encouraging: overall response rate was 48 % in the total cohort and 72 % in patients evaluable according to MDS-IWG-2006 response criteria, respectively. Median OS was 9.6 (95 % CI 8.53–10.7)?months. A clinically relevant OS benefit was observed with any form of disease stabilization (marrow stable disease (8.1 months), hematologic improvement (HI) (9.7 months), or the combination thereof (18.9 months)), as compared to patients without response and/or without disease stabilization (3.2 months). Age, white blood cell count, and BM blast count at start of therapy did not influence OS. The baseline factors LDH >225 U/l, ECOG ≥2, comorbidities ≥3, monosomal karyotype, and prior disease-modifying drugs, as well as the response-related factors hematologic improvement and further deepening of response after first response, were significant independent predictors of OS in multivariate analysis. Azacitidine seems effective in WHO-AML, including patients with >30 % BM blasts (currently off-label use). Although currently not regarded as standard form of response assessment in AML, disease stabilization and/or HI should be considered sufficient response to continue treatment with azacitidine.     

Covalent agonists for studying G protein-coupled receptor activation

Structural studies on G protein-coupled receptors (GPCRs) provide important insights into the architecture and function of these important drug targets. However, the crystallization of GPCRs in active states is particularly challenging, requiring the formation of stable and conformationally homogeneous ligand-receptor complexes. Native hormones, neurotransmitters, and synthetic agonists that bind with low affinity are ineffective at stabilizing an active state for crystallogenesis. To promote structural studies on the pharmacologically highly relevant class of aminergic GPCRs, we here present the development of covalently binding molecular tools activating G_s-, G_i-, and G_q-coupled receptors. The covalent agonists are derived from the monoamine neurotransmitters noradrenaline, dopamine, serotonin, and histamine, and they were accessed using a general and versatile synthetic strategy. We demonstrate that the tool compounds presented herein display an efficient covalent binding mode and that the respective covalent ligand-receptor complexes activate G proteins comparable to the natural neurotransmitters. A crystal structure of the β₂-adrenoreceptor in complex with a covalent noradrenaline analog and a conformationally selective antibody (nanobody) verified that these agonists can be used to facilitate crystallogenesis.One of the major obstacles to the investigation of the structural basis of G protein-coupled receptor (GPCR) activation is the flexibility of their seven-transmembrane core, particularly in the active state (1), and the resulting biochemical instability of the solubilized protein (2, 3). Protein crystallography, the most powerful tool for the study of GPCR structure, requires the formation of stable and conformationally homogeneous ligand-receptor complexes (4). High-affinity agonists with dissociation constants in the low to subnanomolar range and low off-rates facilitate stabilization of the protein throughout the process of expression, purification, and crystallogenesis (2); however, endogenous neurotransmitters usually show poor binding affinity. Low binding affinity with rapid association and dissociation rates leads to conformational heterogeneity that prevents the formation of diffraction-quality crystals. The rapid dissociation rate of agonists also makes it difficult to generate active-state stabilizing proteins, such as the camelid antibodies (nanobodies) that have been used to obtain active-state structures of the β₂-adrenergic receptor (β₂AR) (5) and M2 muscarinic receptor (6).To prevent ligand dissociation, irreversible ligation of electrophilic moieties like halomethylketones, isothiocyanates, Michael acceptors, or aziridinium groups of small-molecule ligands with a suitably positioned nucleophilic residue in the receptor has been used (7–16). However, irreversible ligands often suffer from incomplete cross-linking (15) and reduced receptor activation when covalent binding leads to loss of agonist efficacy (10, 16). Furthermore, their highly electrophilic nature and the abundance of nucleophilic groups in biological systems may lead to a low coupling selectivity (7, 8).Disulfide-based cross-linking approaches (17, 18) offer the advantage that the covalent binding of disulfide-containing compounds is chemoselective for cysteine and enforced by the affinity of the ligand-pharmacophore rather than by the electrophilicity of the cross-linking function (19). We refer to the described ligands as covalent rather than irreversible agonists because cleavage may be promoted by reducing agents and the disulfide transfer process is a reversible chemical reaction in general.Structural information on the target protein facilitates the development of covalent ligand-receptor pairs. Mutation of H93^2.64 in the β₂AR to cysteine introduced an anchor for the disulfide-based covalent agonist FAUC50, which does not perturb ligand binding or the activation of the receptor, and thus enabled, to our knowledge, the first agonist-bound GPCR structure (20). Taking advantage of the high structural homology among aminergic GPCRs, we reasoned that the introduction of cysteine into position X^2.64 should also result in a covalently binding receptor mutant for other aminergic GPCRs.We here report a methodology to generate disulfide-based covalent ligand-receptor pairs to promote structural and functional studies on GPCRs. We demonstrate that even the low-affinity endogenous agonists noradrenaline, dopamine, and serotonin can be converted into efficient covalently binding molecular tools for the β₂AR, the dopamine D₂ receptor (D₂R), and the 5-hydroxytryptamine 2A (5-HT_2A) serotonergic subtype representing G_s-, G_i-, and G_q-coupled GPCRs, respectively. Analogous studies were conducted starting from histamine and the receptor subtype H₁. We applied this strategy to obtain an active-state crystal structure of the β₂AR^H93C and a covalent (nor)adrenaline analog.     

Antigen affinity and antigen dose exert distinct influences on CD4 T-cell differentiation

Cumulative T-cell receptor signal strength and ensuing T-cell responses are affected by both antigen affinity and antigen dose. Here we examined the distinct contributions of these parameters to CD4 T-cell differentiation during infection. We found that high antigen affinity positively correlates with T helper (Th)1 differentiation at both high and low doses of antigen. In contrast, follicular helper T cell (T_FH) effectors are generated after priming with high, intermediate, and low affinity ligand. Unexpectedly, memory T cells generated after priming with very low affinity antigen remain impaired in their ability to generate secondary Th1 effectors, despite being recalled with high affinity antigen. These data challenge the view that only strongly stimulated CD4 T cells are capable of differentiating into the T_FH and memory T-cell compartments and reveal that differential strength of stimulation during primary T-cell activation imprints unique and long lasting T-cell differentiation programs.Following infection, T-cell receptor (TCR) interactions with foreign peptide/MHC (pMHC) drive the rapid clonal expansion and differentiation of T cells into distinct effector subsets specialized against different classes of microbes. An early bifurcation in CD4 T-cell responses results in the generation of T helper (Th)1 effectors, which regulate innate cell microbicidal function and follicular helper T (T_FH) cells, which migrate to B-cell follicles to regulate germinal center (GC) responses and antimicrobial antibody production (1). After pathogen is cleared, T cells undergo a contraction phase during which the majority of effectors die by apoptosis, leaving behind a population of long-lived memory cells to provide protection upon subsequent reinfection. The decision to differentiate into Th1 and T_FH lineages appears to occur very early in the immune response (2, 3). Initial T-cell priming by dendritic cells (DCs) is sufficient to induce fate-committed Th1 and T_FH cells as early as 3 d after infection, whereas maintenance and further expansion of the T_FH compartment depends on T-cell interactions with B cells (2). Similarly, memory T-cell differentiation occurs very early after infection and is critically dependent on B-cell interactions for optimal priming (4, 5). Importantly, CD4 T-cell differentiation is coupled to division, and unlike CD8 T-cell differentiation, requires constant antigen recognition (6, 7).Although the strength of TCR–pMHC interactions has been shown to directly modulate T-cell expansion and clonal dominance within the Th cell compartment (8, 9), how this influences CD4 T-cell fate is not well understood. Cumulative TCR signaling can be influenced by both antigen affinity and antigen dose (10). In terms of proliferation, higher antigen dose can compensate for lower antigen affinity to some extent, but several reports have shown independent effects on T-cell responses both in vitro and in vivo (10–12). These data indicate that antigen affinity and antigen dose may promote qualitatively distinct TCR signals. Recently, modulation of the overall TCR signal by varying either TCR affinity or antigen dose was shown to influence the pattern of effector T-cell differentiation, with higher affinity ligands or higher antigen dose promoting T_FH generation (13–15). However, another study examining high and low avidity CD4 T-cell responses during viral infection found significant differences in Th1 but not T_FH generation (16). Sustained TCR–pMHC interactions have also been shown to promote memory T-cell differentiation, which is associated with increased TCR avidity (17, 18). These studies, however, have focused on the development of the Th1 memory compartment, which is phenotypically and functionally distinct from the T_FH memory compartment (19, 20). Thus, although strong TCR signals resulting from high antigen affinity or high antigen dose can clearly affect the extent and quality of T-cell differentiation, whether or not T cells can discriminate these signals, and how this contributes to T-cell differentiation during infection, has not been determined.To address this question, we infected mice with varying concentrations of Listeria expressing either high or low affinity antigens for the TCR. By normalizing the degree of proliferation induced by high and low affinity antigens we were able to discern distinct influences of antigen affinity and antigen dose on Th cell differentiation. We observed a strong positive correlation between antigen affinity and Th1 differentiation that occurs early and is dose independent. Importantly, high antigen dose does not compensate for the low efficiency of Th1 differentiation induced by low affinity antigen. In contrast, early T_FH effector generation was observed after priming with high, intermediate, and low affinity antigen, but was not maintained at later time points under conditions of low antigen dose. In addition, we found that T cells activated by either high or low affinity antigen are equally capable of memory T-cell differentiation. Surprisingly, memory T cells generated by either low antigen affinity or low antigen dose maintained their biased effector lineages following recall activation with high affinity antigen. These data indicate that differential strength of stimulation during primary T-cell activation can imprint unique and long lasting T-cell differentiation programs.     

Structural and Mechanical Repair of Diffuse Damage in Cortical Bone In Vivo

Physiological wear and tear causes bone microdamage at several hierarchical levels, and these have different biological consequences. Bone remodeling is widely held to be the mechanism by which bone microdamage is repaired. However, recent studies showed that unlike typical linear microcracks, small crack damage, the clusters of submicron‐sized matrix cracks also known as diffuse damage (Dif.Dx), does not activate remodeling. Thus, the fate of diffuse damage in vivo is not known. To examine this, we induced selectively Dif.Dx in rat ulnae in vivo by using end‐load ulnar bending creep model. Changes in damage content were assessed by histomorphometry and mechanical testing immediately after loading (ie, acute loaded) or at 14 days after damage induction (ie, survival ulnae). Dif.Dx area was markedly reduced over the 14‐day survival period after loading (p < 0.02). We did not observe any intracortical resorption, and there was no increase in cortical bone area in survival ulnae. The reduction in whole bone stiffness in acute loaded ulnae was restored to baseline levels in survival ulnae (p > 0.6). Microindentation studies showed that Dif.Dx caused a highly localized reduction in elastic modulus in diffuse damage regions of the ulnar cortex. Moduli in these previously damaged bone areas were restored to control values by 14 days after loading. Our current findings indicate that small crack damage in bone can be repaired without bone remodeling, and they suggest that alternative repair mechanisms exist in bone to deal with submicron‐sized matrix cracks. Those mechanisms are currently unknown and further investigations are needed to elucidate the mechanisms by which this direct repair occurs. © 2014 American Society for Bone and Mineral Research     

Warmed,humidified carbon dioxide insufflation versus standard carbon dioxide in laparoscopic cholecystectomy: a double-blinded randomized controlled trial

Background

During laparoscopic cholecystectomy (LCHE), the insufflation with warmed and humidified carbon dioxide (CO₂) may reduce postoperative pain. The aim of the study was to evaluate the positive effects of heated and humidified carbon dioxide gas on patients with regard to postoperative pain after LCHE.

Patients and methods

This is a prospective, randomized, double-blinded, controlled clinical trial. 148 patients (female = 98, male = 50) scheduled for elective LCHE were randomized into two groups: receiving either heated humidified carbon dioxide, or standard gas. Intraoperative core temperature was measured. The perioperative management was identical for both groups. Postoperative pain intensity was assessed using a visual analog pain scale, and the amount of analgesic consumption was recorded. The postoperative pain management was also standardized and equal for both groups.

Results

67 out of 148 received standard gas (group A), and 81 received warmed, humidified gas (group B). The groups were comparable demographically. The amount of analgesic consumption was recorded. Intraoperative core temperature was significant higher in group B than in group A. Pain was significantly less in group B (p = 0.025) 6 h postoperatively. On the first postoperative day, no significant difference in pain between the two groups was detectable (p = 0.437).

Conclusion

The use of warmed and humidified carbon dioxide during LCHE reduces postoperative pain at the day of operation.