Remote ischaemic preconditioning modifies serum apolipoprotein D,met‐enkephalin,adenosine, and nitric oxide in healthy young adults

Remote ischaemic preconditioning (RIPC) has been employed as a non‐invasive protective intervention against myocardial ischaemia–reperfusion injury in animal studies. However, the underlying mechanisms are incompletely defined in humans and its clinical efficacy has been inconclusive. As advanced age, disease, and drugs may confound RIPC mechanisms in patients, our aim is to measure whether RIPC evokes release of adenosine, bradykinin, met‐enkephalin, nitric oxide, and apolipoproteins in healthy young adults. Healthy subjects (n = 18, 9 males, 23 ± 1.5 years old; 9 females, 23 ± 1.8 years old) participated after informed consent. RIPC was applied using a blood pressure cuff to the dominant arms for four cycles of 5‐minute cuff inflation (ischaemia) and 5‐minute cuff deflation (reperfusion). Blood was sampled at baseline and immediately after the final cuff deflation (Post‐RIPC). Baseline and Post‐RIPC plasma levels of adenosine, bradykinin, met‐enkephalin, apolipoprotein A‐1 (ApoA‐1), apolipoprotein D (ApoD), and nitric oxide (as nitrite) were measured via ELISA and high‐performance liquid chromatography. Mean (±SD) baseline levels of adenosine, bradykinin, met‐enkephalin, ApoA‐1, ApoD, and nitrite in healthy young adults were 13.8 ± 6.5 ng/mL, 2.6 ± 1.9 μg/mL, 594.1 ± 197.4 pg/mL, 3.0 ± 0.7 mg/mL, 22.2 ± 4.0 μg/mL, and 49.8 ± 13.4 nmol/L, respectively. Post‐RIPC adenosine and nitrite levels increased (59.5 ± 37.9%, P < .0001; 32.2 ± 19.5%, P < .0001), whereas met‐enkephalin and ApoD levels marginally decreased (5.3 ± 14.0%, P = .04; 10.8 ± 20.5%, P = .04). Post‐RIPC levels were not influenced by sex, age, blood pressure, waist circumference, or BMI. RIPC produces increased levels of adenosine and nitrites, and decreased met‐enkephalin and ApoD in the plasma of young healthy adults.     

CK2 activates kinesin via induction of a conformational change

Kinesin is the canonical plus-end microtubule motor and has been the focus of intense study since its discovery in 1985. We previously demonstrated a time-dependent inactivation of kinesin in vitro that was fully reversible by the addition of purified casein kinase 2 (CK2) and showed that this inactivation/reactivation pathway was relevant in cells. Here we show that kinesin inactivation results from a conformational change that causes the neck linker to be positioned closer to the motor domain. Furthermore, we show that treatment of kinesin with CK2 prevents and reverses this repositioning. Finally, we demonstrate that CK2 treatment facilitates ADP dissociation from the motor, resulting in a nucleotide-free state that promotes microtubule binding. Thus, we propose that kinesin inactivation results from neck-linker repositioning and that CK2-mediated reactivation results from CK2’s dual ability to reverse this repositioning and to promote ADP release.Intracellular microtubule-based transport is crucial for the creation and maintenance of cellular order, and altered transport is implicated in both neurodegeneration and cancer. Frequently, in vivo cargos are moved by multiple microtubule-based molecular motors (1–6), and changing the number of active motors on the cargo can change cargo force production (4) and also potentially the mean travel distance for predominantly unidirectionally moving cargos (7). However, until recently, it has been unclear how activity of cargo-bound motors might be regulated.Transport is frequently regulated by signaling cascades [see, e.g., cAMP control of pigment granule transport (8) or APP transport (9)]. Thus, multiple signaling pathways might contribute to control of transport under different conditions, and signaling altered in disease might affect transport, which could then contribute to disease progression. Nonetheless, mechanistic understanding of such effects is limited. For these reasons, we would like to understand transport roles of specific disease-relevant kinases. One such kinase is casein kinase 2 (CK2), which is involved in development (10), is up-regulated in various cancers (11), and is decreased in neurodegeneration (12). We found that, over time, kinesin loses its ability to bind microtubules (becomes “inactive”) and that this loss of activity could be reversed by CK2 (13).Mechanistically, how kinesin became inactive—and what CK2 did to reactivate it—was unknown. Here we discover that kinesin’s inactivation results from a conformational change involving repositioning of the neck linker (NL) and that reactivation reverses this conformational change. Intriguingly, the conformational change that results in reactivation causes release of ADP, converting kinesin from a weak microtubule-interacting state (K⋅ADP) to a strong one (K), so that in some ways CK2 acts like a small G-protein nucleotide-exchange factor.     

IL-25 and type 2 innate lymphoid cells induce pulmonary fibrosis

Disease conditions associated with pulmonary fibrosis are progressive and have a poor long-term prognosis with irreversible changes in airway architecture leading to marked morbidity and mortalities. Using murine models we demonstrate a role for interleukin (IL)-25 in the generation of pulmonary fibrosis. Mechanistically, we identify IL-13 release from type 2 innate lymphoid cells (ILC2) as sufficient to drive collagen deposition in the lungs of challenged mice and suggest this as a potential mechanism through which IL-25 is acting. Additionally, we demonstrate that in human idiopathic pulmonary fibrosis there is increased pulmonary expression of IL-25 and also observe a population ILC2 in the lungs of idiopathic pulmonary fibrosis patients. Collectively, we present an innate mechanism for the generation of pulmonary fibrosis, via IL-25 and ILC2, that occurs independently of T-cell–mediated antigen-specific immune responses. These results suggest the potential of therapeutically targeting IL-25 and ILC2 for the treatment of human fibrotic diseases.Disease conditions associated with pulmonary fibrosis are often progressive and have a poor long-term prognosis (1). In the context of developing new treatments for pulmonary fibrosis, the cytokines associated with the pathogenic milieu that can lead to aberrant extracellular matrix deposition are key targets, in particular interleukin (IL)-13, TGF-β, and, more recently, IL-17A (2). However, to develop more effective therapeutics for fibrotic lung diseases a greater understanding of the pathogenesis and the underlying mechanisms that lead to pulmonary fibrosis is needed (3, 4).The cytokine IL-13 was first implicated in fibrosis using profibrotic eggs from the type 2 cytokine-inducing pathogen Schistosoma mansoni, in the presence of a soluble IL-13Rα2-Fc fusion protein (5) and in Il13^−/− mice (6). IL-13 is now widely linked to a range of fibrotic conditions (7) including asthma, where IL-13 is being targeted as a therapy (8). In the context of the cellular source of IL-13 in the generation of fibrosis, CD4⁺ T helper (h) 2 cells are implicated (9). However, more recently innate lymphoid cells (ILC) are emerging as an important source of IL-13 (10, 11). In this context, the type 2 cytokine IL-25 is implicated in the generation of the recently identified IL-13–expressing ILC, termed ILC2 (11–14).Recent studies have implicated IL-25 and ILC2 in the pathogenesis of pulmonary conditions in both murine models and human conditions such as allergic asthma (12, 13, 15, 16). In murine studies intranasal administration of IL-25 results in evidence of pulmonary tissue remodeling including development of perivascular fibrosis, and intratracheal administration results in increased pulmonary Th2 cytokines and airways hyper-reactivity (AHR) (17, 18), whereas blocking IL-25 reduces AHR severity (19). Herein we describe a potential role for IL-25 in the generation of pulmonary fibrosis in experimental mouse models, via the activation of IL-13–producing ILC2. We also observe increases in both IL-25 and ILC2 in the lung of patients with idiopathic pulmonary fibrosis (IPF). These data suggest unique mechanisms for the generation of pulmonary fibrosis and identify an interesting area for further research on the role of IL-25 and ILC2 in the treatment of pulmonary fibrosis.     

Tolvaptan for Volume Management in Heart Failure

Volume management in acute decompensated and chronic heart failure (HF) remains a significant challenge. Although progress has been made in the development of mortality-reducing neurohormonal regimens in the reduced ejection fraction population, no clinical trial has yet demonstrated anything more than symptomatic relief or biomarker reduction with pharmacotherapeutic volume-based interventions made in the acutely decompensated individual or those with evolving outpatient congestion. As the number of patients with HF continues to grow, in addition to HF-related hospitalizations, identifying therapies that have the potential to aid in diuresis more safely and efficaciously is paramount to decreasing inpatient length of stay and preventing unnecessary admissions. More recently, a significant amount of research has been dedicated to the use of vasopressin antagonists, specifically tolvaptan, as adjunctive therapy to loop and thiazide diuretics. Although these agents do not seem to have a pervasive role in fluid management in the acute decompensated and chronic HF populations, they are effective tools to have available for specific clinical situations. This review summarizes the literature surrounding the use of tolvaptan for volume management in congestive HF, as well as offering practical guidance for use of this agent.     

JAK2V617F-positive endothelial cells contribute to clotting abnormalities in myeloproliferative neoplasms

The Janus kinase 2 (JAK2) V₆₁₇F mutation is the primary pathogenic mutation in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Although thrombohemorrhagic incidents are the most common causes of morbidity and mortality in patients with MPNs, the events causing these clotting abnormalities remain unclear. To identify the cells responsible for the dysfunctional hemostasis, we used transgenic mice expressing JAK2V₆₁₇F in specific lineages involved in thrombosis and hemostasis. When JAK2V₆₁₇F was expressed in both hematopoietic and endothelial cells (ECs), the mice developed a significant MPN, characterized by thrombocytosis, neutrophilia, and splenomegaly. However, despite having significantly higher platelet counts than controls, these mice showed severely attenuated thrombosis following injury. Interestingly, platelet activation and aggregation in response to agonists was unaltered by JAK2V₆₁₇F expression. Subsequent bone marrow transplants revealed the contribution of both endothelial and hematopoietic compartments to the attenuated thrombosis. Furthermore, we identified a potential mechanism for this phenotype through JAK2V₆₁₇F-regulated inhibition of von Willebrand factor (VWF) function and/or secretion. JAK2V₆₁₇F⁺ mice display a condition similar to acquired von Willebrand syndrome, exhibiting significantly less high molecular weight VWF and reduced agglutination to ristocetin. These findings greatly advance our understanding of thrombohemorrhagic events in MPNs and highlight the critical role of ECs in the pathology of hematopoietic malignancies.Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders, characterized by significant increases in one or more myeloid-cell lineages. Mutations in the Janus kinase 2 (JAK2) and MPL genes are common in the majority of Philadelphia chromosome-negative (Ph⁻) MPNs, which include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). By far the most frequent mutation in MPNs is JAK2V₆₁₇F (1–4), which occurs in the highly conserved autoinhibitory JAK homology (JH) 2 domain, causing hyperactive kinase activity and hyperproliferation of myeloid progenitor cells, leading to overproduction of red blood cells (RBCs), platelets, and leukocytes. Although ET and PV have a relatively benign clinical course, patients’ life expectancy can be severely reduced by bleeding or thrombosis, the manifestations of which are significantly more common than other MPN-related complications such as myelofibrosis and acute leukemic transformation (5). The frequency and nature of thrombotic and hemorrhagic events vary greatly depending on disease phenotype and patient history. Data taken from a number of previous studies indicate that the probability of major thrombosis ranges between 8–29% (ET) and 11–39% (PV) whereas the incidence of bleeding at initial presentation is less frequent than thrombosis, ranging between 3–18% (ET) and 3–8% (PV) (6–8).A number of abnormalities that could potentially contribute to this prothrombotic phenotype have been identified in the blood and vascular cells of JAK2V₆₁₇F⁺ MPN patients. Much work has focused on defining platelet abnormalities, including increased expression of membrane proteins such as P-selectin and tissue factor (TF), which would prime platelets for activation and increase levels of platelet-activation markers and platelet factor 4 (PF4) in the plasma (9–12). Interestingly, however, aggregation studies show a decreased response to ADP and epinephrine in platelets isolated from patients with ET and PV compared with controls (10). Furthermore, no correlation has been made between severity of thrombocytosis in ET patients and increased risk of thrombosis (6, 13). In contrast, extreme thrombocytosis (platelets >1,500 × 10⁹/L) is thought to contribute to a hemorrhagic phenotype in ET patients, and is commonly attributed to the development of acquired von Willebrand syndrome (AVWS) (11, 12, 14), where the increased platelets bind to highly prothrombotic, ultralarge von Willebrand factor (VWF) multimers, removing them from the plasma (15).Recent studies suggest that leukocytosis is a potential thrombotic risk factor in young PV and ET patients, possibly through the interactions of leukocytes, especially neutrophils, with platelets and endothelial cells (ECs) (16, 17) or the production of prothrombotic molecules such as TF. Increased basal activation of neutrophils has been shown in PV and ET patients, including elevated expression of CD11b and levels of neutrophil proteases in the plasma, both of which are prothrombotic (9, 18, 19). Studies have also shown increased activation of vascular ECs in JAK2V₆₁₇F⁺ MPN patients. Increased P- and E-selectin levels in the plasma, coupled with decreased levels of nitric oxide (NO), could conceivably contribute to a prothrombotic phenotype. Furthermore, JAK2V₆₁₇F⁺ ECs have recently been reported in a subpopulation of MPN patients, and EC expression was coupled with an increased risk of thrombosis (20, 21). Taken together, previous studies describe physiological abnormalities in a number of cell types in JAK2V₆₁₇F⁺ MPN patients, all of which could contribute toward increased thrombosis and/or bleeding. However, these data are often contradictory and fail to definitively explain the mechanism/s responsible for the development of thrombohemorrhagic disease.Here, we used FF1 transgenic mice (22) to express human JAK2V₆₁₇F in specific lineages to determine which cells are responsible for the thrombohemorrhagic manifestations seen in patients with MPNs. FF1 mice were crossed with Pf4-Cre or Tie2-Cre mice to express JAK2V₆₁₇F specifically in platelets alone, or in hematopoietic cells and ECs, respectively (23–28). These models have provided us with an unparalleled opportunity to determine the specific role/s of JAK2V₆₁₇F in pathological thrombosis and hemostasis.     

Phase I/II multisite trial of optimally dosed clofarabine and low-dose TBI for hematopoietic cell transplantation in acute myeloid leukemia

Clofarabine is an immunosuppressive purine nucleoside analog that may have better anti-leukemic activity than fludarabine. We performed a prospective phase I/II multisite trial of clofarabine with 2 Gy total body irradiation as non-myeloablative conditioning for allogeneic hematopoietic cell transplantation in adults with acute myeloid leukemia who were unfit for more intense regimens. Our main objective was to improve the 6-month relapse rate following non-myeloablative conditioning, while maintaining historic rates of non-relapse mortality (NRM) and engraftment. Forty-four patients, 53 to 74 (median: 69) years, were treated with clofarabine at 150 to 250 mg/m², of whom 36 were treated at the maximum protocol-specified dose. One patient developed multifactorial acute kidney injury and another developed multiorgan failure, but no other grade 3 to 5 non-hematologic toxicities were observed. All patients fully engrafted. The 6-month relapse rate was 16% (95% CI, 5%-27%) among all patients and 14% (95% CI, 3%-26%) among high-risk patients treated at the maximum dose, meeting the pre-specified primary efficacy endpoint. Overall survival was 55% (95% CI, 40%-70%) and leukemia-free survival was 52% (95% CI, 37%-67%) at 2 years. Compared to a historical high-risk cohort treated with the combination of fludarabine at 90 mg/m² and 2 Gy TBI, protocol patients treated with the clofarabine-TBI regimen had lower rates of overall mortality (HR of 0.50, 95% CI, 0.28-0.91), disease progression or death (HR 0.48, 95% CI, 0.27-0.85), and morphologic relapse (HR 0.30, 95% CI, 0.13-0.69), and comparable NRM (HR 0.85, 95% CI 0.36-2.00). The combination of clofarabine with TBI warrants further investigation in patients with high-risk AML.