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Obstructive sleep apnea (OSA) is associated with increased cardiovascular morbidity and mortality. This study systematically reviews the effects of oral appliance therapy (OAT) on a broad spectrum of cardiovascular outcomes. A literature search was performed up to December 31st 2016. Twenty-five relevant full-text articles were retrieved. Sixteen articles were considered methodologically sufficient, including 11 randomized controlled trials. Pooled data of the RCTs showed significant reductions in daytime systolic and diastolic blood pressure compared to baseline, but no significant reductions in heart rate, except for daytime heart rate when compared to inactive/placebo OAT. OAT and continuous positive airway pressure (CPAP) were equally effective in reducing blood pressure. Studies assessing the effect of OAT on heart rate variability, circulating cardiovascular biomarkers, and endothelial function and arterial stiffness, generally involved small numbers of patients, and were heterogeneous and inconclusive. Studies assessing the effect of OAT on cardiac function showed no effects on echocardiographic outcomes. One observational study showed that OAT was as effective as CPAP in reducing cardiovascular death. It could be speculated that OAT may lead to a reduction in long-term cardiovascular morbidity and mortality in OSA patients. However, further methodologically high quality, longitudinal studies are warranted to address this key question.

To investigate underlying mechanisms of thrombocytopenia in myelodysplastic syndrome (MDS), radiolabeled platelet studies were performed in 30 MDS patients with platelets

To investigate whether altered megakaryocyte morphology contributes to reduced platelet production in idiopathic thrombocytopenic purpura (ITP), ultrastructural analysis of megakaryocytes was performed in 11 ITP patients. Ultrastructural abnormalities compatible with (para-)apoptosis were present in 78% ± 14% of ITP megakaryocytes, which could be reversed by in vivo treatment with prednisone and intravenous immunoglobulin. Immunohistochemistry of bone marrow biopsies of ITP patients with extensive apoptosis showed an increased number of megakaryocytes with activated caspase-3 compared with normal (28% ± 4% versus 0%). No difference, however, was observed in the number of bone marrow megakaryocyte colony-forming units (ITP, 118 ± 93/105 bone marrow cells; versus controls, 128 ± 101/105 bone marrow cells; P = .7). To demonstrate that circulating antibodies might affect megakaryocytes, suspension cultures of CD34+ cells were performed with ITP or normal plasma. Morphology compatible with (para-)apoptosis could be induced in cultured megakaryocytes with ITP plasma (2 of 10 samples positive for antiplatelet autoantibodies). Finally, the plasma glycocalicin index, a parameter of platelet and megakaryocyte destruction, was increased in ITP (57 ± 70 versus 0.7 ± 0.2; P = .009) and correlated with the proportion of megakaryocytes showing (para-) apoptotic ultrastructure (P = .02; r = 0.7). In conclusion, most ITP megakaryocytes show ultrastructural features of (para-) apoptosis, probably due to action of factors present in ITP plasma.

Idiopathic thrombocytopenic purpura (ITP) is a heterogeneous disease, whereby it is unclear if and in which way prednisone and splenectomy affect the platelet kinetics leading to a complete remission. To determine the effects of prednisone and splenectomy on the mean platelet life (MPL) and platelet production, platelet kinetic studies with Indium-111 tropolonate-labeled autologous platelets were performed in patients with ITP ( n=41). In 17 patients platelet kinetic studies were performed before and during prednisone treatment, and in 24 patients before and after splenectomy. MPL increased after prednisone therapy only in patients ( n=13) with a full recovery (FR, platelets150 x 10(9)/l) and partial recovery (PR, 50 x 10(9)/lplatelets150 x 10(9)/l) from 2.1+/-1.5 days to 4.9+/-1.3 days in FR patients (p=0.03) and from 1.1+/-0.8 days to 2.4+/-1.1 days in PR patients, which is significantly shorter than in normal controls (9.2+/-1.2, p0.0001). The platelet production demonstrated an impressive increase (threefold) during prednisone treatment in all responding and nonresponding patients. These results suggest that the clinical response to prednisone is more related to the effect on MPL than on platelet production. In contrast, the group of splenectomized patients showed that a full recovery of platelet count was associated with near normalization of the MPL (2.4+/-1.6 days vs 8+/-1.4 days) and platelet production (119+/-60 vs 162+/-35 x 10(9)/day). These results demonstrate that prednisone as well as splenectomy increase MPL and production in patients with ITP. However, only after splenectomy is a complete remission obtained, defined as a normal platelet count, mean platelet life, and platelet production.

Recent studies in erythroid cells have shown that autophagy is an important process for the physiological clearance of mitochondria during terminal differentiation. However, autophagy also plays an important role in removing damaged and dysfunctional mitochondria. Defective mitochondria and impaired erythroid maturation are important characteristics of low-risk myelodysplasia. In this study we therefore questioned whether the autophagic clearance of mitochondria might be altered in erythroblasts from patients with refractory anemia (RA, n=3) and RA with ringed sideroblasts (RARS, n=6). Ultrastructurally, abnormal and iron-laden mitochondria were abundant, especially in RARS patients. A large proportion (52+/-16%) of immature and mature myelodysplastic syndrome (MDS) erythroblasts contained cytoplasmic vacuoles, partly double membraned and positive for lysosomal marker LAMP-2 and mitochondrial markers, findings compatible with autophagic removal of dysfunctional mitochondria. In healthy controls only mature erythroblasts comprised these vacuoles (12+/-3%). These findings were confirmed morphometrically showing an increased vacuolar surface in MDS erythroblasts compared to controls (P

To investigate whether the type of programmed cell death of myelodysplastic erythroid cells depends on their cellular context, we performed studies on cells from patients with low-risk myelodysplastic syndromes. We compared erythroid cells (and their precursor cells) from the mononuclear cell fraction with those from the hematon fraction, which are compacted complexes of hematopoietic cells surrounded by their own micro-environment. In directly fixed materials, erythroblasts exhibited signs of autophagy with limited apoptosis (3%) based on ultrastructural characteristics and immunogold labeling for activated caspase-3. After 24 h in culture, myelodysplastic erythroblasts exhibited a significant increase in apoptosis (22 ± 7% vs. 3 ± 2%, p = 0.001). In contrast, the myelodysplastic erythroblasts from the hematon fraction did not exhibit an increased tendency toward apoptosis after culture (7 ± 3.3% vs. 1.8 ± 2.3%), which was in line with results for normal bone marrow cells. The same dependency on the micro-environment was noted for immature erythroid progenitor cells. Myelodysplastic hematons exhibited distinct numbers of erythroid burst-forming units in association with an extensive network of stromal cells, whereas small numbers of erythroid burst-forming units were generated from the myelodysplastic mononuclear cells compared with normal mononuclear cells (10.2 ± 9 vs. 162 ± 125, p0.001). Co-culture of erythroid myelodysplastic cells in the presence of growth factors (vascular endothelial growth factor, leukemia inhibitory factor) or on the MS-5 stromal layer did not restore the expansion of erythroid precursor cells. These data indicate that surviving myelodysplastic erythroid progenitors become more vulnerable to programmed cell death when they are detached from their own micro-environment.

Platelet production requires compartmentalized caspase activation within megakaryocytes. This eventually results in platelet release in conjunction with apoptosis of the remaining megakaryocyte. Recent studies have indicated that in low-risk myelodysplastic syndromes (MDS) and idiopathic thrombocytopenic purpura (ITP), premature cell death of megakaryocytes may contribute to thrombocytopenia. Different cell death patterns have been identified in megakaryocytes in these disorders. Growing evidence suggests that, besides apoptosis, necrosis and autophagic cell death, may also be programmed. Therefore, programmed cell death (PCD) can be classified in apoptosis, a caspase-dependent process, apoptosis-like, autophagic and necrosis-like PCD, which are predominantly caspase-independent processes. In MDS, megakaryocytes show features of necrosis-like PCD, whereas ITP megakaryocytes demonstrate predominantly characteristics of apoptosis-like PCD ( para-apoptosis). Triggers for these death pathways are largely unknown. In MDS, the interaction of Fas/Fas-ligand might be of importance, whereas in ITP antiplatelet autoantibodies recognizing common antigens on megakaryocytes and platelets might be involved. These findings illustrate that cellular death pathways in megakaryocytes are recruited in both physiological and pathological settings, and that different forms of cell death can occur in the same cell depending on the stimulus and the cellular context. Elucidation of the underlying mechanisms might lead to novel therapeutic interventions.

Platelet kinetic studies in idiopathic thrombocytopenic purpura (ITP) have shown that in a subgroup of patients a shortened mean platelet life (MPL) is associated with a decreased platelet production rate (PPR). Other methods of studying certain aspects of thrombocytopoiesis are the plasma concentrations of thrombopoietin and glycocalicin.

Platelets play an important role in haemostasis and thrombosis. For an understanding of the pathophysiology and treatment of thrombocytopenia, it is not sufficient to measure only the platelet count. Platelet kinetic parameters, such as platelet survival and turnover, might be useful because many thrombocytopenia related disorders result from the interaction between production, utilization or destruction, and sequestration of platelets. Therefore, measuring platelet turnover with radiolabelled platelets could be a sensitive and qualitative tool for clinicians. However, the method does not enjoy widespread use because it has some serious drawbacks, such as the problems associated with the manipulation of blood and platelets, and the use of radioactivity. Recently, other useful assays for measuring platelet fluxes have been described in the literature, including plasma thrombopoietin and glycocalicin. In this review, these new tests will be described, compared with the classical method using radiolabelled platelets, and finally evaluated for their usefulness in clinical practice.

Abstract 2932 Several studies have shown that hematopoietic precursor cells in low-risk myelodysplastic syndromes (MDS) undergo premature cell death. The mode of cell death is cell type specific and might include apoptosis, necrosis, and/or autophagy. Studies in MDS erythroblasts have shown enhanced apoptosis and enhanced autophagy (Houwerzijl EJ et al. Leukemia 2009), while megakaryocytes undergo a caspase-3 independent nonapoptotic cell death (Houwerzijl EJ et al. Blood 2005). Increasing data suggest that in MDS the bone marrow microenvironment is also affected. Some stromal cells, including mesenchymal and circulating endothelial cells, can carry similar chromosomal abnormalities as the neoplastic hematopoietic clone (Della Porta MG et al. Leukemia 2008, Lopez-Villar O et al. Leukemia 2009). In addition, recent data show that hematopoietic and endothelial cells have a common precursor, the hemangioblast (Lancrin C et al. Nature 2009). These findings raise the question whether endothelial cells in MDS also die prematurely. To define in more detail the underlying cell death pathways in MDS endothelial cells and to quantify vascularisation, immunohistochemical staining and electron microscopic analysis were performed on bone marrow samples of patients with refractory anemia (RA, n=6) and RA with ringed sideroblasts (RARS, n=6), and healthy controls (n=4). According to the MDS International Prognostic Scoring System (IPSS) the patients were categorized as low risk (n=6) and intermediate risk-1 (n=6). Immunohistochemistry of the MDS bone marrow biopsies demonstrated increased bone marrow microvessel density (MVD) determined by FVIII staining. In both RA and RARS MVD was increased compared to normal (number of vessels: 4 ± 1.6 and 3.8 ± 1.1/powerfield respectively, versus 0.4 ± 0.5 in healthy controls, magnification × 400). The increased number of endothelial cells stained strongly positive for VEGF in both RA and RARS compared to normal bone marrow. The elevated VEGF expression of these cells might be related to a significantly enhanced expression of hypoxia inducible factor-2α (HIF-2α) compared to normal controls, which was especially found in RARS endothelial cells. In contrast, immunostaining for HIF-1α was negative in MDS patients as well as controls. To expand these results ultrastructural analysis was performed on hematons of a subgroup of these patients (n=10). Hematons are compact spherical particles which contain hematopoietic progenitor cells residing within a finely arborized stromal framework, including adipocytes, mesenchymal cells, resident macrophages and endothelial cells (Blazsek I et al. Blood 2000). Hematons can be isolated from the bone marrow aspirate light density fraction. The ultrastructural analysis revealed irregularly shaped endothelial cells without pericytes and degradation of the basal membrane. Cytoplasmic vacuolization was present in endothelial cells, especially in RARS patients. The majority of these vacuoles were double membraned, a main characteristic of autophagy. Mitochondria were normal. No features of apoptosis were found, which was confirmed by a negative immunostaining for caspase-3 and caspase-8. Microvessels were irregularly shaped and showed frequent sprouting. These results indicate that endothelial cells from low-risk MDS patients are ultrastructurally abnormal, showing features of autophagy, but no apoptosis. Autophagy in these cells may be a type of cell death or a cell-rescue reaction to nutrient- and/or growth factor depletion in the microenvironment. On the other hand, in agreement with previous studies, angiogenesis, in low-risk MDS is increased, and appears to be HIF-2α and not HIF-1α mediated. Together, these findings may suggest that endothelial cells in MDS, similar to hematopoietic cells, show a high rate of cellular proliferation, which coincides with an increased susceptibility for premature cell death, i.e. autophagy. The findings might contribute to knowledge of the defective make-up of the stem cell compartment in MDS which requires a strong interaction between hematopoietic stem cells and the microenvironment. Disclosures: No relevant conflicts of interest to declare.

Anemia in myelodysplasia (MDS) is partially ascribed to enhanced programmed cell death (PCD) of committed erythroid cells in the bone marrow compartment. Especially, enhanced apoptosis has been described. However, nonapoptotic forms of PCD have been demonstrated in MDS megakaryocytes characterized by the absence of chromatin condensation and caspase-3 and -8 activation (Houwerzijl EJ et al. Blood2005;105:3472–3479). Recent studies have indicated that besides apoptosis, necrosis and autophagic cell death can be recognized as PCD, whereby cells are capable to switch between the different types of PCD dependent on their cellular context. To define in more detail the underlying cell death pathways in MDS erythroblasts, immunohistochemical staining and ultrastructural and immunolabeling analysis were performed on bone marrow samples of low-risk MDS patients and normal controls (n=4). Immunohistochemistry of MDS bone marrow biopsies (n=23) demonstrated no positive staining of the erythroblasts for active caspase -3 and -8. To confirm these results ultrastructural analysis and immunoelectron microscopy was performed on mononuclear cells (MNC) and hematons of a subgroup of these patients (n=9). Hematons are compact hematopoietic complexes in which hematopoietic cells, including erythroblasts, are embedded in their own microenvironment. The ultrastructural analysis revealed that only a small fraction of erythroid cells of the MNC and hematon fraction of both MDS patients and healthy controls demonstrated features of apoptosis (2 ± 2% vs 0%). However, 52 ± 16% of immature and mature MDS erythroblasts contained cytoplasmic vacuoles in contrast to normal erythroblasts, in which vacuoles were only shown in the matured stage (12 ± 3%). These vacuoles were partly double-membraned and stained positive for the lysosomal marker LAMP (lysosome associated membrane protein)-2, catalase and the mitochondrial inner membrane protein (immunogold staining) underscoring the presence of autophagy of mitochondria and other cytoplasmic components. Morphometric analysis confirmed that the vacuolar surface in the cytoplasm of MDS erythroblasts was increased compared to controls (P

A 58-year-old man was admitted to the hospital with long-term relapsing fevers, malaise, weight loss, and inflammatory laboratory abnormalities. His medical history revealed abdominal aortic aneurysm repair with a bifurcated prosthetic graft 6 months before presentation. An extensive diagnostic workup, including transesophageal echocardiography, thoracic and abdominal computed tomography (CT) scans (A), and Tc leucocyte scanning, showed no abnormalities—in particular, no signs of endocarditis or graft infection. Because serology for chronic Q fever was positive (anti–Coxiella burnetii immunoglobulin G phase I, 1:10,000), targeted antibiotic therapy was started, without clinical response. Subsequent fluorine 18–fluorodeoxyglucose positron emission tomography (FDG-PET) scanning showed an area of increased intensity is the abdomen. FDG accumulates in organ tissues with a high rate of glycolysis. Besides neoplastic cells, lesions with a high concentration of activated inflammatory cells show increased uptake of FDG. Fusion of PET and CT images located the increased uptake in the vascular graft, suggesting infection of the prosthetic graft (cover image and B). The graft was removed, a neoaortoiliac system was created with autologous veins, and antibiotics were given. Coxiella burnetii polymerase chain reaction and culture of the removed graft remained negative (D. Raoult, Unite des Rickettsies, Marseille). The sensitivity of these diagnostic tests, however, is low, whereas an anti–C burnetii immunoglobulin G phase I titer of 1:1600 or more has a sensitivity for chronic Q fever of 100%. After surgery, the patient recovered completely. An FDG-PET/CT scan 9 months later was normal (C). Antibiotic treatment will be continued for at least 3 years. Second to cardiac valves, vascular grafts are the most frequent site of chronic Q fever. Treatment consists of a combination of surgical removal of the infected graft and prolonged antibiotic therapy. Despite these measures, mortality remains high. 1 By combining functional and morphologic information, PET/CT fusion images result in a higher diagnostic accuracy than the separate imaging modalities. 2 Therefore, FDGPET/CT scanning can be a promising tool for identification of occult infectious foci, especially in culture-negative infected cardiovascular devices such as Coxiella burnetii-infected vascular grafts, as this case demonstrates.

Anemia in myelodysplasia (MDS) is partially ascribed to enhanced cell death of committed erythroid cells in the bone marrow compartment. To define in more detail the underlying type of cell death in MDS, immunohistochemical staining and ultrastructural analysis were performed on MDS and normal bone marrow samples. Immunohistochemistry of MDS bone marrow biopsies (n=20) demonstrated no positive staining of the erythroid lineage for active caspase -3 or -8. Ultrastructural analysis was performed in low-risk MDS patients (Refractory anemia (RA; n=3); RA with ringed sideroblasts (RARS; n=4) on isolated bone marrow hematons, which are compact hematopoietic complexes containing adipocytes, mesenchymal and endothelial cells, and hematopoietic cells. In vitro culture assays have demonstrated that these hematons contain a high number of progenitor and primitive cells. Ultrastructural analysis of the hematons of the MDS patients as well as the healthy controls demonstrated no signs of apoptosis in erythroid cells. In contrast, abnormalities compatible with autophagy were present in >60% of the MDS cells. Especially, cytoplasmic vacuoles with double membranes were noticed in basophilic and polychromatic normoblasts. Morphometric analysis of the erythroid cells showed no differences between healthy controls and MDS patients with regard to cell volume, mitochondria content or nuclear surface, but a significant increase was shown for the ratio area of vacuoles/cytoplasm in the MDS basophilic and polychromatic normoblasts (healthy controls vs MDS: 0.003 vs 0.013 (p

The thrombocytopenia in ITP is predominantly caused by autoantibodies that recognize antigens on platelets and megakaryocytes resulting in accelerated platelet destruction and a decreased platelet production rate (PPR). ITP patients with a predominantly suppressed PPR might respond differently to therapy than patients with predominantly peripheral platelet destruction. Tests and/or patient characteristics that can make a distinction between a reduced platelet half life and a reduced PPR could therefore influence diagnostic and therapeutic strategy in ITP. Therefore, in the present study the predictive value of clinical and platelet kinetic parameter for treatment outcome in idiopathic thrombocytopenic purpura (ITP) was investigated. Seventy-five patients with severe ITP (platelets ≤20 × 109/L) were studied. Median age was 46 (range 16–89) years and 34 (45%) patients were males. Median platelet count was 8 (1–20) × 109/L. The mean platelet life was 1 (0.1–6.5) days, and the PPR 160 (2–4670) × 109/day (normal 223 (100–355) × 109/day, p = 0.7). PPR was decreased (355 × 109/day) in 33%, 48%, and 19% of the patients, respectively. All patients started with prednisone at diagnosis (1 mg/kg/day). Initial complete and partial response (CR/PR) was 84% and a durable CR/PR (defined as CR/PR for ≥6 months without treatment) was attained in 44% of the patients. Apart from a higher proportion of patients with a decreased PPR in the group that responded to prednisone therapy (p=0.03), there were no significant differences regarding clinical and platelet kinetic parameters between responders and nonresponders. A durable CR/PR was noticed in 64% of the patients with a decreased PPR (median follow-up of 81 months (18–92)), compared to 34% of the patients with normal or increased PPR (median follow-up 141 (10–284) months (HR: 0.47 [95% CI (0.24–0.92)], p = 0.03). Splenectomy was performed in 32% of the patients with decreased PPR and in 62% of patients with normal or increased PPR (p = 0.03). In addition, patients with a decreased PPR showed significantly less splenic sequestration and a significantly longer mean platelet life than patients with a normal or increased PPR, which underscores that in these patients peripheral destruction of platelets contributes relatively less to thrombocytopenia than suppressed platelet production. Thirty-nine of 42 nonresponders to prednisone underwent splenectomy. Durable CR/PR postsplenectomy was reached in 74%. There were no significant differences with regard to patient characteristics between responders and nonresponders to splenectomy. In conclusion, ITP patients with suppressed PPR have a significant higher durable CR/PR rate to prednisone therapy and are less frequently exposed to splenectomy, than those with a normal or increased PPR.