Chromatin regulation by Histone H4 acetylation at Lysine 16 during cell death and differentiation in the myeloid compartment.

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death.

Red blood cell distribution width is not related with inflammatory parameters in morbidly obese patients.

OBJECTIVE: Red blood cell distribution width (RDW) is a hematological parameter that has been studied in several clinical settings and has been found to be related to both anemia and inflammatory status. As obesity is related to increased inflammatory pattern, we aimed to analyze the RDW in this setting. METHODS: We determined hematological and inflammatory parameters in morbidly obese patients before bariatric surgery (n=142) and normo-weight controls (n=144). RESULTS: RDW was higher in patients than in controls (p<0.001), along with C-reactive protein (p<0.001) and fibrinogen, (p<0.001) while hemoglobin (p=0.026), serum iron (p<0.001), MCH (p=0.002) and MCHC (p<0.001) were lower in morbidly obese patients. The logistic correlation analysis revealed that only low serum iron (<62 µg/dL) and MCH (<28.14 pg) levels were associated with RDW>14% (OR 7.61, 95% CI: 1.93-30.04, p=0.004; OR 5.67, 95% CI: 1.98-16.24, p=0.001; respectively). CONCLUSIONS: These data indicate that the elevated RDW in morbidly obese patients reflects a mild red blood cell hypochromia that does not relate to inflammatory parameters, but to hyposideremia and, consequently, to lower erythrocyte indices, possibly as a result of being on a very low-calorie diet before bariatric surgery. Therefore, RDW should not be considered as an inflammatory marker in this clinical setting.

Hemorheological parameters as independent predictors of venous thromboembolism.

The role played by hemorheological alterations in the development of deep vein thrombosis (DVT) has often been overlooked. Although marked rheological alterations and the relationship with thromboembolic events are well-defined in patients with hematological diseases such as myelom, Waldenström disease and polycythemia vera, the relationship is not so clear in patients without hematological diseases. In the present review, we analyzed studies evaluating the rheological profile in DVT patients. Among the cardiovascular risk factors, only hyperlipidemia, metabolic syndrome, tobacco and obesity increase DVT risk and, in addition, a disturbed rheological profile is shown which could further increase this risk. The significance of hematocrit and fibrinogen, the main factors influencing blood viscosity, is not sufficient to increase blood viscosity in any of the studies analyzed. DVT patients show increased fibrinogen levels and erythrocyte aggregation throughout all the studies despite patients not being in an acute reactant phase. In addition to rheological alterations, it is necessary to consider local conditions at pockets of venous valves which undergo deterioration with aging and play an important role equally to alterations in the rheological profile. Moreover, it is necessary to take into account that systemic rheological alterations are not comparable to those in low shear rate areas where minimum disturbances could be more relevant. It would be convenient to perform multicentric studies with the same rheological methodology and pre-analytical procedures to evaluate, in order to obviate the effect of thrombophilic and circumstantial risk factors, rheological parameters in patients with spontaneous DVT to elucidate their real contribution to the development of thromboembolic events.

Rheological alterations and thrombotic events in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterised by increased venous and arterial thrombotic risk. Nevertheless, how hemorheological alterations contribute to thrombotic risk remains a question of debate. We aimed to determine the rheological profile in 105 patients with SLE (24 with a thrombotic event) and 105 healthy controls. We determined blood viscosity and erythrocyte aggregation along with plasma lipids and fibrinogen. Although SLE patients showed lower blood viscosity at 230 s(-1) at a native hematocrit when compared with controls (p < 0.001), differences disappeared after adjusting the hematocrit to 45% (p = 0.095). When comparing SLE patients with and without thrombotic events, no differences in any rheological parameter were found (p > 0.05), except in fibrinogen which was higher in patients with thrombosis (p = 0.013). No differences in the rheological parameters were observed when venous and arterial thrombotic events were compared, although a tendency for higher fibrinogen was observed in patients with venous thrombosis (p = 0.053). Only hematocrit, fibrinogen and triglycerides were independent predictors of native blood viscosity in the multivariate regression analysis, even after adjusting for continuous variables and for tobacco and hypertension: beta coefficient: 0.727 p < 0.001; beta coefficient: 0.152 p = 0.003 and beta coefficient: 0.133 p = 0.015, respectively. The logistic regression analysis revealed that neither increased native blood viscosity (BVn > 4.33) nor increased erythrocyte aggregation (EA1 > 7.85) increased thrombotic risk: OR 0.636, CI 0.313-3.12, p = 0.578 and OR 2.01, CI 0.77-5.20, p = 0.152, respectively. However, hyperfibrinogenemia (Fbg > 342 mg/dL) increased thrombotic risk by around three times: OR 3.44 CI 1.32-8.96, p = 0.011. Our results suggest that the role of blood viscosity and erythrocyte aggregation in thrombotic risk in SLE patients fails to demonstrate any association.

Erythrocyte deformability in morbid obesity before bariatric surgery. Influence of abdominal obesity.

Although there are several studies dealing with erythrocyte deformability (ED) in obese patients, research on this topic in morbidly obese subjects is scarce. In these studies ED seems to be decreased, although the cause remains unknown. A case-control study in 76 morbid obese subjects (23 women and 53 men, aged 44 ± 13 years) and in 79 normal-weight controls (30 women and 49 men, aged 43 ± 13 years) was undertaken. ED has been determined by ektacytometric techniques in a Rheodyn SSD, by means of the elongation index (EI) at 12, 30 and 60 Pascals, along with anthropometric, lipidic, metabolic and inflammatory parameters. EI was statistically lower in morbidly obese subjects than in controls at all the shear stresses tested (EI12: 47.3 ± 2.14 vs. 47.9 ± 2.07; p = 0.047, EI30: 52.16 ± 2.1 vs. 53.12 ± 1.4; p = 0.007, EI60: 53.9 ± 2.4 vs. 55.2 ± 2.50; p = 0.001) as were anthropometric lipidic and inflammatory parameters (p < 0.001). In the bivariate correlation EI60 correlated negatively with most anthropometric, lipidic and inflammatory parameters. However, in the multivariate analysis, the case-control status was not significantly associated with EI60 and only triglycerides, glucose, hs-CRP and waist circumference were independently associated with EI60, constituting independent predictors of altered ED although, waist circumference, showed the highest statistical significance (p = 0.007). ED is decreased in morbidly obese subjects associated with insulin resistance and inflammation parameters although abdominal obesity seems to be of paramount importance in altering this rheological parameter.

Mutations causing Greenberg dysplasia but not Pelger anomaly uncouple enzymatic from structural functions of a nuclear membrane protein.

The lamin B receptor (LBR) is an inner nuclear membrane protein with a structural function interacting with chromatin and lamins, and an enzymatic function as a sterol reductase. Heterozygous LBR mutations cause nuclear hyposegmentation in neutrophils (Pelger anomaly), while homozygous mutations cause prenatal death with skeletal defects and abnormal sterol metabolism (Greenberg dysplasia). It has remained unclear whether the lethality in Greenberg dysplasia is due to cholesterol defects or altered nuclear morphology.To answer this question we characterized two LBR missense mutations and showed that they cause Greenberg dysplasia. Both mutations affect residues that are evolutionary conserved among sterol reductases. In contrast to wildtype LBR, both mutations failed to rescue C14 sterol reductase deficient yeast, indicating an enzymatic defect. We found no Pelger anomaly in the carrier parent excluding marked effects on nuclear structure. We studied Lbr in mouse embryos and demonstrate expression in skin and the developing skeletal system consistent with sites of histological changes in Greenberg dysplasia. Unexpectedly we found in disease-relevant cell types not only nuclear but also cytoplasmatic LBR localization. The cytoplasmatic LBR staining co-localized with ER-markers and is thus consistent with the sites of endogeneous sterol synthesis. We conclude that LBR missense mutations can abolish sterol reductase activity, causing lethal Greenberg dysplasia but not Pelger anomaly. The findings separate the metabolic from the structural function and indicate that the sterol reductase activity is essential for human intrauterine development.

Parameters of inflammation in morbid obesity: lack of effect of moderate weight loss.

BACKGROUND: Obesity has been associated with a chronic activation of the acute-phase response. The aims of our study were to investigate whether levels of inflammatory cytokines are higher in obese patients, to evaluate their relationship with metabolic syndrome, and to analyze the effect of moderate weight loss upon their levels. METHODS: Sixty-seven severe or morbid obese patients were compared with 67 controls. Patients were submitted to a 4-week very low calorie diet followed by a low calorie diet for 2 months. Exclusion criteria were organic disease, ischemic heart disease or stroke, diabetes mellitus, hyperlipidemia, and hypertension. An evaluation was performed before and after the diet, in which fibrinogen, blood count, high-sensitive C-reactive protein (CRP), interleukin 6 (IL-6), and tumoral necrosis factor alpha (TNF-alpha) were measured. The Student t test was employed to compare differences between the groups and Pearson correlation coefficients were calculated. RESULTS: Obese patients showed higher levels of CRP (P < 0.001), IL-6 (P < 0.001), TNF-alpha (P < 0.001), leukocyte (P = 0.001), and neutrophil count (P < 0.001) than controls. In obese patients, inflammatory parameters were significantly correlated with anthropometric parameters and did not differ between obese subjects with or without metabolic syndrome. Moderate weight loss (excess weight loss 19.6%) was achieved through dieting, but no change was observed in any inflammatory parameter. CONCLUSIONS: Obesity is associated to a chronic inflammatory state that seems to be due to an increased secretion of cytokines, and this state is not related to the presence of metabolic syndrome. Moderate weight loss does not ameliorate this inflammatory state in the short term.

Plasma viscosity and related cardiovascular risk factors in a Spanish Mediterranean population.

INTRODUCTION: Plasma viscosity (PV) constitutes an independent important predictor of initial and recurrent cardiac events and mortality. It has been suggested that there is a geographical variation in PV values related to coronary event rates. Little information exists regarding PV in Spain. Therefore, our objective was to determine PV in a large sample of randomly selected subjects from the Spanish population and to study which demographic or cardiovascular risk factors (CVRF) influence levels of PV in this population. MATERIALS AND METHODS: 1277 subjects (503 males, 774 females) aged 43+/-14 years (range: 20-70) were randomly selected from an Eastern Spanish population. These subjects were free of cardiovascular diseases and other major diseases. PV was measured at 37 degrees C by means of the Fresenius GmbH plasma viscosimeter. In addition, total cholesterol, triglycerides, glucose and fibrinogen were measured. RESULTS: In the crude analysis, no differences in PV were observed regarding gender (males: 1.235+/-0.061 cP; females: 1.236+/-0.059 cP, P=0.952). Women older than 50 years showed higher PV:1.248+/-0.057 cP than those aged less than 50 years: 1.232+/-0.059 cP, P=0.001. No differences in PV by age groups were observed in men (P=0.842). Furthermore, we evaluated the prevalence of the following CVRF: hypercholesterolemia, hypertriglyceridemia, hypertension, diabetes, tobacco smoking, obesity, and high fibrinogen levels. Prevalence of these factors was: 28%, 4.3%, 8%, 2.3%, 33%, 8.8% and 15% respectively. As some of these CVRF were correlated with PV levels, we carried out a multivariate analysis to adjust PV levels for the potential confounding effect of each one of these factors. After multivariate adjustment, PV was positively associated with high fibrinogen (>320 mg/dL) levels in both men and women (P<0.001). In addition, in women, but not in men, PV levels were independently associated with obesity (P<0.001) and hypertriglyceridemia (P=0.01). After the multivariate adjustment, the association between PV and age older than 50 in women did not remain statistically significant, revealing a confusing effect of the CVRF in the crude analysis.

Long-term prospective study of recurrent venous thromboembolism in patients younger than 50 years.

Long-term incidence of recurrent venous thromboembolism (VTE) in patients younger than 50 years, not affected by a malignancy or chronic diseases, are poorly characterized. After the initial episode of VTE and cessation of oral anticoagulation, 98 patients, mean age 32.2+/-9.2 years were followed for a median of 117 months (range 6-165). Congenital risk factors for VTE were present in 36% of patients, acquired persistent (positive antiphospholipid antibodies during the whole follow-up) in 19%, and acquired transitory in 44%. Thirty episodes of recurrent VTE were documented. The cumulative incidence of VTE after 1 year of follow-up was 5.1%, 9.8% after 2 years, 14% after 4 years, and 34.2% after 8 years. In the univariate analysis, the relative risk of recurrent VTE was 2.66 [95% confidence interval (CI) 1.03-6.90] for congenital risk factors, 4.97 (95% CI 1.75-14.0) for persistent acquired (antiphospholipid antibodies), 2.64 (95% CI 1.23-5.66) for male gender and 2.27(1.00-5.15) for body mass index>30 kg/m2. In the multivariate analysis, male gender [hazard ratio (HR) 4.23, 95% CI 1.88-9.77) the presence of congenital factors (HR 3.28, 95% CI 1.25-8.63) and acquired persistent factors (HR 8.50, 95% CI 2.84-25.50) were independent risk factors for recurrent VTE. In patients under 50 years of age without malignancy or underlying chronic disease, hospitalized for an acute thromboembolic event, the presence of antiphospholipid antibodies, congenital defects of coagulation, male sex, and obesity were risk factors for recurrent VTE. These data raise the possibility that selected patients with VTE may require prolonged anticoagulation to prevent recurrent disease.

Red blood cell deformability in iron deficiency anaemia.

In patients with iron deficiency anaemia (IDA) it has been suggested that the shortened erythrocyte lifespan may be in part due to decreased erythrocyte deformability. In order to know whether erythrocyte deformability is decreased in IDA patients, we have determined the erythrocyte Elongation Index (EI) by means of ektacytometric techniques (Rheodyn SSD, Myrenne Gmbh, Germany), in 50 IDA patients and 100 well age and sex matched healthy controls. At the three shear stresses tested, 12, 30 and 60 Pa, IDA patients show statistically lower EI than controls (37.4+/-6.7 vs 48.6+/-2.9; 45.0+/-6.0 vs 54.5+/-2.8; 48.7+/-5.8 vs 57.0+/-2.9 mPa.s, respectively; p<0.001). A statistically significant correlation was found between EI at 12, 30, and 60 Pa and the hematimetric indices (MCV, MCH and MCHC), suggesting that the alteration in surface/volume ratio (shape) which characterizes this kind of microcytic hypocromic anaemia, accounts in part for the decreased EI. Rheodyn SSD, as an ektacytometric technique, is very sensitive to alterations in red blood cell geometry, for what seems to be a useful tool for detecting diminished erythrocyte deformability in IDA patients.

[Risk factors and clinical characteristics of thromboembolic venous disease in young patients: a prospective study]. / Estudio prospectivo de los factores de riesgo y las características clínicas de la enfermedad tromboembólica en pacientes jóvenes.

BACKGROUND AND OBJECTIVE: We aimed to determine the risk factors of thromboembolic disease in young patients and to study the clinical characteristics according to the etiology. PATIENTS AND METHOD: A prospective study of 100 patients under 50 years who were not affected by neoplasias or chronic diseases and who required hospitalization due to thromboembolic disease. The morphological diagnosis was performed by eco-Doppler, flebography, lung gammagraphy or CT scan. Risk factors assessed were antithrombin, protein C and S deficiency, presence of factor V Leiden, prothrombin G20210A, hyperhomocisteinemia, increased PAI-I, increased factor VIII, and presence of antiphospholipid antibodies (APAs). Acquired factors were also evaluated. RESULTS: In 87% of patients, a venous thrombosis was observed in lower limbs. 37% of patients had congenital risk factors and 19% had APAs, whereas in the remaining patients only acquired factors were demonstrated. Most frequent congenital factors were factor V Leiden, pothrombin G20210A, and protein C and S deficiency. Most patients presented several risk factors. A family thrombotic history was significantly more frequent in the group with congenital risk factors. CONCLUSIONS: In 56% of young patients with thromboembolic disease, a congenital etiology or APAs are identified. In these patients the number of acquired factors needed to trigger thrombosis is fewer than in patients in whom a cause is not identified.

Mutations in the gene encoding the lamin B receptor produce an altered nuclear morphology in granulocytes (Pelger-Huët anomaly).

Pelger-Huët anomaly (PHA; OMIM *169400) is an autosomal dominant disorder characterized by abnormal nuclear shape and chromatin organization in blood granulocytes. Affected individuals show hypolobulated neutrophil nuclei with coarse chromatin. Presumed homozygous individuals have ovoid neutrophil nuclei, as well as varying degrees of developmental delay, epilepsy and skeletal abnormalities. Homozygous offspring in an extinct rabbit lineage showed severe chondrodystrophy, developmental anomalies and increased pre- and postnatal mortality. Here we show, by carrying out a genome-wide linkage scan, that PHA is linked to chromosome 1q41-43. We identified four splice-site, two frameshift and two nonsense mutations in LBR, encoding the lamin B receptor. The lamin B receptor (LBR), a member of the sterol reductase family, is evolutionarily conserved and integral to the inner nuclear membrane; it targets heterochromatin and lamins to the nuclear membrane. Lymphoblastoid cells from heterozygous individuals affected with PHA show reduced expression of the lamin B receptor, and cells homozygous with respect to PHA contain only trace amounts of it. We found that expression of the lamin B receptor affects neutrophil nuclear shape and chromatin distribution in a dose-dependent manner. Our findings have implications for understanding nuclear envelope-heterochromatin interactions, the pathogenesis of Pelger-like conditions in leukemia, infection and toxic drug reactions, and the evolution of neutrophil nuclear shape.