Hydroxyurea attenuates activated neutrophil-mediated sickle erythrocyte membrane phosphatidylserine exposure and adhesion to pulmonary vascular endothelium

Activated neutrophils increase erythrocyte phosphatidylserine (PS) exposure. PS-exposed sickle red blood cells (SSRBCs) are more adhesive to vascular endothelium than non-PS-exposed cells. An increase in SSRBC fetal hemoglobin (HbF) concentration has been associated with improved rheology and decreased numbers of vasoocclusive episodes. This study examined the effects of HbF, PS-exposed SSRBCs, and chronic hydroxyurea (HU) treatment on activated neutrophil-mediated SSRBC retention/adherence in isolated-perfused rat lungs. Lungs were perfused with erythrocyte suspensions from 1) individuals homozygous for hemoglobin S with 0-7% HbF (SS), 2) with > or =8% HbF (SS + F), and 3) individuals homozygous for hemoglobin S treated with HU therapy for > or =1 yr (SS + HU). Retention of SSRBCs from the SS + HU group was significantly less than that seen in both the SS and SS + F groups. No difference was observed between the SS and SS + F groups. The percentage of HbF and F-cells did not differ between the SS + F and SS + HU groups. At baseline, the proportion of PS-exposed SSRBCs was not different between the SS and SS + HU groups. However, SSRBC treatment with activated neutrophil supernatant caused a twofold increase in PS-exposed SSRBCs in the SS control and no change in the SS + HU group. We conclude that 1) HU attenuates SSRBC retention/adherence in the pulmonary circulation seen in response neutrophil activation, 2) HU stabilizes SSRBC membrane PS, and 3) HU attenuation SSRBC retention/adherence in the pulmonary circulation occurs through a mechanism(s) independent of HbF.     

Anatoxin‐a occurrence and potential cyanobacterial anatoxin‐a producers in Spanish reservoirs1

The presence of anatoxin‐a (ANA) in Mediterranean region freshwaters has been reported only recently, and this work presents the first survey for ANA on a national scale in such waters. Fourteen reservoirs were sampled over a distance of 550 km from northeast to southwest Spain. Genera of cyanobacteria with ANA‐producing members in other countries were present in all of the Spanish reservoirs, but this toxin was detected in only one reservoir. The maximum ANA concentration detected was 0.31 μg · L^?1, and the most probable producer was Anabaena flos‐aquae (Lyngb.) Bréb. ex Bornet et Flahault. These findings suggest that in spite of the abundant cyanobacterial populations, ANA may be of lower occurrence in the Spanish reservoirs investigated than in other European freshwaters, and different strains of potential ANA producers occur in different ecoregions.     

Hindlimb unloading induces a collagen isoform shift in the soleus muscle of the rat

To determine whether hindlimb unloading (HU) alters the extracellular matrix of skeletal muscle, male Sprague-Dawley rats were subjected to 0 (n = 11), 1 (n = 11), 14 (n = 13), or 28 (n = 11) days of unloading. Remodeling of the soleus and plantaris muscles was examined biochemically for collagen abundance via measurement of hydroxyproline, and the percentage of cross-sectional area of collagen was determined histologically with picrosirius red staining. Total hydroxyproline content in the soleus and plantaris muscles was unaltered by HU at any time point. However, the relative proportions of type I collagen in the soleus muscle decreased relative to control (Con) with 14 and 28 days HU (Con 68 +/- 5%; 14 days HU 53 +/- 4%; 28 days HU 53 +/- 7%). Correspondingly, type III collagen increased in soleus muscle with 14 and 28 days HU (Con 32 +/- 5%; 14 days HU 47 +/- 4%; 28 days HU 48 +/- 7%). The proportion of type I muscle fibers in soleus muscle was diminished with HU (Con 96 +/- 2%; 14 days HU 86 +/- 1%; 28 days HU 83 +/- 1%), and the proportion of hybrid type I/IIB fibers increased (Con 0%; 14 days HU 8 +/- 2%; 28 days HU 14 +/- 2%). HU had no effect on the proportion of type I and III collagen or muscle fiber composition in plantaris muscle. The data demonstrate that HU induces a shift in the relative proportion of collagen isoform (type I to III) in the antigravity soleus muscle, which occurs concomitantly with a slow-to-fast myofiber transformation.     

Urease enhances the formation of iron nitrosyl hemoglobin in the presence of hydroxyurea

Although it has been shown that hydroxyurea (HU) therapy produces measurable amounts of nitric oxide (NO) metabolites, including iron nitrosyl hemoglobin (HbNO) in patients with sickle cell disease, the in vivo mechanism for formation of these is not known. Much in vitro data and some in vivo data indicates that HU is the NO donor, but other studies suggest a role for nitric oxide synthase (NOS). In this study, we confirm that the NO-forming reactions of HU with hemoglobin (Hb) or other blood constituents is too slow to account for NO production measured in vivo. We hypothesize that, in vivo, HU is partially metabolized to hydroxylamine (HA), which quickly reacts with Hb to form methemoglobin (metHb) and HbNO. We show that addition of urease, which converts HU to HA, to a mixture of blood and HU, greatly enhances HbNO formation.     

Cell kinetics-dependent antitumor effect of irinotecan hydrochloride induced by the synchronizing effect of hydroxyurea: cell kinetics and dosing time

Influence of hydroxyurea (HU) on the antitumor effect of irinotecan hydrochloride (CPT-11) was investigated in ICR male mice transplanted with sarcoma 180 cells (S-180). A single dose of CPT-11 (100 mg/kg) was injected at various times after a single dose of HU (300 mg/kg). The relative tumor weight varied significantly depending on the timing of CPT-11 injection after HU injection (P < 0.01). The higher antitumor effect of CPT-11 was observed when DNA synthesis of S-180 cells increased (20 hr), and the lower effect was observed when the DNA synthesis decreased (0 hr). The loss of body weight also varied significantly depending on the timing of CPT-11 injection after HU injection (P < 0.01). The toxicity of CPT-11 was higher when the inhibitory effect of HU on DNA synthesis of bone marrow cells was stronger (15 hr), and the lower toxicity was observed when the inhibitory effect was not observed (0 hr). The plasma SN-38 concentration at 2 hr after CPT-11 injection was higher at 20 hr after HU injection than at 0 hr after HU injection. The difference in plasma esterase activity between 0 hr and 20 hr after HU injection was regarded as the mechanism underlying the dosing time-dependent difference of the SN-38 concentration. These experiments suggest that HU can produce a different phase of cell cycle between tumor cells and normal cells. This leads to increase the antitumor effect of CPT-11 without increasing the adverse effect of the drug. It is essential to consider the dosing time in the two-drug combination therapy.     

Anacetrapib promotes reverse cholesterol transport and bulk cholesterol excretion in Syrian golden hamsters

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) and triglyceride between HDL and apoB-containing lipoproteins. Anacetrapib (ANA), a reversible inhibitor of CETP, raises HDL cholesterol (HDL-C) and lowers LDL cholesterol in dyslipidemic patients; however, the effects of ANA on cholesterol/lipoprotein metabolism in a dyslipidemic hamster model have not been demonstrated. To test whether ANA (60 mg/kg/day, 2 weeks) promoted reverse cholesterol transport (RCT), 3H-cholesterol-loaded macrophages were injected and (3)H-tracer levels were measured in HDL, liver, and feces. Compared to controls, ANA inhibited CETP (94%) and increased HDL-C (47%). 3H-tracer in HDL increased by 69% in hamsters treated with ANA, suggesting increased cholesterol efflux from macrophages to HDL. 3H-tracer in fecal cholesterol and bile acids increased by 90% and 57%, respectively, indicating increased macrophage-to-feces RCT. Mass spectrometry analysis of HDL from ANA-treated hamsters revealed an increase in free unlabeled cholesterol and CE. Furthermore, bulk cholesterol and cholic acid were increased in feces from ANA-treated hamsters. Using two independent approaches to assess cholesterol metabolism, the current study demonstrates that CETP inhibition with ANA promotes macrophage-to-feces RCT and results in increased fecal cholesterol/bile acid excretion, further supporting its development as a novel lipid therapy for the treatment of dyslipidemia and atherosclerotic vascular disease.     

Experimental Study of Low Dose Ultrashortwave Promoting Nerve Regeneration after Acellular Nerve Allografts Repairing the Sciatic Nerve Gap of Rats

Objectives To observe the effect of ultrashortwave (USW) therapy on nerve regeneration after acellular nerve allografts(ANA) repairing the sciatic nerve gap of rats and discuss its acting mechanisms. Methods Sixteen Wistar rats weighing 180–220 g were randomly divided into four groups with four rats in each group: normal control group; acellular group (ANA, treated by hypotonic-chemical detergent, was applied for bridging a 10 mm-long sciatic nerve defect); USW group (After 24 h of ANA repairing the sciatic nerve gap, low dose USW was administrated for 7 min, once a day, 20 times a course of treatment, three courses of treatment in all); and autografts group. 12 weeks after operation, a series of examinations was performed, including electrophysiological methods, the restoring rate of tibialis anterior muscle wet weight, histopathological observation (myelinated nerve number, myelin sheath thickness, and axon diameter), vascular endothelial growth factor (VEGF) mRNA expression of spinal cord, and muscle at injury site, and analyzed statistically. Results Compared to acellular nerve allografts alone, USW therapy can increase nerve conductive velocity, the restoring rate of tibialis anterior muscle wet weight, myelinated nerve number, axon diameter, VEGF mRNA expression of spinal cord, and muscle at injury site, the difference is significant. There were no differences between USW group and autografts group except myelin sheath thickness. Conclusions USW therapy can promote nerve axon regeneration and Schwann cells proliferation after ANA repairing the sciatic nerve gap of rats, the upregulation of VEGF mRNA expression of spinal cord and muscle may play an important role.     

Deoxyribonucleoside triphosphate metabolism and the mammalian cell cycle. Effects of hydroxyurea on mutant and wild-type mouse S49 T-lymphoma cells

DNA precursor synthesis can be blocked specifically by the drug hydroxyurea (HU) which has therefore been used for anticancer therapy. High concentrations of HU, however, affect other processes than DNA synthesis; nevertheless, most studies on the biological action of HU have been made with concentrations at least one order of magnitude higher than those needed for cell-growth inhibition. In this study we characterized the effects of low concentrations of HU (i.e. concentrations leading to 50% inhibition of cell growth in 72 h) on cell cycle kinetics and nucleotide pools in mouse S49 cells with various defined alterations in DNA precursor synthesis. The effect of 50 microM HU on deoxyribonucleoside triphosphate pools was a 2-3-fold decrease in the dATP and dGTP pools, with no change in the dCTP pool and a certain increase in the dTTP pool. Addition of deoxycytidine or thymidine led to a partial reversal of the growth inhibition and cell-cycle perturbation caused by HU, and was accompanied by an increased level of the deoxyribonucleoside triphosphates. Addition of purine deoxyribonucleoside gave no protection, indicating that salvage of these nucleosides could not supply precursors for DNA synthesis in T-lymphoma cells. We observed a higher sensitivity to HU of cells lacking purine nucleoside phosphorylase or with a ribonucleotide reductase with altered allosteric regulation. Cells lacking thymidine kinase or deoxycytidine kinase were just as sensitive as wild-type cells.     

Clinical and Serological Features of Patients Referred through a Rheumatology Triage System because of Positive Antinuclear Antibodies

Background

The referral of patients with positive anti-nuclear antibody (ANA) tests has been criticized as an inappropriate use of medical resources. The utility of a positive ANA test in a central triage (CT) system was studied by determining the autoantibody profiles and clinical diagnoses of patients referred to rheumatologists through a CT system because of a positive ANA test.

Methods

Patients that met three criteria were included: (1) referred to Rheumatology CT over a three year interval; (2) reason for referral was a “positive ANA”; (3) were evaluated by a certified rheumatologist. The CT clinical database was used to obtain demographic and clinical information and a serological database was used to retrieve specific ANA and/or extractable nuclear antigen (ENA) test results. Clinical information was extracted from the consulting rheumatologist''s report.

Results

15,357 patients were referred through the CT system; 643 (4.1%) of these because of a positive ANA and of these 263 (40.9%) were evaluated by a certified rheumatologist. In 63/263 (24%) of ANA positive patients, the specialist provided a diagnosis of an ANA associated rheumatic disease (AARD) while 69 (26.2%) had no evidence of any disease; 102 (38.8%) had other rheumatologic diagnoses and 29 (11%) had conditions that did not meet AARD classification criteria. Of ANA positive archived sera, 15.1% were anti-DFS70 positive and 91.2% of these did not have an AARD.

Conclusions

This is the first study to evaluate the serological and clinical features of patients referred through a CT system because of a positive ANA. The spectrum of autoantibody specificities was wide with anti-Ro52/TRIM21 being the most common autoantibody detected. Approximately 15% of referrals had only antibodies to DFS70, the vast majority of which did not have clinical evidence for an AARD. These findings provide insight into the utility of autoantibody testing in a CT system.     

Brief daily exposure to low-intensity vibration mitigates the degradation of the intervertebral disc in a frequency-specific manner

Hindlimb unloading of the rat causes rapid hypotrophy of the intervertebral disc (IVD) as well as reduced IVD height and glycosaminoglycan content. Here we tested the hypothesis that low-intensity mechanical vibrations (0.2 g), as a surrogate for exercise, will mitigate this degradation. Four groups of Sprague-Dawley rats (4.5 mo, n = 11/group) were hindlimb unloaded (HU) for 4 wk. In two of the HU groups, unloading was interrupted for 15 min/day by placing rats in an upright posture on a platform that was vertically oscillating at 45 or 90 Hz (HU+45, HU+90). Sham control rats stood upright on an inactive plate for 15 min/day (HU+SC). These three experimental groups were compared with HU uninterrupted by weightbearing (HU) and to normally ambulating age-matched controls. In the HU and HU+SC rats, 4 wk of unloading resulted in a 10% smaller IVD height, as well as less glycosaminoglycan in the whole IVD (7%) and nucleus pulposus (17%) and a greater collagen-to-glycosaminoglycan ratio in the whole IVD (17%). Brief daily exposure to 90 Hz mechanical oscillations mitigated this degradation; compared with HU ± SC, the IVD of HU+90 had an 8% larger height and greater glycosaminoglycan content in the whole IVD (12%) and nucleus pulposus (24%). In contrast, the 45 Hz signal failed to mitigate changes in height or glycosaminoglycan content brought with altered spinal loading, but normalized the collagen-to-glycosaminoglycan ratio to levels observed in age-matched controls. In summary, unloading caused marked phenotypic and biochemical changes in the IVD, a deterioration that was not slowed by brief weightbearing. However, low-intensity 90 Hz vibrations superimposed on weightbearing largely preserved the morphology and biochemistry of the IVD and suggest that these biomechanically based signals may help protect the IVD during long bouts of nonambulation.     

Trichomonas vaginalis: identification of a phospholipase A-dependent hemolytic activity in a vesicular subcellular fraction

Trichomonad total extracts (TTE), or vesicular (P30) and soluble (530) subcellular fractions from 3 pathogenic Trichomonas vaginalis strains (GT-3. GT-13. and GT-15), lysed both human and Sprague-Dawley rat erythrocytes in a time- and dose-dependent manner. The entire hemolytic activity of TTE was located in P30, showing 2 peaks of maximum activity, one at pH 6.0 and another at pH 8.0. in the presence of 1 mM Ca2+. Hemolytic activity on rat erythrocytes was greater at pH 6.0 16.71 +/- 0.33 hemolytic units IHU]/mg/hr to 11.60 +/- 0.24 HU/mg/hr) than at pH 8.0 (3.81 +/- 0.30 HU/mg/hr to 5.75 +/- 0.65 HU/mg/hr). and it was greater than that on human red blood cells at pH 6.0 (2.67 +/- 0.19 HU/mg/hr to 4.08 +/- 0.15 HU/mg/hr) or pH 8.0 (2.24 +/- 0.0 9 HU/mg/hr to 2.81 +/- 0.06 HU/mg/hr). The alkaline and acidic hemolytic activity diminished (60-93% at pH 6.0 and 78-93% at pH 8.0) by the effect of 80 microM Rosenthal's inhibitor, which also inhibited 27-45% and 29-54% trichomonad alkaline and acidic phospholipase A activities, respectively. Vesicles, vacuoles, and hydrogenosomes were rich in P30. Trichomonas vaginalis has a hemolytic PLA, which could be involved in its cytopathogenic mechanism.     

Hydroxyurea Use and Hospitalization Trends in a Comprehensive Pediatric Sickle Cell Program

Background

A decline in hospitalizations and pain episodes among those with sickle cell disease (SCD) who take hydroxyurea (HU) has been shown when compared to pre-HU patterns but paradoxically, when compared to those who have never been treated, HU recipients often have more frequent hospitalizations. This analysis evaluates the impact of increasing usage of HU on trends in hospitalizations and blood transfusions within a large SCD treatment program.

Methods

Eligibility was restricted to patients with Hb SS or Hb Sβ⁰-thalassemia who were 2–18 years old between 2006–2010 and received care at St. Jude Children''s Research Hospital (N = 508). Hospitalizations and blood transfusions were calculated for each of the years under study for those exposed and never exposed to HU. Differences in number of hospitalizations before and after HU initiation were compared.

Results

The proportion of patients receiving HU increased by 4% per year on average. In the HU exposed group, a modest decline in mean per-patient hospitalizations and per-patient hospital days occurred, while those never exposed to HU trended toward a slight increase over time. Rates of blood transfusions declined among those on HU but not in patients never exposed to HU. Patients on HU had a median of one fewer hospital admission in the year after initiation of HU, compared to the year prior. Two deaths occurred in the patient population, both of whom were not exposed to HU.

Conclusions

Increasing usage of HU was concurrent with decreased hospitalization rates and blood transfusions. Our results support the utility of HU on decreasing hospitalizations and transfusions for patients with SCD outside of the clinical trial setting.     

The in Vivo Toxicity of Hydroxyurea Depends on Its Direct Target Catalase

Hydroxyurea (HU) is a well tolerated ribonucleotide reductase inhibitor effective in HIV, sickle cell disease, and blood cancer therapy. Despite a positive initial response, however, most treated cancers eventually progress due to development of HU resistance. Although RNR properties influence HU resistance in cell lines, the mechanisms underlying cancer HU resistance in vivo remain unclear. To address this issue, we screened for HU resistance in the plant Arabidopsis thaliana and identified seventeen unique catalase mutants, thereby establishing that HU toxicity depends on catalase in vivo. We further demonstrated that catalase is a direct HU target by showing that HU acts as a competitive inhibitor of catalase-mediated hydrogen peroxide decomposition. Considering also that catalase can accelerate HU decomposition in vitro and that co-treatment with another catalase inhibitor alleviates HU effects in vivo, our findings suggests that HU could act as a catalase-activated pro-drug. Clinically, we found high catalase activity in circulating cells from untreated chronic myeloid leukemia, offering a possible explanation for the efficacy of HU against this malignancy.     

Role of computed tomography in the estimation of the density of the bony block of an injured vertebral column segment at different stages of treatment with an external transpedicular fixation apparatus

A Somatom AR.HP computed tomograph (Siemens) was used to make studies in 54 patients with injury to the vertebral column and spinal cord in order to determine the formation of a bony block and its density in different portions of an injured segment at the stages of treatment. During therapy, the highest values of bony block density were observed in the dorsal portions of an injured segment (mean 377.32 +/- 184.57 HU). This may be accounted for by a slight effect of artefacts of an external transpedicular fixation apparatus, the main components of which are present at the posterior supporting complex. After treatment, the highest values of bony block density were noted in the central portions of an injured segment (mean 353.11 +/- 208.01 HU). This may be associated with rather serious intervertebral disk damage in this portion. The bony block density index was 339.75 +/- 139.41 HU in the left lateral portions and as high as 312.56 +/- 135.07 HU in the right ones. In the late period, the highest bony block density index (mean 397 +/- 193.07 HU) was seen in the right portions of an injured segment, which may be due to slight scoliotic deformity with the angle being open to the right.     

Correlation of antinuclear antibody and anti-double-stranded DNA antibody with clinical response to infliximab in patients with rheumatoid arthritis: a retrospective clinical study

Introduction  

The induction of antinuclear antibodies (ANAs) or anti-double-stranded (ds) -DNA antibodies (Abs) after infliximab (IFX) therapy in rheumatoid arthritis (RA) is a well-known phenomenon, but the correlation of such Abs with the clinical response to IFX has not yet been determined. The aims of this retrospective observational study were to examine the prevalence of positive ANA and anti-ds-DNA Abs before and after IFX therapy in patients with RA and to investigate whether an increased titer of such Abs is associated with the clinical efficacy of IFX.     

Genetic destabilization of Candida albicans by hydroxyurea

Candida albicans is a commensal component of the normal human microflora, but frequently causes infections in persons undergoing treatment for malignancies. The cytotoxicity and genotoxicity of the antineoplastic agent hydroxyurea (HU) for the yeast is demonstrated. Cultivation of the organism on defined complete medium in the presence of HU induced growth inhibition or cell death, gene mutations, segregations of heterozygous loci through reciprocal and nonreciprocal mitotic recombinations, and a special heritable system for high frequency switching between phenotypes reflecting cellular growth characteristics and susceptibilities to HU. Each of these responses is influenced differently by variations in drug concentration and temperature. The biochemical complexity underlying cytotoxic and genetic effects of HU, and the implications for the likelihood of HU induced changes occurring in indigenous yeast populations of persons undergoing therapy with the drug, are discussed.     

Alterations in skeletal perfusion with simulated microgravity: a possible mechanism for bone remodeling.

Bone loss occurs as a consequence of exposure to microgravity. Using the hindlimb-unloaded rat to model spaceflight, this study had as its purpose to determine whether skeletal unloading and cephalic fluid shifts alter bone blood flow. We hypothesized that perfusion would be diminished in the hindlimb bones and increased in skeletal structures of the forelimbs and head. Using radiolabeled microspheres, we measured skeletal perfusion during control standing and after 10 min, 7 days, and 28 days of hindlimb unloading (HU). Femoral and tibial perfusion were reduced with 10 min of HU, and blood flow to the femoral shaft and marrow were further diminished with 28 days of HU. Correspondingly, the mass of femora (-11%, P < 0. 05) and tibiae (-6%, P < 0.1) was lowered with 28 days of HU. In contrast, blood flow to the skull, mandible, clavicle, and humerus was increased with 10 min HU but returned to control levels with 7 days HU. Mandibular (+10%, P < 0.05), clavicular (+18%, P < 0.05), and humeral (+8%, P < 0.1) mass was increased with chronic HU. The data demonstrate that simulated microgravity alters bone perfusion and that such alterations correspond to unloading-induced changes in bone mass. These results support the hypothesis that alterations in bone blood flow provide a stimulus for bone remodeling during periods of microgravity.     

Feeding a corn oil/sucrose-enriched diet enhances steatohepatitis in sedentary rats

The current study investigated the combined effects of feeding a high-fat/high-sucrose (HF/HS) diet to rodents rendered sedentary via hindlimb unloading (HU). For 3 wk before HU, male Wistar rats were fed chow or a diet in which 32% of calories were derived from corn oil fat and 48% of calories from sucrose. Feeding continued during an additional 3-wk period of HU. Subsequently, blood samples were collected for determination of circulating leukocyte counts, insulin levels, and portal vein endotoxin. Inflammation, necrosis, and steatosis were assessed in formalin-fixed liver sections. No biochemical or histological evidence of injury was observed in control rats fed chow or HF/HS. HU increased circulating neutrophils and resulted in hyperinsulinemia. Mild hepatic fat accumulation and minimal focal necroinflammation were observed in this group. Feeding HF/HS during HU exacerbated hyperinsulinemia, hepatic steatosis, Kupffer cell content, and cytokine expression. Significant portal endotoxemia was noted in HU rats but was not influenced by HF/HS diet. On the other hand, feeding HF/HS significantly enhanced lipid peroxidation end products in liver of HU rats by approximately threefold compared with chow-fed rats. In summary, these findings demonstrate that feeding a high-calorie diet potentiates steatosis and injury in sedentary HU rats. Mechanisms underlying enhanced injury most likely involved lipid peroxidation. Importantly, these findings suggest that dietary manipulation combined with physical inactivity can be used to model steatohepatitis.