Development of a simple method for rapid isolation of polymorphonuclear leukocytes from human blood

Polymorphonuclear leukocytes (PMNs; commonly known as neutrophils) play essential roles in innate immunity and inflammation. Although there are standardized methods for the isolation of human neutrophils, they are time consuming and demand considerable technical expertise, making them unfeasible for many clinical applications. Here, we describe a simple and time-efficient technique for the isolation of human neutrophils, which adapts a readily available commercial cell preparation tube (CPT) currently in use for isolation of peripheral blood mononuclear cells (PBMC) and plasma and is now adapted to also yield neutrophils. The total time required for neutrophil isolation was less than 1 hr. Neutrophils isolated by this method were highly purified (> or =97%) as assessed by surface expression of the neutrophil specific marker, CD66b. Neutrophils isolated by this method were functional as demonstrated by their ability to secrete interleukin-1 receptor antagonist (IL-1RA). Neutrophils isolated using this new technique secreted significant amounts of soluble IL-1RA (929.3+/-197 pg/10(6)cells/mL) in response to lipopolysaccharide (LPS). Use of this adapted CPT method allows simultaneous isolation of functional human neutrophils as well as PBMC and plasma. Adoption of this new method will allow the conduct of different neutrophil assays at any clinical site without requiring trained laboratory personnel or a large staff time commitment.     

Crystalline and disordered state of poly(dihexylsilylene) copolymers

A systematic comparison of random copolymers, derived from poly(dihexylsilylene) (PDHS) by incorporation of monomeric units with shorter unbranched alkyl side chains, has been carried out based on calorimetry, variable temperature UV spectroscopy, and ²⁹Si MAS (magic angle spinning) solid state NMR investigations. Also, hexylmethylsilylene units and branched monomers have been copolymerized. Up to 10% comonomer with shorter linear side chains (i. e., pentyl to propyl) could be incorporated into PDHS without impeding the all-trans order of the crystalline phase. In this case, the UV absorption maximum of the crystalline low-temperature phase was affected only slightly according to the length and fraction of the comonomer side chains. A less ordered crystal structure (λ_max = 345–355 nm) was observed when the content of comonomers with shorter side chains was about 20%. Yet, all these materials form conformationally disordered mesophases. A clear disordering transition and corresponding thermochromism was not observed any more when 50% of propyl side chains were incorporated. The order of the crystalline and the mesophase is also strongly perturbed if only a small fraction (4%) of the side chains are branched at C².     

Nitric oxide mediates intestinal hyperaemic responses to intraluminal bile-oleate

It has long been recognized that intestinal blood flow increases at mealtimes. Mesenteric hyperaemia is also evoked by activation of sensory peptidergic nerves. Our studies explored the possible role of endogenous nitric oxide (NO) in the rat intestinal vasodilator response to luminal instillation of an oleic acid plus bile mixture before and after acute intrajejunal instillation of capsaicin and after chronic pretreatment with capsaicin. In anaesthetized rats we measured jejunal blood flow (BF) with an ultrasonic Doppler flowmeter and systemic arterial pressure (AP) with a pressure transducer. Intestinal perfusion with 80 mM oleic acid in bile increased BF by 98±12%. Instillation of 4 mg of capsaicin into the jejunal lumen initially increased BF by 42±9% but was followed by vasoconstriction. Inhibition of NO synthase with 25 mg/kg i.v. N-nitro-L-arginine (L-NNA) decreased BF by 27±5% and increased AP by 37±11%. After treatment with L-NNA and after acute and chronic administration of capsaicin, the bile-oleate-induced maximal increases in BF above control levels were 42±7%, 65±12%, and 58±8%, respectively. The observed inhibitory effect of L-NNA on the intestinal hyperaemic response to the bile-oleate mixture was reversed by pretreatment with L-arginine (100 mg/kg i.V.). In capsaicin pretreated rats the subsequent bile-oleate-induced hyperaemia was reduced in magnitude but the inhibitory effects of L-NNA were proportionately the same as in animals not receiving capsaicin. These findings support the hypothesis that NO is involved with bile-oleate-induced mesenteric hyperaemia.     

The activity of erythrocyte enzymes in rats subjected to running exercises

Summary The studies were carried out on male Wistar rats subjected to running within an electric rotating drum. The animals were divided into four experimental groups, differing one from another as to the duration of training. Each training session lasted 30 days. In the first group the daily run lasted 3 min, in the second group 5 min; in the third group, a 1 min run on the first day, and one min longer on each successive day; in the fourth group a 2 min run on the first day and for two min longer on each successive day.The determinations made prior to and after training included the peripheral blood erythrocyte (Er) and reticulocyte (Ret.) count, the hemoglobin concentration (Hb) and packed cell volume (PCV) and, determined by spectrophotometric methods, the activity of pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR). Training induced an improvement of all enzymatic activities. The heavier the physical exertion, the more intensive was the enzymatic activity of red blood cells, due to the intensification of bone marrow erythropoetic activity under physical exertion and the appearance of young red cells in peripheral blood. All the experimental groups revealed a drop in erythrocyte count (Er), hemoglobin concentration (Hb), and hematocrit values (PCV), as well as an increase in the reticulocytes count (Ret) and in the activity of all the enzymes investigated. In the fourth group anemia was detected: prolonged endurance training decreased the RBC by 24.2%, Hb by 31.1%, PCV by 26.2% and increased the reticulocyte count by 881.6%. Pronounced loading with physical effort leads to shifts in the glucose utilization ratio along particular erythrocyte metabolic pathways. This change in enzymatic activities may prove to be one of the causes of faster elimination of old RBCs.     

The impact of location and patency of the arteriovenous fistula on quality of life of kidney transplant recipients

BackgroundArteriovenous fistulae (AVFs) may remain patent after kidney transplantation (KTx), contributing to maladaptive cardiac remodeling. The flow in AVFs is associated with the diameter of its vessels and thus with the AVF location. The main objective of this study is to assess the influence of AVF location and its patency on the self-reported quality of life (QOL) of kidney transplant recipients (KTRs) with past history of hemodialysis.MethodsTo gain clinical data, during a scheduled visit, 353 KTRs were asked to fill out an anonymous questionnaire. From this group, 284 respondents were found eligible for analysis. The outcome was defined as prevalence of symptoms and health status, measured with the Left Ventricular Dysfunction-36 (LVD-36) Questionnaire in symptomatic patients.ResultsThe hemodialysis patients (n = 243) were divided into two groups according to AVF location, i.e., DAVF – distally located AVF – (n = 174) and PAVF – proximally located AVF – (n = 69). The proportion of patients with heart failure (HF) was higher in PAVF group (24% vs. 12%, p = 0.0482). In the multivariable regression, PAVF, serum creatinine levels, and the presence of HF or coronary artery disease (CAD) remained independent predictors of lower functional capacity. Among patients with heart disease, the presence of active AVF was independently associated with worse functional outcome (higher LVD-36 scores).ConclusionsThe influence of persistent PAVF in KTRs seems to be unfavorable, especially when coexisting with CAD or HF. Abbreviations: AVF arteriovenous fistula; BMI body mass index; CAD coronary artery disease; D-AVF distally-located arteriovenous fistula; EC exercise capacity; HD hemodialysis; HF heart failure; KTx kidney transplantation; KTR kidney transplant recipient; LVD-36 Left Ventricle Disfunction – 36; LVEF left ventricle ejection fraction; LVH left ventricle hypertrophy; NYHA New York Heart Association; P-AVF proximally located arteriovenous fistula; PD peritoneal dialysis; PRO patient-reported outcomes; QOL quality of life.     

Zernike representation of corneal topography height data after nonmechanical penetrating keratoplasty

PURPOSE: To demonstrate a mathematical method for decomposition of discrete corneal topography height data into a set of Zernike polynomials and to demonstrate the clinical applicability of these computations in the postkeratoplasty cornea. METHODS: Fifty consecutive patients with either Fuchs' dystrophy (n = 20) or keratoconus (n = 30) were seen at 3 months, 6 months, and 1 year (before suture removal) and again after suture removal following nonmechanical trephination with the excimer laser. Patients were assessed using regular keratometry, corneal topography (TMS-1, simulated keratometry [SimK]), subjective refraction, and best-corrected visual acuity (VA) at each interval. A set of Zernike coefficients with radial degree 8 was calculated to fit two model surfaces: a complete representation (TOTAL) and a representation with parabolic terms only to define an approximate spherocylindrical surface (PARABOLIC). The root mean square error (RMS) was calculated comparing the corneal raw height data with TOTAL (TOTALRMS) and PARABOLIC (PARABOLICRMS). The cylinder of subjective refraction was correlated with the keratometric readings, the SimK, and the respective Zernike parameter. Visual acuity was correlated with the tilt components of the Zernike expansion. RESULTS: The measured corneal surface could be approximated by the composed surface 1 with TOTALRMS < or = 1.93 microm and by surface 2 with PARABOLICRMS < or = 3.66 microm. Mean keratometric reading after suture removal was 2.8+/-0.6 D. At all follow-up examinations, the SimK yielded higher values, whereas the keratometric reading and the refractive cylinder yielded lower values than the respective Zernike parameter. The correlation of the Zernike representation and the refractive cylinder (P = 0.02 at 3 months, P = 0.05 at 6 months and at 1 year, and P = 0.01 after suture removal) was much better than the correlation of the SimK and refractive cylinder (P = 0.3 at 3 months, P = 0.4 at 6 months, P = 0.2 at 1 year, and P = 0.1 after suture removal). Visual acuity increased from 0.23+/-0.10 at the 3-month evaluation to 0.54+/-0.19 after suture removal. After suture removal, there was a statistically significant inverse correlation between VA and tilt (P = 0.02 in patients with keratoconus and P = 0.05 in those with Fuchs' dystrophy). CONCLUSIONS: Zernike representation of corneal topography height data renders a reconstruction of clinically relevant corneal topography parameters with a marked reduction of redundance and a small error. Correlation of amount/axis of refractive cylinder with respective Zernike parameters is more accurate than with keratometry or respective SimK values of corneal topography analysis.     

"Orientation teeth" in non-mechanical laser corneal trephination for penetrating keratoplasty: 2.94 microm Er:YAG v 193 nm ArF excimer laser.

BACKGROUND/AIMS: "Orientation teeth" at the donor trephination margin and correspondent "notches" at the host margin facilitate graft orientation and avoid "horizontal torsion" induced by asymmetric suture placement. In this study the quality and reproducibility of these structures created by non-mechanical laser corneal trephination were compared using two laser emissions. METHODS: The procedure was performed in 20 enucleated pigs' eyes using open metal masks with eight "orientation teeth/notches" (0.3 x 0.15 mm, base x height), an automated globe rotation device, and either a 193 nm ArF excimer laser or a Q switched 2.94 microm Er:YAG laser. "Teeth/notches" were analysed by planimetry and scanning electron microscopy (SEM). RESULTS: Mean size was 0.30 (0.027) x 0. 16 (0.017) mm for "teeth" and 0.30 (0.035) x 0.15 (0.021) mm for "notches" (excimer), and 0.31 (0.022) x 0.16 (0.015) mm and 0.30 (0.031) x 0.14 (0.021) mm respectively (Er:YAG). Overall, variability of notches was higher than that of teeth. By SEM, comparable cut regularity and sustained ablation profile were observed with both lasers. However, the corneal surface at the cut edge appeared slightly elevated (相似文献   

Nuclear Pedigree Criteria for the Identification of Individuals Suspected to Be at Risk of an Inherited Predisposition to Gastric Cancer

Gastric cancer is the second most frequently diagnosed malignancy worldwide and therefore represents a significant healthcare burden. Environmental and genetic factors are involved in the development of gastric cancer. To date only one clear genetic predisposition has been identified involving mutations in the E-cadherin gene. The disease phenotype in patients harbouring E-cadherin mutations appears to be specifically related to diffuse gastric cancer. Little is known genetically about the other forms of gastric cancer. Since there is a growing awareness about the necessity of early intervention criteria have been developed that aid the identification of hereditary forms of gastric cancer. The aim of the current study was to identify minimal inclusion criteria so that nuclear pedigree families can be provided with risk assessment and/or genetic testing.The results reveal that inclusion features described herein such as (a) gastric cancer diagnosed before 46 years of age; (b) two gastric cancers among first degree relatives diagnosed over the age of 50 are useful in identifying suspected hereditary gastric cancer patients.     

Using the Smith-Watson-Topper Parameter and Its Modifications to Calculate the Fatigue Life of Metals: The State-of-the-Art

The Smith-Watson-Topper parameter (SWT) in its original form was designed to estimate the fatigue life of metal materials in a uniaxial load state (tension–compression) in the range up to fatigue crack initiation, with non-zero mean values. This parameter is based on the analysis of both stress and strain. Therefore, the stress–strain criterion is the focus, rather than the energy criterion. This paper presents the original SWT model and its numerous modifications. The first part presents different versions of this parameter defined by the normal parameters. Then, it presents versions defined through the tangent parameter and the most promising parameter defined through the tangent and normal parameters. It was noted that the final form of the equivalent value is defined either by stress or by an energy parameter. Therefore, the possible characteristics from which the fatigue life can be determined are also presented.