Nylon-fiber-induced neutrophil fragmentation

We have investigated the effects of mechanical elution of neutrophils from nylon-wool fiber (NWF) using the scanning electron microscope and biochemical analysis of elution fractions. We have determined that mechanical removal of neutrophils from nylon-wool fiber disrupts neutrophils adherent to nylon-wool fiber and augments release of granules, release of peripheral cytoplasmic fragments, and release of lactic dehydrogenase, a soluble cytoplasmic enzyme. Mechanical shearing of the adherent cell, and not adherence per se, causes the fragmentation. The extent of fragmentation is proportional to the NWF surface area available to neutrophils and is maximal at the temperature for optimal adherence and spreading. Agents that decrease cell spreading (n-ethylmaleimide and cold) diminish fragmentation. Cytochalasin B, an agent that destabilizes the neutrophil cortex, increases fragmentation. Fragmentation may be an important contributing cause of the abnormal morphology, function, and in vivo survival of nylon-wool-fiber procured human neutrophils. The prevention of fragmentation would appear to be necessary to insure the procurement of optimally functioning cells. Elution of NWF-adherent neutrophils in the cold might be a practical way to diminish neutrophil damage during clinical filtration leukapheresis.     

The relationship between CR3 deficiency and neutrophil actin assembly

Polymorphonuclear leukocytes (PMN) with a deficiency of the complement receptor type 3 (CR3) membrane glycoprotein family have impairments in the ability to adhere to surfaces as well as chemotactic and phagocytic defects, processes that require a functional contractile apparatus. PMN from the patient with neutrophil actin dysfunction (NAD) displayed similar functional characteristics to those with CR3 deficiency suggesting the two disorders may be the same disease. In order to evaluate the relationship between CR3 deficiency and actin assembly, actin filament assembly was measured in PMN from six previously reported homozygotes (two severe and four moderate CR3-deficient patients) as well as five heterozygotes for CR3 deficiency. PMN from all patients had normal unstimulated concentrations of F-actin and after exposure to the chemotactic peptide FMLP (5 x 10(-7) mol/L for 5 to 40 seconds at 25 degrees C) assembled actin normally. Pretreatment of normal PMN with concentrations of monoclonal anti-alpha CR3 antibody, capable of blocking PMN adherence, also failed to impair FMLP- induced actin filament assembly. CR3 glycoprotein expression was measured in PMNs from the mother, father, and older sister of the NAD patient (N Engl J Med 291:1093, 1974). Actin filament assembly was recently shown to be defective in PMNs from all three family members. The total concentrations of the alpha and beta CR3 subunits were below normal in PMN detergent extracts from the mother (25% of simultaneous controls) and older sister (56% of control). PMN surface expression of these two subunits was also found to be depressed (mother, 50%; older sister, 63% of control). These findings suggest these two NAD family members are heterozygote carriers for CR3 deficiency as well as NAD. Simultaneous studies of the father, however, demonstrated normal total concentrations of both the alpha and beta CR3 subunits (126% of controls) as well as normal surface expression of both subunits after phorbol myristate acetate stimulation and incubation at 37 degrees C (mean, 112% of controls) but slightly lower than normal levels after FMLP stimulation (mean, 83%). These findings indicate that CR3 deficiency generally is not associated with defective actin filament assembly and support the conclusion that NAD represents a unique kindred in which PMN actin function differs from previously reported genotypes of CR3 deficiency.     

Prevention of degradation of human polymorphonuclear leukocyte proteins by diisopropylfluorophosphate

Proteases can complicate the characterization of proteins from cells, especially human polymorphonuclear leukocytes (PMN), which contain abundant neutral proteases. We tested the ability of agents to inhibit proteolysis, with special reference to the subunit polypeptides of the contractile proteins actin, myosin, and actin-binding protein (ABP). Phenylmethylsulfonyl fluoride (PMSF), O-phenanthroline, EGTA, EDTA, N- ethylmaleimide, alone or in combinations, failed to prevent extensive proteolysis of the PMN proteins during solubilization of cells with dodecyl sulfate. These inhibitors and also alpha-1-antitrypsin and soybean trypsin inhibitor similarly could not prevent proteolysis during homogenization of cells in cold isosomolar sucrose. Treatment of PMN with greater than or equal to mM diisopropylfluorophosphate (DFP) prior to solubilization or homogenization markedly inhibited proteolysis. PMSF and DFP were equally effective in inhibiting proteolysis in PMN extracts, suggesting that the efficacy of DFP may result from its permeation of intact cells and granules before barriers are disrupted by detergents or homogenization. Treatment of PMN with DFP under conditions inhibiting proteolysis did not affect their rate of phagocytosis. We recommend the use of DFP in future studies correlating functions and protein structure of PMN.     

The severity of immune neutropenia correlates with the maturational specificity of antineutrophil antibodies

Sera from 15 patients with antineutrophil antibodies against peripheral blood neutrophils were incubated with normal bone marrow, and the reaction with marrow precursors was determined. Serum from controls did not deposit IgG on either peripheral blood or marrow myeloid cells. Serum from patients with moderate immune neutropenia with myeloid hyperplasia deposited IgG on neutrophils, bands and metamyelocytes, but not on earlier precursors. Serum from one patient with severe neutropenia and myeloid hypoplasia deposited IgG on myelocytes and promyelocytes as well as more mature cells. Serum from a patient with antineutrophil antibodies which impaired chemotaxis but who did not have neutropenia deposited IgG only on neutrophils and occasional bands. Immune neutropenia may become increasingly severe as antibodies become directed against earlier precursors.     

Readability of patient education materials available at the point of care

Background

Many patient education materials (PEMs) available on the internet are written at high school or college reading levels, rendering them inaccessible to the average US resident, who reads at or below an 8^th grade level. Currently, electronic health record (EHR) providers partner with companies that produce PEMs, allowing clinicians to access PEMs at the point of care.

Objective

To assess the readability of PEMs provided by a popular EHR vendor as well as the National Library of Medicine (NLM).

Design

We included PEMs from Micromedex, EBSCO, and MedlinePlus. Micromedex and EBSCO supply PEMs to Meditech, a popular EHR supplier in the US. MedlinePlus supplies the NLM. These PEM databases have high market penetration and accessibility.

Measurements

Grade reading level of the PEMs was calculated using three validated indices: Simple Measure of Gobbledygook (SMOG), Gunning Fog (GFI), and Flesch–Kincaid (FKI). The percentage of documents above target readability and average readability scores from each database were calculated.

Results

We randomly sampled 100 disease-matched PEMs from three databases (n = 300 PEMs). Depending on the readability index used, 30-100% of PEMs were written above the 8^th grade level. The average reading level for MedlinePlus, EBSCO, and Micromedex PEMs was 10.2 (1.9), 9.7 (1.3), and 8.6 (0.9), respectively (p ≤ 0.000) as estimated by the GFI. Estimates of readability using SMOG and FKI were similar.

Conclusions

The majority of PEMS available through the NLM and a popular EHR were written at reading levels considerably higher than that of the average US adult.KEY WORDS: readability, health literacy, patient education materials, electronic health records     

In vitro function and phagocytosis of galactosylated platelet concentrates after long-term refrigeration

BACKGROUND: Short-term refrigeration of platelets (PLTs) in the absence of plasma results in their rapid clearance after transfusion. Blocking beta-N-acetylglucosamine (beta-GlcNAc) residues of glycoprotein Ibalpha (GPIbalpha) with galactose prevents binding of refrigerated human and mouse PLTs to macrophages and prolongs the circulation times of refrigerated mouse PLTs. PLT-associated galactosyltransferase efficiently galactosylates chilled PLTs in the presence of its substrate UDP-galactose is added to PLT-rich plasma. STUDY DESIGN AND METHODS: To characterize the hemostatic function of refrigerated and galactosylated human PLTs processed in the blood bank, PLT aggregation was studied in vitro under static and flow conditions and expression of integrin beta3 (CD61), CD62P (P-selectin), GPIbalpha (CD42b), annexin V binding, and integrin alphaIIbeta3 activation with flow cytometry. Affinity of macrophages for galactosylated refrigerated PLTs was evaluated with THP-1 cells, which recognize and phagocytize refrigerated PLTs. RESULTS: PLTs refrigerated and galactosylated for 14 days 1) maintained their ability to aggregate when exposed to agonists in a standard aggregometry assay, 2) showed less pronounced changes in surface expression of GPIbalpha compared with room temperature (RT)-stored PLTs, 3) increased P-selectin expression, and 4) were poorly phagocytized by differentiated THP-1 cells in vitro. In addition, it is shown that refrigeration of PLTs does not affect their adhesive properties under in vitro flow conditions. CONCLUSION: It is shown that refrigerated human PLTs retain in vitro function better than RT PLTs during storage and demonstrate that galactosylation prevents recognition of stored refrigerated PLTs by macrophages in vitro.     

Influence of hemoglobin precipitation on erythrocyte metabolism in alpha and beta thalassemia

Certain aspects of the metabolism of centrifuged young and old erythrocytes in hemoglobin H disease have been examined and compared with similar studies of beta thalassemia and normal cells. Glycolysis, hexose monophosphate shunt activity (HMPS), potassium flux, and glutathione (GSH) content were measured. The distributions of hemoglobins H and F, as well as the activities of erythrocyte glucose-6-phosphate dehydrogenase (G6PD) and glutamic oxalacetic transaminase (GOT), were utilized for estimations of the relative ages of the cell samples. The young erythrocytes in hemoglobin H disease differed in several respects from older hemoglobin H cells. They contained more soluble hemoglobin H and GSH and, after splenectomy, fewer inclusions. HMPS activity was subnormal in hemoglobin H young cells and rose to normal activity in old cells. Potassium flux tended to increase in old cells when inclusions were present.Beta thalassemia young cells contained less hemoglobin F and, after splenectomy, more inclusions than old cells. In addition, they had markedly increased glycolysis and HMPS activity. GSH was randomly distributed. Potassium flux was increased in younger cells and particularly increased when inclusions appeared in younger cells after splenectomy.The results are interpreted to indicate that inclusion formation is associated with increased erythrocyte cation permeability in the thalassemia syndromes. This is not related to the level of intracellular GSH.The decreased HMPS activity in young hemoglobin H cells may be due to the presence of the extra thiols of soluble hemoglobin H which can act as a reducing agent. The substitution of hemoglobin H for glutathione in this capacity would then spare the NADPH-requiring glutathione reductase system. As a consequence, HMPS activity would decline. However, in older cells the oxidized hemoglobin H precipitates; these must rely upon GSH and glutathione reductase activity for thiol reduction capacity. Accordingly, HMPS activity increases to normal in the old cell population.     

Detection, Pathogenesis, and Prevention of Damage to Human Granulocytes Caused by Interaction with Nylon Wool Fiber: IMPLICATIONS FOR FILTRATION LEUKAPHERESIS

Granulocytes collected by reversible adhesion to nylon wool fiber (NWF) function relatively well in standard in vitro tests; however, they have an abnormally shortened survival time in the circulation. Assuming that this rapid disappearance represents clearance and that recognition by phagocytes is important for such clearance, we used an autologous in vitro cell:cell recognition assay to determine whether phagocytes can detect cellular changes induced by exposure of normal granulocytes to NWF. Human granulocytes incubated with NWF 1 h at 37 degrees C, eluted with 20% acid citrate dextrose plasma, and washed stimulated the hexose monophosphate shunt activity of normal granulocytes an average of twofold (193+/-40% of controls), indicating a recognition response. NWF-induced granulocyte recognition was not dependent on plasma factors or activated complement components but was dependent on the time that the granulocyte was on the NWF and was maximal by 60 min of exposure. After elution from NWF, granulocytes demonstrated resting glucose oxidation rates only slightly higher than normal; however, during the first 20 min of exposure to NWF, granulocytes increased their rate of (14)CO(2) production from [1-(14)C]glucose three- to five-fold. Therefore, experiments were performed to determine whether toxic oxygen metabolites produced by NWF-adherent cells might contribute to recognition. The results showed that (a) normal granulocytes exposed to NWF in the presence of scavengers of superoxide anion (superoxide dismutase) or free radicals (ascorbate, mannitol, or benzoate) and washed before assay did not stimulate glucose oxidation of indicator granulocytes; and (b) NWF granulocytes prepared from cells unable to generate high levels of toxic oxygen metabolites, i.e. cells prepared anaerobically or from a patient with chronic granulomatous disease, also failed to stimulate indicator granulocytes. Human granulocytes placed in contact with NWF show an oxidative burst and become recognizable to other phagocytes. Free radical scavengers are effective in minimizing this recognition conferred on NWF-procured granulocytes.