Heterogeneity of human whole blood platelet subpopulations. III. Density-dependent differences in subcellular constituents

Structurally intact platelet cohorts of differing densities can be isolated from normal subjects by the use of isosmolar arabinogalactan density gradients. Using platelets separated in this fashion, we have studied the density-dependent distribution of four subcellular organelles: mitochondria, lysosomes, dense bodies, and alpha granules. Mitochondria, which are not secreted during platelet release, demonstrate a slow decline in monoamine oxidase activity within the gradient. Lysosomal beta-glucuronidase does not vary significantly with platelet density. In contrast, dense body number and endogenous serotonin content decrease significantly with decreasing platelet density, primarily as the result of differences in the number of storage organelles. Platelet factor 4 content declines rapidly in comparison to lysosomal activities (P less than .001 from bottom to top of the gradient); but beta-thromboglobulin, also an alpha granule component, exhibits considerably less change than platelet factor 4 (P less than .001). Thus, specific platelet subcellular constituents have different density distributions. We postulate that these density differences may be due to differential in vivo loss of selective biochemical constituents from unique subcellular compartments.     

Immunologic status of hemophilia patients treated with cryoprecipitate or lyophilized concentrate

We evaluated 37 patients with moderate or severe hemophilia A and six patients with severe factor IX deficiency for clinical or laboratory evidence of immune abnormalities. Patients were assigned to one of four groups according to the type of clotting factor replacement. Twenty patients had received only cryoprecipitate during the two years preceding the evaluation (group I); 11 additional patients were treated predominantly with cryoprecipitate but had also received up to nine bottles of factor VIII concentrate (group II); six patients received factor VIII concentrate (group III); six patients received factor IX concentrate (group IV). There was no clinical or laboratory evidence of immunodeficiency among the 43 patients. The mean absolute number of Th cells was normal in all patient groups, but the mean absolute number of Ts cells was increased compared with controls, both in patients treated with cryoprecipitate and in patients treated with factor VIII or factor IX concentrate. There was no correlation between the Th/Ts ratio and patient age, alanine aminotransferase level, hepatitis serology, in vitro lymphocyte function, or amount of clotting factor administered. Our observations demonstrate that the volunteer or commercial origin of clotting factor replacement cannot fully explain the alterations in lymphocyte subset distribution previously described in patients with hemophilia A.     

Enhancement of chemotactic factor-stimulated neutrophil oxidative metabolism by leukotriene B4

Leukotriene B4 (LTB4) is a potent primary stimulator of neutrophil chemotaxis, aggregation, and degranulation and induces superoxide production at higher concentrations. In order to determine whether LTB4 modulates neutrophil responses to oxidative stimuli, human neutrophils (PMNs) were incubated with LTB4 prior to stimulation with f-Met-Leu-Phe (fMLP, 10(-7) mol/L), opsonized zymosan (OZ, 250 micrograms/mL), or phorbol myristate acetate (PMA, 32 nmol/L). Superoxide (O2-) production by stimulated PMNs was assessed by the superoxide dismutase-inhibitable reduction of cytochrome c. LTB4 alone did not stimulate O2- production in concentrations below 10(-7) mol/L and had no effect on the O2- assay. In the concentration range of 10(-12) to 10(-8) mol/L, LTB4 did not alter O2- release induced by OZ or PMA. In contrast, LTB4-treated cells demonstrated enhanced O2- production following exposure to fMLP, and in the presence of 10 nmol/LLTB4, generated 180% +/- 41% of O-2 quantities produced by control cells (n = 23). Enhancement was LTB4 dose-dependent, was maximal in the range of 1 to 10 nmol/L LTB4, was not reversed by removal of the lipid from the medium prior to fMLP stimulation, and was not dependent on the presence of Ca++ or Mg++ in the suspending medium. Chemiluminescence of fMLP-stimulated neutrophils was increased to 323% of controls in neutrophils preincubated with 10 nmol/L LTB4. Unlike augmentation of oxidative responses to fMLP seen with other degranulating stimuli, enhancement by LTB4 was not correlated with an increase in 3H-fMLP receptor binding. These results indicate that, in addition to its primary effects on neutrophil function, LTB4 modulates PMN oxidative responses to the chemotactic peptide and, thus, may amplify the release of oxygen metabolites at inflammatory foci.     

Protein kinase C inhibits epidermal growth factor-dependent tyrosine phosphorylation of phospholipase C gamma and activation of phosphoinositide hydrolysis.

Recent studies have shown that the receptor for epidermal growth factor (EGF) can associate with and tyrosine-phosphorylate the gamma-isozyme of phosphoinositide (PtdIns)-specific phospholipase C (PLC gamma), suggesting a possible mechanism for activation of PtdIns hydrolysis by EGF. In the present study, the coupling between PtdIns hydrolysis and PLC gamma tyrosine phosphorylation in WB liver epithelial cells was examined. Peak levels of [P-Tyr]PLC gamma, measured by anti-P-Tyr immunoblotting, occurred at 0.5-2 min of EGF treatment and coincided with the onset of [3H]inositol phosphate production. The termination of PtdIns hydrolysis after EGF stimulation was accompanied by return of [P-Tyr]PLC gamma to near-basal levels. Activation of protein kinase C (PKC) with a phorbol ester inhibited (IC50 = 3-10 nM) both EGF-dependent PtdIns hydrolysis and PLC gamma phosphorylation by more than 90%. Both EGF-stimulated responses were potentiated in cells depleted of PKC by prolonged phorbol ester treatment. At physiological ionic strength, monoclonal antibodies to PLC gamma specifically precipitated (in addition to PLC gamma) the EGF receptor and at least six other [P-Tyr]proteins from extracts of EGF-treated cells. PKC activation had differential effects on the tyrosine phosphorylation of these coprecipitating proteins, i.e. the relative abundance of certain [P-Tyr] proteins decreased, whereas that of another protein increased. In conclusion, EGF-stimulated tyrosine phosphorylation of PLC gamma is broadly correlated with stimulation of PtdIns hydrolysis, consistent with a role for tyrosine phosphorylation in PLC activation. The attendant diacylglycerol release and activation of PKC may terminate PLC gamma activation, in part by inhibiting PLC gamma phosphorylation by the EGF receptor. Our results suggest further that PKC may exert regulatory effects by altering the relationship of PLC gamma to its associated [P-Tyr]proteins.     

Pulsatile masses surrounding vascular prostheses: real-time US color flow imaging

A prospective evaluation of color flow mapping and real-time ultrasound was performed to determine if pseudoaneurysms could be distinguished from other causes of masses surrounding vascular grafts of the lower extremities. Twelve palpable pulsatile masses were imaged. Diagnoses were confirmed at angiography (n = 11), computed tomography (n = 7), aspiration biopsy (n = 5), and operative intervention (n = 6). A swirling pattern of blood flow was seen in six of seven cases of pseudoaneurysm. Lack of flow signals was noted in four of the five collections representing hematoma (n = 2) or infection (n = 2). The seventh case was later shown to be an infected, thrombosed pseudoaneurysm. The single false-positive diagnosis was made early in the series when the flow signals detected were due to transmitted arterial pulsations. The authors conclude that color Doppler flow imaging is useful in the differential diagnosis of pulsatile masses associated with prosthetic grafts. Prosthetic graft pseudoaneurysms have a specific appearance of swirling blood flow arising from a wide neck and are distinguishable from traumatic or iatrogenic pseudoaneurysms of the native vascular tree.     

New universal precaution aspiration tray

A new tray has been designed for use during procedures involving needles and other sharp objects. The tray includes a foam adhesive pad, marked into 10 sections, into which the sharp objects can be placed point first. After the procedure, the objects can be safely withdrawn by their handles and then discarded. The tray has been used in more than 250 procedures.     

Outcome of mid-urethral sling procedures in Korean women with stress urinary incontinence according to body mass index

BACKGROUND: The objective of this study was to determine whether the outcome of mid-urethral sling procedures is influenced by the body mass index of Korean women suffering from stress urinary incontinence (SUI). METHODS: A total of 285 women, ranging in age from 28 to 80 years (mean 55.4), all of whom were followed up for at least 6 months, were ultimately included in this study. The patients were classified as follows: normal weight, 18.5-23 kg/m2; overweight, 23-27.5 kg/m2; obesity, 27.5 kg/m2 or higher. RESULTS: We noted bladder perforations in 11 cases (4.9%, 3.8% and 2.2% in the normal weight, overweight, and obesity groups, respectively; P = 0.449). We determined there to be no significant differences among the three groups with regard to cure rate (P = 0.173). The rates of postoperative urinary retention were 9.9% in the normal weight group, 10.1% in the overweight group, and 15.6% in the obesity group (P = 0.396). We determined there to be no significant differences among the three groups with regard to the persistence of urgency (P = 0.312). Seventy-nine patients (27.7%) exhibited symptoms indicative of voiding disorder (hesitancy, poor flow, or sensations of incomplete emptying). The postoperative development of these voiding symptoms was not significantly different among the three groups (P = 0.106). CONCLUSION: Our results demonstrate both the feasibility and the safety of mid-urethral sling procedures for obese Korean women who suffer from SUI. Additional studies, including prospective randomized trials with longer follow-up periods, will be required in order to confirm these findings.