Infusible platelet membrane microvesicles: a potential transfusion substitute for platelets

BACKGROUND: Several substitutes for intact, viable platelets have been used for transfusion, both to people and in animal models, with varied success. Infusible platelet membrane (IPM) is prepared from human platelets. IPM retains the glycoprotein (GP)lb receptor and has platelet factor 3 activity (procoagulant activity). However, factor V, serotonin, a cytoplasmic marker enzyme (purine nucleotide phosphorylase), GPIIb/IIIa complex, and HLA class I and II antigens are all absent in IPM. STUDY DESIGN AND METHODS: IPM is prepared from outdated platelets. The platelets were disrupted by freezing and thawing; they were washed and heated to inactivate possible viral contaminants, and then the sonicated membrane microvesicle fraction was separated and lyophilized. The hemostatic activity of IPM was measured by its ability to reduce the prolonged bleeding time in thrombocytopenic rabbits. RESULTS: Administration of IPM at a dose of 2 mg per kg results in a substantial reduction in the bleeding time. In a series of 23 experiments, a median preinjection bleeding time of 15 minutes was reduced to 6 minutes within 4 hours after IPM administration. Administration of IPM did show a mild enhancement in the thrombogenicity index, as measured in the Wessler rabbit model. This enhancement is, however, not significant, as a thrombogenicity index value of up to 0.6 is clinically acceptable. CONCLUSION: IPM may have clinical potential as a substitute for platelets in the treatment of bleeding due to thrombocytopenia.     

Alveolar inflation during generation of a quasi-static pressure/volume curve in the acutely injured lung

OBJECTIVE: Lower and upper inflection points on the quasi-static curve representing a composite of pressure/volume from the whole lung are hypothesized to represent initial alveolar recruitment and overdistension, respectively, and are currently utilized to adjust mechanical ventilation in patients with acute respiratory distress syndrome. However, alveoli have never been directly observed during the generation of a pressure/volume curve to confirm this hypothesis. In this study, we visualized the inflation of individual alveoli during the generation of a pressure/volume curve by direct visualization using in vivo microscopy in a surfactant deactivation model of lung injury in pigs. DESIGN: Prospective, observational, controlled study. SETTING: University research laboratory. SUBJECTS: Eight adult pigs. INTERVENTIONS: Pigs were anesthetized and administered mechanical ventilation, underwent a left thoracotomy, and were separated into two groups: control pigs (n = 3) were subjected to surgical intervention, and Tween lavage pigs (n = 5) were subjected to surgical intervention plus surfactant deactivation by Tween lavage (1.5 mL/kg 5% solution of Tween in saline). The microscope was then attached to the lung, and the size of each was alveolus quantified by measuring the alveolar area by computer image analysis. Each alveolus in the microscopic field was assigned to one of three types, based on alveolar mechanics: type I, no visible change in alveolar size during ventilation; type II, alveoli visibly change size during ventilation but do not totally collapse at end expiration; and type III, alveoli visibly change size during tidal ventilation and completely collapse at end expiration. After alveolar classification, the animals were disconnected from the ventilator and attached to a super syringe filled with 100% oxygen. The lung was inflated from 0 to 220 mL in 20-mL increments with a 10-sec pause between increments for airway pressure and alveolar confirmation to stabilize. These data were utilized to generate both quasi-static pressure/volume curves and individual alveolar pressure/area curves. MEASUREMENTS AND MAIN RESULTS: The normal lung quasi-static pressure/volume curve has a single lower inflection point, whereas the curve after Tween has an inflection point at 8 mm Hg and a second at 24 mm Hg. Normal alveoli in the control group are all type I and do not change size appreciably during generation of the quasi-static pressure/volume curve. Surfactant deactivation causes a heterogenous injury, with all three alveolar types present in the same microscopic field. The inflation pattern of each alveolar type after surfactant deactivation by Tween was notably different. Type I alveoli in either the control or Tween group demonstrated minimal change in alveolar area with lung inflation. Type I alveolar area was significantly (p <.05) larger in the control as compared with the Tween group. In the Tween group, type II alveoli increased significantly in area, with lung inflation from 0 mL (9666 +/- 1340 microm2) to 40 mL (12,935 +/- 1725 microm2) but did not increase further (220 mL, 14,058 +/- 1740 microm2) with lung inflation. Type III alveoli initially recruited with a relatively small area (20 mL lung volume, 798 +/- 797 microm2) and progressively increased in area throughout lung inflation (120 mL, 7302 +/- 1405 microm2; 220 mL, 11,460 +/- 1078 microm2) CONCLUSION: The normal lung does not increase in volume by simple isotropic (balloon-like) expansion of alveoli, as evidenced by the horizontal (no change in alveolar area with increases in airway pressure) pressure/area curve. After surfactant deactivation, the alveolar inflation pattern becomes very complex, with each alveolar type (I, II, and III) displaying a distinct pattern. None of the alveolar pressure/area curves directly parallel the quasi-static lung pressure/volume curve. Of the 16, only one type III atelectatic alveolus recruited at the first inflection point and only five recruited concomitant with the second inflation point, suggesting that neither inflection point was due to inflection point was due to massive alveolar recruitment. Thus, the components responsible for the shape of the pressure/volume curve include all of the individual alveolar pressure/area curves, plus changes in alveolar duct and airway size, and the elastic forces in the pulmonary parenchyma and the chest wall.     

Partial redirection of transgenic human growth hormone secretion from rat salivary glands

Regulated secretory pathway proteins, when delivered as transgenes to salivary glands, are secreted predominantly into saliva. This is not useful for those proteins whose therapeutic function is required systemically, for example, human growth hormone (hGH). One strategy to improve the efficiency of hGH secretion into the bloodstream involves manipulation of existing sorting signals. The C terminus of hGH is highly conserved and contains a domain similar to the regulated pathway sorting domain of pro-opiomelanocortin (POMC). We hypothesized that, similar to POMC, mutation of this domain would divert hGH secretion from the regulated to the constitutive pathway, which in salivary glands leads to the bloodstream. Several mutations were made in the C terminus of the hGH cDNA and tested in vitro. One biologically active mutant containing E174A and E186A substitutions, and with an included C-terminal extension, was studied in greater detail. Compared with wild-type hGH, we found that this mutant hGH accumulated in the Golgi/trans-Golgi network and showed increased basal secretion in AtT20 cells, a model endocrine cell line. Importantly, in vivo, the mutant hGH displayed a relative increase in the proportion of constitutive pathway secretion seen from rat salivary glands, with a significantly lower saliva-versus-serum secretion ratio (p=0.03). Although this mutant is unlikely to be therapeutically beneficial, these results suggest that the final destination of a transgenic secretory protein may be controlled by reengineering its sorting determinants.     

Effects of Macroplastique® Implantation System for stress urinary incontinence and urethral hypermobility in women

A study was carried out to evaluate efficacy of Macroplastique® (MPQ) Implantation System (MIS) in women with urodynamic stress urinary incontinence (SUI) and urethral hypermobility after an unsuccessful conservative treatment. This is a prospective randomized controlled trial in women without previous incontinence surgery. Twenty-four women received MPQ. Twenty-one controls underwent a pelvic floor muscle exercises home program. Follow-up was at 3 months and the MPQ group also at 12 months. At 3 months, pad usage decreased significantly more in the MPQ group than in the control group (p?=?0.015). According to physician and patient self-assessment, respectively, 71% and 63% women in the MPQ group were considered cured or markedly improved. This was significantly higher compared to controls. There was a significant higher increase of Incontinence Quality-of-Life questionnaire score in the MPQ group compared to controls (p?=?0.017). Improvements in MPQ group at 3 months are sustained to 12 months. Adverse events were mild and transient. MIS is an acceptable option for women with SUI and urethral hypermobility.     

The expression of type III hyperlipoproteinemia: involvement of lipolysis genes

Type III hyperlipoproteinemia (HLP) is mainly found in homozygous apolipoprotein (APO) E2 (R158C) carriers. Genetic factors contributing to the expression of type III HLP were investigated in 113 hyper- and 52 normolipidemic E2/2 subjects, by testing for polymorphisms in APOC3, APOA5, HL (hepatic lipase) and LPL (lipoprotein lipase) genes. In addition, 188 normolipidemic Dutch control panels (NDCP) and 141 hypertriglyceridemic (HTG) patients were genotyped as well. No associations were found for four HL gene polymorphisms and two LPL gene polymorphisms and type III HLP. The frequency of the rare allele of APOC3 3238 G>C and APOA5 −1131 T>C (in linkage disequilibrium) was significantly higher in type III HLP patients when compared with normolipidemic E2/2 subjects, 15.6 vs 6.9% and 15.1 vs 5.8%, respectively, (P<0.05). Furthermore, the frequencies of the APOA5 c.56 G>C polymorphism and LPL c.27 G>A mutation were higher in type III HLP patients, though not significant. Some 58% of the type III HLP patients carried either the APOA5 −1131 T>C, c.56 G>C and/or LPL c.27 G>A mutation as compared to 27% of the normolipidemic APOE2/2 subjects (odds ratio 3.7, 95% confidence interval=1.8–7.5, P<0.0001). The HTG patients showed similar allele frequencies of the APOA5, APOC3 and LPL polymorphisms, whereas the NDCP showed similar allele frequencies as the normolipidemic APOE2/2. Patients with the APOC3 3238 G>C/APOA5 −1131 T>C polymorphism showed a more severe hyperlipidemia than patients without this polymorphism. Polymorphisms in lipolysis genes associate with the expression and severity of type III HLP in APOE2/2.     

Daily-life activities and in-shoe forefoot plantar pressure in patients with diabetes

OBJECTIVE: To assess differences regarding in-shoe forefoot plantar pressure (PP) in patients with diabetes during various daily-life activities. RESEARCH DESIGN AND METHODS: In-shoe PP was measured in 93 patients during: level walking, ramp and stair walking, turning in different settings and while performing the Up & Go test. Separate PPs were determined for the big toe and metatarsal (mt) regions one to five. RESULTS: Across all activities, similar PPs were measured in the big toe and mt-1 to mt-3 region. Lower PPs were measured in mt-4 and mt-5 region. PPs during level walking were mostly higher when compared to the other activities (p相似文献   

Radioimmunoassay and tandem mass spectrometry measurement of bedtime salivary cortisol levels: a comparison of assays to establish hypercortisolism

CONTEXT: Although bedtime salivary cortisol measurement has been proposed as the optimal screening test for the diagnosis of Cushing's syndrome, its performance using commercially available assays has not been widely evaluated. OBJECTIVE: Our objective was to compare RIA and tandem mass spectrometry (LC-MS/MS) measurement of salivary cortisol in obese subjects and healthy volunteers. DESIGN AND SETTING: We conducted a cross-sectional prospective study of outpatients. SUBJECTS AND METHODS: We studied 261 obese subjects (186 female) with at least two additional features of Cushing's syndrome and 60 healthy volunteers (30 female). Subjects provided split bedtime salivary samples for cortisol measurement by commercially available RIA and LC-MS/MS. Results were considered normal or abnormal based on the laboratory reference range. Subjects with abnormal results underwent evaluation for Cushing's syndrome. RESULTS: In paired samples, RIA gave a lower specificity than LC-MS/MS in obese subjects (86 vs. 94%, P = 0.008) but not healthy volunteers (86 vs. 82%, P = 0.71). Among subjects with at least one abnormal result, both values were abnormal in 44% (confidence interval 26-62%) of obese and 75% (confidence interval 33-96%) of healthy volunteers. In obese subjects, salivary cortisol concentrations were less than 4.0 to 643 ng/dl (<0.11-17.7 nmol/liter; normal, < or =100 ng/dl, 2.80 nmol/liter) by LC-MS/MS and less than 50 to 2800 ng/dl (1.4-77.3 nmol/liter; normal, < or =170 ng/dl, 4.7 nmol/liter) by RIA. Cushing's syndrome was not diagnosed in any subject. CONCLUSION: Salivary cortisol levels should not be used as the sole test to diagnose Cushing's syndrome if laboratory-provided reference ranges are used for diagnostic interpretation.     

Mouse behavioral mutants have neuroimaging abnormalities

Impaired cognitive, memory, or motor performance is a distinguishing characteristic of neurological diseases. Although these symptoms are frequently the most evident in human patients, additional markers of disease are critical for proper diagnosis and staging. Noninvasive neuroimaging methods have become essential in this capacity and provide means of evaluating disease and tracking progression. These imaging methods are also becoming available to scientists in the research laboratory for assessment of animal models of neurological disease. Imaging in mouse models of neurological disease is of particular interest, owing to the availability of inbred strains and genetic manipulation tools that permit detailed investigation of the roles of various genes and gene products in disease pathogenesis. However, the relative prevalence of neuroimaging abnormalities in mice exhibiting neurological symptoms has not been reported. This prevalence has both theoretical and practical value because it is influenced by both the sensitivity of macroscopic anatomical measures to underlying genetic and disease processes and by the efficiency of neuroimaging in detecting and characterizing these effects. In this paper, we describe a meta-analysis of studies involving behavioral mouse mutants at our laboratory. In summary, we have evaluated 15 different mutant genotypes, of which 13 showed abnormal neuroimaging findings. This indicates a surprisingly high prevalence of neuroimaging abnormalities (87%) and suggests that disease processes affecting behavior generally alter neuroanatomy as well. As a consequence, neuroimaging provides a highly sensitive marker of neurological disease in mice exhibiting abnormal behavior.     

Effect of NSAID on muscle injury and oxidative stress

Indirect markers of muscle damage and delayed onset muscle soreness were examined and correlated to changes in oxidative stress, plasma antioxidant potential, and use or nonuse of non-steroidal anti-inflammatory drugs in 60 ultra-marathoners following the Western States Endurance Run. Blood was collected prior to and immediately following the race and analyzed for muscle damage by creatine phosphokinase and oxidative stress by F (2)-isoprostanes, protein carbonyls, and lipid hydroperoxides and antioxidant potential by the ferric reducing ability of plasma. Subjects recorded delayed onset muscle soreness during the week following the race. Lipid hydroperoxide concentrations were unchanged, but F (2)-isoprostanes, protein carbonyls, ferric reducing ability of plasma, creatine phosphokinase, and delayed onset muscle soreness increased significantly postrace. Protein carbonyls were significantly higher postrace in nonsteroidal anti-inflammatory drug users versus nonusers (p < 0.05). However, there was no difference between users and non-users for all other markers. Postrace creatine phosphokinase concentrations were not correlated with oxidative stress markers but were correlated with changes in delayed onset muscle soreness. Based upon these findings, caution should be used when consuming nonsteroidal anti-inflammatory drugs during ultra distance events.