Isolation and characterization of murine cell surface components. I. Purification of milligram quantities of Thy-1.1

The Thy-l.1 molecule was isolated from the BW5147 murine lymphoblastoid cell line. The initial step in purification was the preparation of a crude plasma membrane fraction followed by acetone precipitation. The acetone pellet was solubilized using deoxycholate (DOC) and Thy-1.1 was purified by use of a Lens culinaris lectin affinity column and an AcA-34 gel filtration column. The purified glycoprotein with Thy-1.1 activity had a mol wt of approximately 25,000 daltons. The isolation of this molecule was effected by detecting Thy-I activity utilizing rabbit anti- mouse brain serum tested on rat thymocytes. Congenic anti-Thy-l.1 serum was ineffective in detecting Thy-l.1 after DOC solubilization. An antiserum prepared in rabbits to the purified Thy-1.1 was found to be cytotoxic to mouse and rat thymocytes. The cytotoxic activity of this antisera could be completely absorbed with AKR/Jax brain and thymus but was not absorbed by liver. In addition, AKR/Jax thymocytes totally absorbed all cytotoxic activity of the rabbit anti-purified Thy-1 serum for BW5147 cells suggesting that the cell line shares identical specificities with normal thymocytes. The purified Thy-1.1 molecule was able to totally absorb the cytotoxic activity of mouse congenic anti-Thy-1. These studies serve as a model for the isolation of other murine lymphoid cell surface components in quantities for detailed structural and functional analysis.     

Mild hemophilia A with factor VIII assay discrepancy: using thrombin generation assay to assess the bleeding phenotype

Summary.  Introduction:  In some patients with mild hemophilia A, there are discrepancies between 1-stage (1-st) and 2-stage (2-st) factor VIII (FVIII) clotting assays, and also chromogenic assays for FVIII activity (FVIII:C). We examined whether thrombography could provide a better evaluation of the hemostatic status of these patients. Methods:  Two families with such discrepancies and markedly contrasting clinical histories were studied. Family X had no serious bleedings, in contrast to family Y. Sixty-one moderate/mild hemophiliacs without discrepancy and 15 healthy subjects served as controls. Calibrated automated thrombography was performed with platelet-rich plasma after one freeze-thawing cycle and low tissue factor concentration. Results:  The chromogenic FVIII:C levels were higher (0.90 ± 0.15 and 0.47 ± 0.13 IU mL⁻¹) than the 1-st clotting ones (0.14 ± 0.05 and 0.10 ± 0.05 IU mL⁻¹) in family X and Y, respectively ( P  < 0.001). Mean endogenous thrombin potential (ETP) was 1579 ± 359 n m  min⁻¹ and 1060 ± 450 for healthy controls and hemophilic controls, respectively. For members of family X, the ETP values were 1188, 1317 and 2277 n m  min⁻¹, whereas for those of family Y they ranged from 447 to 1122 n m  min⁻¹. Two novel missense point mutations were evidenced: p.Ile369Thr in family X and p.Phe2127Ser in family Y. In family X, we postulate that the mutation is responsible for a delayed but non-deleterious FVIII activation. Conclusions:  Our results suggest that the hemostatic phenotype assessed by thrombography may be clinically relevant in moderate/mild hemophilic patients with discrepant FVIII:C results.     

Advanced magnetic resonance imaging in benign hereditary chorea: Study of two familial cases

No brain abnormalities are usually detected on conventional magnetic resonance imaging (MRI) in benign hereditary chorea (BHC); there are currently no studies with advanced techniques in literature. We investigated whether conventional and advanced MRI techniques could depict regional brain abnormalities in two familial BHC patients and 24 healthy controls. No brain abnormalities on conventional scans were detectable; also, no significant differences in fractional anisotropy of the basal nuclei were observed. Volumetric analysis showed a decreased volume of the striatum bilaterally compared with controls, whereas spectroscopy demonstrated a significant increased myoinositol/creatine ratio bilaterally, a reduction of choline/creatine ratio bilaterally, and of N‐acetyl‐aspartate/creatine in the right putamen. With the limits of the small sample size in the patient group, these data show that, despite the absence of macroscopic changes on conventional MRI, volumetric and metabolic abnormalities are present in the basal nuclei of BHC patients. © 2010 Movement Disorder Society.     

Impact of patient characteristic factors on the dynamics of liver glucose metabolism: Evaluation of multiparametric imaging with dynamic whole-body 18F-fluorodeoxyglucose-positron emission tomography

Aims

To assess the impact of various patient characteristics on the dynamics of liver glucose metabolism using automated multiparametric imaging with whole-body dynamic ¹⁸F-fluorodeoxyglucose (FDG)-positron emission tomography (PET).

Materials and Methods

We retrospectively enrolled 540 patients who underwent whole-body dynamic FDG-PET. Three quantitative indices representing hepatic glucose metabolism [mean standardized uptake value normalized by lean body mass (SULmean), metabolic glucose rate (kinetic index) and distribution volume (DV)] were measured from multiparametric PET images produced automatically based on the Patlak plot model. Patient characteristics including age, sex, body mass index, fasting time, blood glucose level, and the presence of diabetes mellitus (DM) or hepatic steatosis (HS) were collected. We examined the correlations between the characteristic factors and three quantitative indices using multiple regression analysis.

Results

The success rate of kinetic analysis using multiparametric PET imaging was 93.3% (504/540). Hepatic SULmean was significantly correlated with age (p < .001), sex (p < .001) and blood glucose level (p = .002). DV was significantly correlated with age (p = .033), sex (p < .001), body mass index (p = .002), fasting time (p = .043) and the presence of HS (p = .002). The kinetic index was significantly correlated with age (p < .001) and sex (p = .004). In the comparison of the healthy, DM and HS groups, patients with DM had a significantly increased SULmean, whereas patients with HS had a significantly decreased DV.

Conclusions

Our results showed that liver glucose metabolism was influenced by various patient characteristic factors. Multiparametric FDG-PET imaging can be used to analyse the kinetics of liver glucose metabolism in routine clinical practice.     

Doppler velocimetry determined redistribution of fetal blood flow: Correlation with growth restriction in diamniotic monochorionic and dizygotic twins

OBJECTIVE: Our purpose was to study fetal growth and blood flow distribution in diamniotic monochorionic compared with dizygotic (diamniotic dichorionic) twins by use of Doppler velocimetry of the umbilical artery and middle cerebral artery. STUDY DESIGN: Study candidates were divided into group A, consisting of 33 pairs (66 fetuses) of diamniotic monochorionic twins, and group B, 50 pairs (100 fetuses) of diamniotic dichorionic twins. Diamniotic monochorionic placentation was confirmed by microscopic placental examination for group A. Diamniotic dichorionic placentation was ensured for group B by selecting only twins with different-sex pairs (dizygotic twins). Targeted ultrasonography with biometry was performed in each twin, and Doppler recordings of the umbilical artery and middle cerebral artery were obtained. Waveforms were analyzed and the systolic/diastolic ratio, the resistance index, and a measure of blood flow redistribution (brain-sparing effect), the cerebral/placental ratio, was calculated for each fetus. Growth status at birth was assessed by the number of small-for-gestational-age infants (≤10th percentile), low-birth-weight infants (≤25th percentile), and percent of growth discordance between twins. Intertwin differences were assessed by Δ values (value of larger twin minus value of smaller twin). RESULTS: Diamniotic monochorionic compared with dizygotic twins demonstrated a significantly greater probability of blood flow redistribution. For the study population as a whole, the brain-sparing effect was noted in 67% of small-for-gestational-age babies and only 7% of non-small-for-gestational-age infants (p ≤ 0.001). For the diamniotic monochorionic pregnancies blood flow redistribution occurred in 6 of 10 small-for-gestational-age infants (60%) and 6 of 46 non-small-for-gestational-age infants (13%). In the diamniotic monochorionic group small-for-gestational-age compared with non-small-for-gestational-age infants were more likely to show blood flow redistribution, which was the result of significantly decreased resistance in the middle cerebral artery and significantly increased resistance in the umbilical artery. Small-for-gestational-age infants (≤10th percentile) occurred much less frequently in the dizygotic group. Two of two small-for-gestational-age infants in the dizygotic group showed blood flow redistribution. Although the extremes of birth weight were more common in the diamniotic monochorionic group, both groups had relatively large numbers of small babies with birth weights in the lower 25th percentile (50.0% for diamniotic monochorionic and 44.0% for dizygotic twins, not significant). However, 42.3% (11/26) of diamniotic monochorionic twins who were in the low-birth-weight group showed blood flow redistribution compared with only 3.3% (1/30) whose birth weights were ≥25th percentile (p ≤ 0.001). In the dizygotic twins 10% of lower-birth-weight infants redistributed blood flow compared with 1% in the higher-birth-weight group, a nonsignificant difference. Diamniotic monochorionic compared with dizygotic twins were delivered earlier (32.9 weeks vs 34.8 weeks, p ≤ 0.001), were smaller (1832 gm vs 2304 gm, p ≤ 0.001), showed higher birth weight discordance (29.8% vs 14%, p ≤ 0.05), and had greater numbers (19.7% vs 2.3%, p ≤ 0.01) of infants at ≤10th percentile birth weight. CONCLUSIONS: Diamniotic monochorionic twins from the lower-birth-weight groups more often show blood flow redistribution compared with dizygotic twins of similar low birth weights. Placental vascular connections and the attendant hemodynamic changes in the fetuses of diamniotic monochorionic twins probably account for this difference. Brain-sparing events occur commonly without clinical twin transfusion syndrome in this group. These findings have implications for management. (Am J Obstet Gynecol 1998;178:1359-67.)