Influence of oxygen tension on the viscoelastic behavior of red blood cells in sickle cell disease

Although the rheological behavior of sickle cell suspensions and of hemoglobin S solutions is known to be strongly dependent on oxygen tension (PO2), little data exist concerning the influence of PO2 on the viscoelasticity of individual HbSS RBC. We have used micropipette aspiration techniques to test the deformation response of both HbSS and control HbAA RBC over a wide range of PO2 at 23 degrees C. Sickled, spiculed HbSS cells were present for PO2 approximately less than 35 mm Hg; for a number of these cells, the deformation response was essentially elastic and an effective membrane rigidity (EMR) was calculated. EMR increased with decreasing PO2 and was approximately 5 to 50 times higher than the equivalent rigidity of oxygenated HbSS RBC. In addition, the rate of membrane deformation was very slow for sickled cells; the half-time for the deformation process increased as PO2 was lowered and was about two orders of magnitude longer than the equivalent time for normal RBC. Other sickled cells exhibited plastic deformation when subjected to comparable deforming forces and experienced irreversible membrane deformation and budding. At all PO2 levels tested, some HbSS RBC remained as discocytes; these cells had normal membrane elasticity and membrane viscosity. Furthermore, changes in PO2 did not affect the membrane properties of HbAA RBC. Thus, gross abnormalities in the deformation response of HbSS RBC were only detected after morphological sickling had occurred. These abnormalities most likely arose from changes in the cytoplasmic HbS viscoelasticity and, if present in vivo, would be expected to impair the flow of HbSS cells in the microcirculation.     

Dihydrotestosterone and Leptin Regulate Gonadotropin-Releasing Hormone (GnRH) Expression and Secretion in Human GnRH-Secreting Neuroblasts

IntroductionThe reversal of hypogonadotropic hypogonadism (HH), occurring after discontinuation of testosterone therapy in adolescents with delayed puberty and in a small percentage of adults with congenital HH, suggests a role for androgens in favoring a spontaneous recovery of reproductive function.AimWe investigated the effect of androgens and leptin on gonadotropin-releasing hormone (GnRH) expression and secretion in human GnRH-secreting neuroblasts (FNC-B4).MethodsQuantitative real-time polymerase chain reaction RT-PCR for mRNA expression and radioimmunoassay for GnRH secretion were used. Immunohistochemical studies assessed GnRH protein expression. FNC-B4 migration was analyzed with multiwell Boyden chamber technique.Main Outcome MeasuresEffects of the non-aromatizable androgen dihydrotestosterone (DHT) and leptin in FNC-B4 were tested after 24 and 48 hours.ResultsExposure to increasing concentrations of DHT after 24 hours significantly stimulated GnRH mRNA in FNC-B4. This effect was still present after prolonged exposure (48 hours). Similarly, treatment with leptin significantly induced GnRH mRNA after 24 hours, but not at 48 hours. Interestingly, mRNA for leptin receptors (LEPR) was significantly reduced after 48 hours of leptin, while, at this time point, it was stimulated by DHT. Coincubation for 48 hours with leptin and DHT maintained the stimulatory effect on both GnRH and LEPR mRNA, suggesting that DHT could stabilize the leptin effect by preventing downregulation of LEPR. Similar results were obtained for GnRH protein expression analysis. Moreover, both DHT and leptin increased GnRH release into the culture medium. We also found that DHT or leptin treatment significantly increased FNC-B4 basal migration. As we previously found that GnRH stimulates FNC-B4 migration, we hypothesized that this effect could be mediated by DHT- and leptin-induced GnRH release. Accordingly, the GnRH antagonist cetrorelix inhibited DHT- and leptin-induced migration.ConclusionOur results suggest that androgens (adequate hormonal status) could have a positive effect on GnRH neuronal activity by synergizing with leptin (adequate energy status) in the regulatory mechanisms required for reproductive and sexual fitness. Morelli A, Fibbi B, Marini M, Silvestrini E, De Vita G, Chavalmane AK, Vignozzi L, Filippi S, Forti G, Vannelli GB, and Maggi M. Dihydrotestosterone and leptin regulate gonadotropin-releasing hormone (GnRH) expression and secretion in human GnRH-secreting neuroblasts. J Sex Med 2009;6:397–407.     

壳聚糖/聚乙二醇琥珀酸酯薄膜的制备及其与肌成纤维细胞的相容性

目的：制备防粘连壳聚糖/聚乙二醇琥珀酸酯薄膜并观察其与肌成纤维细胞的相容性。方法：实验于2006-05/11在南方医科大学附属珠江医院中心实验室完成。实验材料：在透析后的壳聚糖与聚乙二醇琥珀酸酯或聚乙二醇共混后置入冻干机冻干制得壳聚糖/聚乙二醇琥珀酸酯薄膜或壳聚糖/聚乙二醇膜，并将新生2～5d的SD大鼠骨骼肌成纤维细胞种植于膜片上。实验评估：①MTT法测定肌成纤维细胞接种在不同膜片上的吸光度值，计算相对贴附率。相对贴附率=不同膜的A490nm/培养板的A490nm×100%。②MTT法测定肌成纤维细胞在不同膜片上的生长1，5d后的吸光度值。③相差显微镜下观察肌成纤维细胞的生长形貌。结果：①肌成纤维细胞在不同膜片上的贴附率：肌成纤维细胞在壳聚糖/聚乙二醇琥珀酸酯薄膜上能良好黏附、增殖，而在壳聚糖/聚乙二醇膜、壳聚糖膜上黏附性差。联合培养12h，5d后MTT法结果显示，壳聚糖/聚乙二醇琥珀酸酯组的A值分别为0.074±0.009，0.141±0.031，分别为壳聚糖组的6.17倍和6.13倍（P〈0.05）。②肌成纤维细胞的生长特性：肌成纤维细胞在壳聚糖/聚乙二醇琥珀酸酯膜上的活性最高，增殖能力最强，增长速度最快，其次为壳聚糖/聚乙二醇膜，但两者差异无显著性意义（P〉0.05），而细胞在壳聚糖膜上的增殖能力较低，膜上细胞数目较少，与其他组比较差异有显著性意义（P〈0.05）。③肌成纤维细胞与不同膜片联合培养1，5d时的生长形貌：壳聚糖膜上细胞未贴壁生长，为透明的圆球形，呈游离状态，未能很好舒展，且有些皱缩，生长活力也不旺盛;细胞与壳聚糖/聚乙二醇膜、壳聚糖/聚乙二醇琥珀酸酯膜片联合培养的生长情况要明显好于壳聚糖膜，细胞相互融合成片，多呈长梭形，细胞间隙狭窄，紧密排列成束，成指纹状结构且聚集生长的趋势也更明显。结论：将接支琥珀酰基的聚乙二醇与壳聚糖共混组成的网状系统改进了膜片的力学性能，提高了膜片的柔韧性，使其成膜性更好;壳聚糖/聚乙二醇琥珀酸酯薄膜具有良好的生物相容性，肌成纤维细胞在壳聚糖/聚乙二醇琥珀酸酯薄膜上的黏附及生长情况明显好于壳聚糖薄膜。     

Synergistic inhibition of platelet activation by plasmin and prostaglandin I2

Endothelial cell prostacyclin (PGI2) inhibits platelet activation by raising platelet cyclic AMP. Previously, platelet activation was also shown to be blocked by plasmin formed by endothelium-derived tissue plasminogen activator (TPA). We have now studied interactions between PGI2 and plasmin in the control of platelet function. PGI2 and plasmin cause synergistic inhibition of thrombin- and ADP-induced aggregation of washed platelets. Inhibition by PGI2 is similarly potentiated by TPA added to platelet-rich plasma to generate plasmin. Thrombin-stimulated rise in platelet cytosolic Ca2+, measured by fura2 fluorescence, and thromboxane A2 formation, measured by radioimmunoassay (RIA), are likewise synergistically inhibited by PGI2 and plasmin. Plasmin neither increases nor potentiates PGI2-stimulated increases in platelet cyclic AMP. Thus, PGI2 and plasmin cause synergistic inhibition of platelet activation by both cyclic AMP-dependent and independent mechanisms. This interaction between two different endothelium-derived products may play an important role in localizing the hemostatic plug to a site of vascular injury by preventing further thrombin-mediated accrual of platelets.     

Sex steroids and odorants modulate gonadotropin-releasing hormone secretion in primary cultures of human olfactory cells

Olfactory neurons and GnRH neurons share a common origin during development. In the nasal epithelia, GnRH neurons persist throughout fetal life and adulthood. The fate and function of these neurons in vivo have remained unknown. In a previous in vitro study, we isolated, cloned, and propagated primary long term cell cultures from the olfactory neuroepithelium of 8- to 12-week-old human fetuses. These cells expressed both neural proteins as well as olfactory genes and were responsive to odorant stimuli. We now report that these human olfactory cells also express the GnRH gene and protein. Combined HPLC and RIA studies have indicated that these cells release authentic GnRH in spent media. The release of GnRH was time dependent and was positively affected by sex steroids and odorants. Immunohistochemical data demonstrated the presence of sex steroid receptors in these cells. The presence of the alpha- and beta-subtypes of the estrogen receptor was also demonstrated by RT-PCR and Western blot analysis. When the cells were stimulated with increasing concentrations of 17beta-estradiol in the presence of a fixed concentration of progesterone (10(-7) mol/L), the combination of the two steroids induced a 3- to 4-fold increase in GnRH secretion. This stimulatory effect was completely blunted by tamoxifen. Neither 17beta-estradiol nor progesterone was effective when tested separately. Treatment with increasing concentrations of the odorant, l-carvone, induced a time- and dose-dependent dramatic increase in GnRH protein release (1000-fold increase) and gene expression. Repeated application of the stimulus resulted in a progressive lower responsiveness of the cells. To our knowledge, this is the first time that primary cell cultures from human fetal olfactory neuroepithelium have been shown to express and release GnRH. Our results also demonstrate that these cultures, which are sensitive to sex steroids and odorants, can be useful models in the study of the complex array of regulatory factors that finely tune GnRH secretion in humans.     

Declining value of alanine aminotransferase in screening of blood donors to prevent posttransfusion hepatitis B and C virus infection. The Retrovirus Epidemiology Donor Study

BACKGROUND: Since the mid-1980s, blood banks in the United States have screened donors for elevated alanine aminotransferase (ALT) in an effort to prevent posttransfusion hepatitis. The present study was designed to quantitate the residual value of ALT screening following the implementation of hepatitis C virus (HCV) assays. STUDY DESIGN AND METHODS: Two approaches were used. First, a database of 2.3 million donations made by 586,507 volunteer blood donors between 1991 and 1993 was used to compare the incidence of seroconversion to hepatitis B virus (HBV) and HCV marker positivity in donors with elevated ALT values and with normal ALT values. Second, the duration of ALT elevation prior to HBV and HCV seroconversion was determined from 34 well-documented cases of posttransfusion HBV and HCV; elevated-ALT window periods were multiplied by rates of HBV and HCV incidence in donors to project the yield of ALT screening. Predictive value and cost- effectiveness analyses were also performed to compare the value of ALT screening before and after HCV screening was implemented. RESULTS: Both approaches indicate that ALT testing does not detect HBV in the window phase but does currently identify approximately 3 HCV window-phase donations per 1 million donations; this contrasts with ALT detection of approximately 1800 HCV-infectious units per 1 million donations prior to anti-HCV screening. Currently, only 8 in 10,000 donated units with elevated ALT (negative anti-HCV) are infected with HCV. The cost of continued ALT screening was estimated at $7,931,000 per quality- adjusted year of life saved. CONCLUSION: The yield, predictive value, and cost-effectiveness of ALT screening of blood donors have declined dramatically with the implementation of progressively improved anti-HCV assays. ALT screening of volunteer blood donors should be discontinued.