Volume regulation and KCl cotransport in reticulocyte populations of sickle and normal red blood cells

The potassium chloride co-transporter (KCC) is a member of the electroneutral cation chloride family of cotransporters found in multiple tissues that are involved in transepithelial ion transport and regulation of intracellular ion content and cell volume. We have shown previously that three of the four KCC genes - KCC1, KCC3, and KCC4 - are expressed in red blood cells (RBC) (Exp. Hem. 33:624, 2005). Functionally, the KCC mediates volume reduction of reticulocytes that establishes the higher cellular hemoglobin concentration (CHC) of mature RBC. KCC activity is higher in reticulocytes and diminishes with age. KCC activity in RBC containing sickle hemoglobin (SS RBC) is elevated compared to normal (AA RBC) in part due to reticulocytosis in SS blood. However, we have demonstrated that SS reticulocytes have abnormal regulation of KCC activity leading to increased CHC upon activation of KCC compared to AA reticulocytes (Blood 104:2954, 2004; Blood 109:1734, 2007). These findings implicate KCC as a factor in the dehydration of SS RBC, which leads to elevated Hb S concentration and enhances Hb S polymerization and hemolysis. Because KCC activity correlates with cell age, standard flux measurements on blood samples with different numbers of reticulocytes or young non-reticulocytes are not comparable. The Advia automated cell counter measures cell volume (MCV) and cellular hemoglobin concentration (CHC) in reticulocytes, an age-defined population of cells, and thus circumvents the problem of variable reticulocyte counts among SS and AA blood samples. In this study, reticulocyte CHC measurements on fresh blood demonstrated a clear difference between AA and SS cells, reflecting in vivo dehydration of SS reticulocytes, although there was significant inter-individual variation, and the CHC distributions of the two groups overlapped. After KCC activation in vitro by cell swelling using the nystatin method, the initial changes in reticulocyte MCV and CHC with time were used to estimate flux rates mediated by KCC, assuming that changes were associated with isotonic KCl movements. After 20-30min a final steady state MCV/CHC (set point) was achieved and maintained, reflecting inactivation of the transporter. CHC set points were 26.5-29g/dl in SS reticulocytes compared to 25-26.5g/dl in AA reticulocytes, reflecting abnormal regulation in SS cells. These results were reproducible in the same individual over time. KCC flux derived from CHC ranged from 5 to 10.3mmolK/kgHb/min in SS reticulocytes, compared to 2.9-7.2mmolK/kgHb/min in AA reticulocytes. Such measures of KCC activity in red cell populations controlled for cell age will facilitate further studies correlating KCC activity with phenotypic or genetic variability in sickle cell disease.     

The value of flow cytometric analysis of platelet glycoprotein expression of CD34+ cells measured under conditions that prevent P- selectin-mediated binding of platelets

In the present study, we show by adhesion assays and ultrastructural studies that platelets can bind to CD34+ cells from human blood and bone marrow and that this interaction interferes with the accurate detection of endogenously expressed platelet glycoproteins (GPs). The interaction between these cells was found to be reversible, dependent on divalent cations, and mediated by P-selectin. Enzymatic characterization showed the involvement of sialic acid residues, protein(s). The demonstration of mRNA for the P-selectin glycoprotein ligand 1 (PSGL-1) in the CD34+ cells by polymerase chain reaction (PCR) analysis suggests that this molecule is present in these cells. Under conditions that prevent platelet adhesion, a small but distinct subpopulation of CD34+ cells diffusely expressed the platelet GPIIb/IIIa complex. These cells were visualized by immunochemical studies. Furthermore, synthesis of mRNA for GPIIb and GPIIIa by CD34+ cells was shown using PCR analysis. The semiquantitative PCR results show relatively higher amounts of GPIIb mRNA than of PF4 mRNA in CD34+CD41+ cells in comparison with this ratio in platelets. This finding is a strong indication that the PCR results are not caused by contaminating adhering platelets. MoAbs against GPIa GPIb alpha, GPV, P- selectin, and the alpha-chain of the vitronectin receptor did not react with CD34+ cells. The number of CD34+ cells expressing GPIIb/IIIa present in peripheral blood stem cell (PBSC) transplants was determined and was correlated with platelet recovery after intensive chemotherapy in 27 patients. The number of CD34+CD41+ cells correlated significantly better with the time of platelet recovery after PBSC transplantation (r = .83, P = .04) than did the total number of CD34+ cells (r = .55). Statistical analysis produced a threshold value for rapid platelet recovery of 0.34 x 10(6) CD34+CD41+ cells/kg. This study suggests that if performed in the presence of EDTA the flow cytometric measurement of GPIIb/IIIa on CD34+ cells provides the most accurate indication of the platelet reconstitutive capacity of the PBSC transplant.     

Long-term follow-up of autoantibody profiles in black female lupus patients and clinical comparison with Caucasian and Asian patients

The aim was to determine whether the autoantibody profile in Black female lupus patients is associated with clinical subsets, fluctuates over time and/or reflects disease activity. A clinical comparison with Caucasian and Asian patients matched for age of onset and disease duration was also undertaken. Up to seven serial bleeds from Black female lupus patients who had been followed up for periods of 3.15 yr were tested for antibodies to Ro/SSA, La SSB. Sm, RNP and ribosomal P using ELISA research assays. Significant differences in both clinical and serological profiles between the ethnic groups were found. Varying aspects of disease activity were linked to anti-DNA (renal, cardiovascular, global score), anti-ribosomal P (musculoskeletal, haematology) and anti-Sm (general) antibodies. There are differences in clinical and serological profiles amongst systemic lupus erythematosus patients of different ethnic origin. However, using the BILAG system, relatively few antibodies were found to reflect disease activity accurately in serial measurements.      

Mouse Strain Differences in Oral Operant Ethanol Reinforcement under Continuous Access Conditions

The present experiment examined ethanol setf-administration in C57BL/6J (C57) and DBA/2J (DBA) mice using a continuous access operant procedure. Adult male C57 and DBA mice were initially trained to perform a lever press response to obtain access to 10% w/v sucrose solution. Subsequently, the mice were placed in operant chambers on a continuous (23 hr/day) basis with access to food (FR1), 10% v/v ethanol (FR4), and water from a sipper tube. C57 mice displayed greater rates of responding on the ethanol-associated lever compared with DBA mice. Responding on the food lever was the same in both strains, but DBA mice consumed greater amounts of water. C57 mice consistently displayed both prandial and nonprandial episodes (bouts) of ethanol responding. DBA mice did not respond for ethanol in bouts. Following 50 consecutive sessions, ethanol concentration was altered every 5 days. Response patterns were determined using 0, 5, 10, 20, and 30% v/v ethanol concentrations. C57 mice displayed concentration-dependent responding on the ethanol lever showing that ethanol was functioning as an effective reinforcer in this strain. In contrast, responding on the ethanol lever by DBA mice did not change as a function of ethanol concentration. Saccharin (0.2% w/v) was subsequently added to the ethanol mixture, and responding was examined at 0, 5, 10, and 20% ethanol concentrations. Overall, ethanol lever responding was increased in both strains. As before, C57 mice showed higher levels of ethanol responding, compared with DBA mice. C57 mice also showed higher responding for saccharin alone. These results are consistent with findings that suggest orally administered ethanol is a more effective reinforcer in C57 mice than in DBA mice. Furthermore, C57 mice engage in ethanol-reinforced responding over a broader range of conditions than DBA mice.     

Application of image-guided surface coil P-31 MR spectroscopy to human liver, heart, and kidney

Localized phosphorus-31 magnetic resonance (MR) spectroscopy in humans has previously been accomplished with surface coils by means of depth-resolved surface coil spectroscopy or rotating frame experiments, in which the extent of tissue sampled critically depends on surface coil placement. The authors' goal was to modify the surface coil image-selected in vivo spectroscopy (ISIS) experiment to accomplish three-dimensional volume selection through application of selective pulses in the presence of B0 gradients. Advantages of ISIS include the ability to use proton images to define the volume of interest (VOI) and reduced dependence on exact positioning of the surface coil. However, rapid replication of the surface coil ISIS experiment can cause spectral contamination from signals originating outside the VOI. A modified version of the ISIS experiment was developed to alleviate contamination under conditions of rapid replication. Applications of localized P-31 MR spectroscopy for observation of high-energy phosphorus metabolites are presented in human liver, heart, and transplanted and normal kidney.     

Regulation of microfilament organization and anchorage-independent growth by tropomyosin 1.

Variants of chemically immortalized Syrian hamster embryo cells that had either retained (supB+) or lost (supB-) the ability to suppress tumorigenicity when hybridized with a fibrosarcoma cell line were subcloned. Both supB cell types are nontumorigenic; however, the supB- but not supB+ cells exhibit conditional anchorage-independent growth. Alterations of actin microfilament organization were observed in supB- but not supB+ cells that corresponded to a significant reduction of the actin-binding protein tropomyosin 1 (TM-1) in subB- cells. To examine the possibility of a direct relationship between TM-1 expression and the subB- phenotype, subB+ cells were transfected with an expression vector containing the TM-1 cDNA in an antisense orientation. The antisense-induced reduction of TM-1 levels in supB+ clones caused a microfilament reorganization and conferred anchorage-independent growth potential that were indistinguishable from those characteristic of supB- cells. These data provide direct evidence that TM-1 regulates both microfilament organization and anchorage-independent growth and suggest that microfilament alterations are sufficient for anchorage-independent growth.     

On the interaction of rabbit antithrombin III with the luminal surface of the normal and deendothelialized rabbit thoracic aorta in vitro

Pure rabbit antithrombin III was isotope labeled (with 125I or 3H) by two different methods; neither procedure caused a loss of antithrombin activity although both methods affected the affinity of the protein for Sepharose-heparin. From segments from freshly excised rabbit aorta, the uptake of isotope-labeled antithrombin III by the endothelium was rapid and saturable, although relatively small compared to the uptake of thrombin; binding of 3H-antithrombin III to the endothelium resembled that of 125I-antithrombin III. Transendothelial passage of antithrombin III into the subendothelial layers (intima-media) was slow and progressive. Endothelium binding was not affected by pretreating the vessel with either heparin, thrombin, or glycosaminoglycan-specific enzymes. Endothelium-bound antithrombin III was not selectively displaced by either heparin or thrombin. In contrast, endothelium-bound thrombin was rapidly dislodged by antithrombin III as a thrombin- antithrombin III complex. The surface of the deendothelialized aorta (ie, subjected to a balloon catheter) bound antithrombin III avidly. Pretreatment of the deendothelialized vessel with glycosaminoglycan- specific enzymes, particularly heparitinase, decreased intima-media binding by up to 80%. 125I-antithrombin III, when bound to the deendothelialized vessel surface, was actively displaced by either heparin, thrombin, or by unlabeled antithrombin III. The relatively poor binding of antithrombin III compared with that of thrombin by the endothelium in vitro supports an earlier proposal (Lollar P, Owen WG: J Clin Invest 66:1222-1230, 1980) that thrombin bound to high-affinity sites, possibly pericellular proteoglycan, of the endothelium is inactivated by plasma antithrombin III in vivo. Such a situation probably holds for large arteries at least.