The effects of additive solution pH and metabolic rejuvenation on anaerobic storage of red cells

BACKGROUND: Red cell (RBC) storage can be extended to 9 weeks under anaerobic or alkaline conditions. Simultaneous use of these approaches has not provided additive benefit. Our objective was to determine whether anaerobic storage with acidified additive solution (AS) coupled with metabolic rejuvenation might further improve the benefits of anaerobic storage. STUDY DESIGN AND METHODS: RBC storage in AS with a pH value of 6.5, 7.4, or 8.3 in aerobic or anaerobic conditions was examined using a panel of in vitro biochemical and RBC markers. RBC rejuvenation during cold storage was also evaluated. A randomized crossover radiolabeled recovery study (eight subjects) evaluated anaerobic RBC storage using AS65 with cold rejuvenation for up to 16 weeks of storage. RESULTS: Adenosine triphosphate (ATP) and diphosphoglycerate acid (DPG) were better maintained in anaerobic storage than in aerobic storage. Acidic or neutral AS preserved ATP concentration better, while a neutral or basic pH AS favored maintenance of DPG levels at higher levels for a longer period. AS pH had less of an effect on exposure of phosphatidylserine (PS), vesicle protein release, and hemolysis. Rejuvenation of RBCs during cold, anaerobic storage resulted in increases in ATP and DPG levels and a reversal of PS exposure. Anaerobic storage of RBCs in pH 6.5 AS rejuvenated at 7 weeks of storage yielded RBC 24‐hour recoveries of 77.3 ± 12.5 percent after 10 weeks' storage time. After a second rejuvenation at Week 11, six subjects' units demonstrated a recovery of 75.9 ± 7.3 percent at 12 weeks of storage. CONCLUSION: Extended RBC storage may be achieved using anaerobic conditions combined with low‐pH AS and rejuvenation during storage.     

Intrinsic factors influencing post stroke brain reorganization

Reorganization of the brain, specifically the motor cortex surrounding the stroke, accounts for much of the observed neurological recovery following stroke. Not surprisingly, size of the stroke lesion has the greatest impact on neurological recovery in both animal and clinical research studies. Spontaneous recovery of lost function is possible after a cortical lesion, particularly if the lesion is small. Age correlates negatively with recovery; older individuals generally demonstrate slower and less complete recovery. However, age by itself is a poor predictor of functional recovery.     

Quantitative evaluation of liver-specific promoters from retroviral vectors after in vivo transduction of hepatocytes

Hepatic gene therapy could be used to treat a number of inherited blood diseases such as hemophilia or thrombophilia. Although liver-directed retroviral transduction can result in long-term gene expression in vivo, the low level of protein production has limited its clinical application. We reasoned that the insertion of liver-specific promoters into retroviral vectors would increase gene expression in vivo. The 347- bp human alpha 1-antitrypsin (hAAT), the 810-bp murine albumin (mAIb), the 490-bp rat phosphoenolpyruvate carboxykinase (rPECK), and the 596- bp rat liver fatty acid binding protein promoters were inserted into a Moloney murine leukemia retroviral backbone containing the hAAT reporter gene. Vectors that produced appropriately sized RNA and hAAT protein in vitro were tested in vivo by transducing regenerating rat livers. Long-term serum expression of the hAAT reporter gene was normalized to retroviral transduction efficiency as determined by using a polymerase chain reaction-based assay of genomic DNA from transduced rat livers. The hAAT, mAIb, and rPEPCK promoters were, respectively, 35- , 8-, and 0.02-fold as strong as the previously studied constitutive Pol-II promoter. We conclude that the hAAT promoter resulted in the highest expression from a retroviral vector and may result in therapeutically significant expression of other clinically significant blood proteins.     

Spinal metastases mimicking low back pain of mechanical origin: a case report

Spinal malignancies are an essential consideration when a patient presents to a chiropractic office with back pain. This single case report exemplifies the importance of patient presentation and physical examination findings. We must also consider the rationale for x-raying patients on an individual case basis. Textbook cases do not always exist and special diagnostic tests do not always provide a definitive diagnosis of underlying pathology. Even though history and examination findings suggest a routine diagnosis, continual re-evaluation and recognition of the need to change the diagnosis on occasion is extremely important. The patient should not only be thoroughly evaluated upon initial presentation, but also each time they present for treatment. The decision to x-ray a patient is considered important. X-ray examination can be used to confirm a diagnosis or to rule out potential pathologies, and not necessarily done as a routine screening procedure.A case report is presented in which the pathologic signs were not evident on plain film x-rays upon initial presentation.     

STUDIES OF PPLO INFECTION : II. THE NEUROTOXIN OF MYCOPLASMA NEUROLYTICUM

Rolling disease has been produced and studied in rats and mice, using the exotoxin of the A strain of Mycoplasma neurolyticum. The primary lesion of the brain consists of spongiform degeneration, associated with vesicle formation in the cortex and underlying white matter of the cerebral hemispheres, and in the molecular layer of the cerebellum. The brains of animals surviving 2 days or longer show extensive necrotizing lesions resembling ischemic necrosis, in both cerebral hemispheres. The brains of rats and mice with rolling disease become deeply stained by intraperitoneally injected trypan blue, indicating early disruption of the blood brain barrier. The toxin appears to be a thermolabile protein with a molecular weight exceeding 200,000. It is only active when injected by vein, and causes no disease when injected intracerebrally, intraperitoneally or subcutaneously, suggesting the existence of specific receptors within the vascular bed of the central nervous system. Protection is afforded by rabbit antibody against the toxin, but only when antibody is injected within less than 3 min after intravenous injection of toxin, indicating rapid fixation to receptors in the brain. The toxin is inactivated by incubation for 10 min at 37°C with suspensions of the sedimentable component of normal brain. The inactivating factor in brain sediment is very thermostable, not affected by trypsin, and eliminated by treatment with periodate. Similar inactivation of toxin is demonstrable with water-soluble gangliosides of brain. A theoretical concept to explain the action of the toxin is proposed.     

Biochemical Characterization and Cytochemical Localization of a Catecholamine-Sensitive Adenylate Cyclase in Isolated Capillary Endothelium

Capillaries were isolated from epididymal fat, and a catecholamine-sensitive adenylate cyclase found in these capillaries was characterized. The effect of various hormones on the accumulation of adenosine 3′:5′-cyclic monophosphate in capillary endothelial cells was determined and the cyclase was found to exhibit mixed alpha and beta characteristics. Cyclase was cytochemically localized in these endothelial cells with 5′-adenylyl-imidodiphosphate as a specific cyclase substrate and alloxan as a specific cyclase inhibitor. Lead imidodiphosphate was precipitated at or near the site of cyclase activity upon hydrolysis of 5′-adenylyl-imidodiphosphate by cyclase. This reaction product was observed primarily on the luminal surface of intact capillaries, in micropinocytic invaginations, in free vesicles within the cytoplasm, and in the intracellular junctions.     

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.     

Human platelet fibrinogen: purification and hemostatic properties

Conditions were developed in which 80% to 90% of platelet fibrinogen could be routinely purified in nondegraded form from the fluid phase of platelet suspensions stimulated with the calcium ionophore, A23187, in the presence of calcium, leupeptin, and prostaglandin E1. Fibrinogen was separated from other released proteins by chromatography on diethylaminoethanol (DEAE)-cellulose using a continuous pH and ionic strength gradient. Purified platelet fibrinogen, greater than 98% homogeneous by immunoelectrophoresis and sodium-dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), consisted of intact A alpha, B beta and gamma A chains, but not gamma' chains, and was 95% to 96% clottable. Platelet fibrinogen was shown to compete for the binding of radiolabeled plasma fibrinogen to ADP-activated platelets in a manner identical to that of unlabeled plasma fibrinogen itself. Also, at equivalent protein concentrations, platelet and plasma fibrinogens supported platelet aggregation to an equivalent extent. Based upon these results, we conclude that there is no significant difference between platelet and plasma fibrinogen with respect to their size, their clottability, their affinity for the activated platelet fibrinogen receptor, or their capacity to support subsequent platelet aggregation.