Effect of hydrogen peroxide exposure on normal human erythrocyte deformability, morphology, surface characteristics, and spectrin-hemoglobin cross-linking.

To further define the conditions for forming spectrin-hemoglobin cross-linking in human erythrocyte membranes and to examine its possible effects on membrane function, we incubated normal human erythrocytes for up to 3 h in concentrations of H2O2, varying from 45 to 180 microM, in an azide phosphate buffer, pH 7.4. The chemical changes observed indicated that methemoglobin formation occurred early and at a low concentration (45 microM). Morphologic changes characterized by increased echinocyte formation occurred in a dose-dependent fashion. In addition, decreased cell deformability commensurate with increased membrane rigidity was found. Finally, an increase in cell recognition as determined by monocyte phagocytosis and adherence in vitro, as well as decreased phosphatidylcholine accessibility to bee venom phospholipase A2, was found in H2O2-treated erythrocytes compared with controls. Both of these latter changes were closely correlated with the extent of spectrin-hemoglobin cross-linking. In addition to these protein-mediated interactions, lipid peroxidation also occurred after H2O2 exposure, as shown by generation of fluorescent amino propene derivatives. The addition of the antioxidant, butylated hydroxytoluene, decreased the fluorescent derivatives, but did not prevent the effects on membrane function. This suggests that lipid peroxidation, though present, was not necessary for the membrane changes found. In contrast, spectrin-hemoglobin aggregation and the alterations in membrane function were completely prevented by prior exposure of the erythrocytes to carbon monoxide.     

Bioeffects of myocardial contrast microbubble destruction by echocardiography

BACKGROUND: Microbubble destruction during contrast echocardiography is known to cause capillary leaks and red blood cell extravasation in skeletal muscle. This study evaluated the biological effects of microbubble destruction on cardiac muscle. METHODS: Contrast echocardiography was performed in 36 rats randomized to receive either Definity or Optison at a mechanical index (MI) of 1.6, 1.2, or 0.8. Myocardial bioeffects were assessed by measuring left ventricular (LV) size and fractional area shortening and histopathology. In addition, blood samples for troponin T were drawn at baseline, postinfusion (30 minutes), day 1, day 4, and day 7. LV size and function were measured at baseline and immediately prior to euthanasia on day 7, after which the heart was removed and sectioned for histopathology. RESULTS: There was no statistical difference in LV size or function regardless of the contrast agent or MI, nor was there any histopathological evidence of myocardial damage. However, troponin T increased over time (F = 3.77, P = 0.012), peaking at 30 minutes and returning to normal by day 4. The difference between Definity and Optison was not statistically significant. However, troponin T values were higher at a higher MI (F = 5.01, P = 0.012). Of 12 rats imaged at a MI of 1.6, 9 (75%) had elevated troponin T as compared to 4 (33%) of 12 at a MI of 1.2. None of the 12 rats imaged at a MI of 0.8 had an elevated troponin T at any time point. CONCLUSIONS: Microbubble destruction at high acoustic power (MI 1.6) can cause mild troponin T elevations that are not associated with LV dysfunction or histopathological evidence of myocardial damage.     

Surface Remodeling vs. Whole-Cell Hemolysis of Reticulocytes Produced With Erythroid Stimulation or Iron Deficiency Anemia

³²P in membrane phosphatidylethanolamine (PE) and red cell ¹⁴C, reflecting cytoplasmic hemoglobin, were measuredsequentially in rats given transfusions ofdoubly-labeled reticulocytes. With reticulocytes from normal rats there was a smalldecline in the levels of both the membraneand the cytoplasmic labels; the changeswere almost parellel, although loss ofmembrane PE-³²P exceeded that of ¹⁴C toa small extent. By contrast, with "stressreticulocytes" from bled donors, there wasa markedly disproportionate loss of themembrane label; this asymmetrical lossof membrane material was diminishedwhen recipients had been splenectomized.With transfusions of doubly-labeled reticulocytes from rats with severe iron deficiency anemia, there was a marked lossof both membrane PE-³²P and red cell ¹⁴Cwhich was only moderately asymmetrical.The asymmetrical loss of the membranelabel found with stress reticulocytes supports the conclusion that these cells undergo a process of surface remodeling duringtheir maturation in the peripheral blood.The spleen is partly responsible for thisprocess. Normal reticulocytes also appearto undergo a minor degree of remodeling.On the other hand, the almost symmetricalloss of membrane and cytoplasmic labelobserved with reticulocytes from iron deficient rats indicates that many of thecells in this model of ineffective erythropoiesis are hemolyzed in their entirety.These experiments demonstrate that stressreticulocytes differ under different conditions and may lose cellular material bytwo, possibly interrelated, mechanisms:surface remodeling or whole-cell hemolysis.

Submitted on January 9, 1974 Accepted on June 19, 1974     

Two novel AIRE mutations in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) among Indians

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare recessive disorder resulting from mutations in the autoimmune regulator ( AIRE ) gene. There is no information on AIRE mutations in Indians. In a cross-sectional study, nine patients (eight families), from four referral hospitals in India, were studied for AIRE mutations by direct sequencing. We screened for new mutations in 150 controls by allele-specific PCR. The patients had 1–7 known components of APECED. Three patients had unusual manifestations: presentation with type 1 diabetes; chronic sinusitis and otitis media; and facial dysmorphism. All patients carried homozygous, probably recessive, AIRE mutations. Two unrelated patients from a small in-bred community (Vanika Vaisya) in south India carried an unreported missense mutation, p.V80G, in the N-terminal caspase recruitment domain. Another unique mutation, p.C302X, resulting in a truncated protein with deletion of both zinc-finger domains, was detected in a patient from Gujarat. Neither mutation was detected in controls. Other mutations, previously described in Caucasians, were: 13 base pair deletion (p.C322fsX372) in 4 (38%), and Finn-major (p.R257X) and p.R139X (Sardinian) mutation in one subject each. In conclusion, in this first series of APECED in Indians, we detected AIRE mutations previously reported in Caucasians, as well as unique mutations. Of these, p.V80G is possibly an ancestral mutation in an in-bred community.     

Advances in approaches to the cranial base: minimizing morbidity

The most innovative and meaningful recent advances regarding surgery of the cranial base involve the ability to perform a complete resection followed by a water- and airtight reconstruction while minimizing facial incisions and morbidity. Perhaps the first step in this direction took place when the subcranial/subfrontal approach was introduced for anterior skull base surgery. Originally developed by Raveh in 1978 for the management of severe skull base injuries, these approaches were later adapted for the treatment of congenital anomalies prior to their utilization for resection of anterior skull base tumors. The endoscopic approaches are quite practical with promising long-term efficacy for the treatment of most benign, infectious, and inflammatory disorders. The minimal recovery time, functional outcomes, and obvious aesthetic advantages are only tempered by the lack of long-term data regarding the efficacy of these approaches in the treatment of malignancies.     

Hereditary lecithin-cholesterol acyltransferase deficiency. Report of 2 new cases and review of the literature

Two new cases of hereditary lecithin-cholesterol acyltransferase (LCAT) deficiency in a brother and sister born to consanguinous parents are reported. Both have corneal opacity, splenomegaly and mild hemolytic anemia. The brother, the older of the 2, also has significant proteinuria. The literature dealing with reported cases of hereditary LCAT deficiency and the clinical, pathological, diagnostic and management aspects of the disorder are reviewed.     

Monovalent cation transport in irreversibly sickled cells.

Using discontinuous density gradients of Stractan II, we have separated sickle cell blood into discrete subpopulations of reticulocytes, mature discoid cells, and irreversibly sickled cells (ISCs). We have measured active and passive fluxes of monovalent cations in mature discoid cells, ISCs, and normal control cells, also separated upon density gradients. These measurements revealed a decreased active cation transport in ISC-rich populations. However, parallel measurements of Na, K-ATPase activity showed normal ouabain-sensitive ATPase activity in ISCs. Passive permeability to external Rb was also normal in ISCs. The observation of depressed pump activity in intact ISCs, contrasted with normal ATPase activity in ISC membranes, suggests the presence of factors in the intact cell which inhibit the active transport of Na and K in ISCs.     

Rheological properties of antibody-coated red cells

In vivo and in vitro antibody-coated nonspherocytic red cells have normal deformability in iso-osmolar media, and their isolated membranes have normal mechanical stability as measured in the ektacytometer, a laser visco-diffractometer. The deformability of red cells from patients with hemolytic anemia was proportionately reduced with the degree of spherocytosis irrespective of whether the direct antiglobulin test was positive. However, when antibody-sensitized nonspherocytic cells were suspended in hyperosmolar media and exposed to constant shear stress in the ektacytometer, they showed reduction of their deformability index in the presence of polyvinylpyrrolidone (PVP; 360,000 Daltons) but not dextran. This reduction was not due to alterations in cell water or cation content. Also, when incubated at 37 degrees C, sensitized red cells reacted specifically with PVP, and underwent a decrease in cell deformability. This appears to be the result of changes either in red cell membrane rigidity or membrane viscosity. The PVP effect was reproducible, time- and temperature- dependent, and predicted reliably the Rh phenotype. These observations were used to develop a novel approach to the antiglobulin test in the blood bank.