Monoclonal antibodies recognizing epitopes on the extracellular face and intracellular N-terminus of the human erythrocyte anion transporter (band 3) and their application to the analysis of South East Asian ovalocytes

This report describes the production and characterization of 13 rodent monoclonal antibodies to the human erythrocyte anion transport protein AE1 (syn. band 3). Eleven antibodies (4 murine and 7 rat) recognize epitopes dependent on the integrity of the third extracellular loop of the protein. Two antibodies (1 murine and 1 rat) recognize epitopes on the N-terminal cytoplasmic domain. Quantitative binding studies using radioiodinated IgG and Fab fragments of antibodies to extracellular epitopes on AE1 ranged from 77,000 to 313,000 (IgG) and from 241,000 to 772,000 (Fab) molecules bound at saturation. The results indicate that the epitopes recognized by different antibodies vary in their accessibility and suggest that there is heterogeneity in the organization of individual AE1 molecules in the red blood cell membrane. Quantitative binding studies on South East Asian ovalocytes using several antibodies to AE1 and an anti-Wrb show a marked reduction in the number of antibody molecules bound at saturation. These results are consistent with the existence of highly cooperative interactions between transmembrane domains of AE1 in normal erythrocytes and the disruption of these interactions in the variant AE1 found in South East Asian ovalocytes.     

Familial pattern of corticosteroids and their metabolism in adult human subjects--the Scottish Adult Twin Study

Corticosteroids are important in the regulation of normal physiology and are key factors in regulating cardiovascular physiology and disease, the development of which is known to have a genetic component. However, there is little information on the extent to which plasma and urine steroid levels are determined by familial and genetic factors. We have examined basal and ACTH-stimulated plasma steroid levels and 24-h corticosteroid metabolite excretion rates in 146 pairs of adult twins [75 monozygotic (MZ); 71 dizygotic (DZ)]. Intraclass correlation coefficients were measured for all variables; several plasma steroid measurements were strongly related in both (MZ) and (DZ) twins, consistent with a familial pattern. These included basal levels of 11-deoxycortisol and aldosterone. ACTH-stimulated plasma aldosterone levels were also significantly correlated, to a significant degree, in both MZ and DZ twins. The index of 11beta-hydroxysteroid dehydrogenase activity (tetrahydrocortisol + allotetrahydrocortisol/tetrahydrocortisone) and of the more specific index of activity of the type 2 isoform of this enzyme (urine free cortisol/cortisone) also correlated, to a similar degree, in DZ and MZ twins. In contrast, for the basal and ACTH-stimulated plasma concentrations and 24-h urine excretion rates of several corticosteroids, there was evidence of significant heritability (H2), in that correlation in MZ twins was greater than in DZ. For example, basal plasma corticosterone concentrations (B) (H2 = 0.44), basal and stimulated 11-deoxycorticosterone concentrations (DOC) (H2 = 0.44 and 0.41, respectively), stimulated 11-deoxycortisol concentrations (H2 = 0.53), and the index of 11beta-hydroxylase activity DOC/B (H2 = 0.49) were all significantly heritable. For the urinary variables, 24-h tetrahydrodeoxycortisol (H2 = 0.59) and free aldosterone (H2 = 0.56) were significantly heritable. Our data provide the first evidence that plasma and urine levels of important glucocorticoids and mineralocorticoids show a strong familial pattern, and in some instances, there is evidence of a genetic component to this. This suggests that corticosteroids have a plausible role in essential hypertension that has a similar heritable component.     

Maternal administration of granulocyte colony-stimulating factor improves neonatal rat survival after a lethal group B streptococcal infection

Maternally administered recombinant human granulocyte colony- stimulating factor (rhG-CSF) has been shown to cross the placenta and induce a peripheral neutrophilia and increases in the marrow and spleen neutrophil storage pools in fetal and newborn rats. In the present study, we have used this model system to investigate the efficacy of prenatally administered rhG-CSF on neonatal defense to a lethal challenge with Group B-beta hemolytic Streptococcus (GBS). Pregnant rats were injected with rhG-CSF twice daily beginning 6 days before parturition. At birth, all pups were infected with a dose of GBS that is lethal for 90% of infected pups (LD90). Survival was monitored daily for 5 days. Survival of infected pups from saline-treated mothers beyond 60 hours after infection was 10%. No difference in survival was observed among pups from mothers treated 2 and 4 days before parturition. In contrast, we determined that survival was 82.5% among infected pups from mothers treated for 6 days before parturition with rhG-CSF. Our results demonstrate that maternal administration of rhG- CSF augments neonatal defenses against a lethal bacterial challenge.     

Homozygosity for a HERG potassium channel mutation causes a severe form of long QT syndrome: identification of an apparent founder mutation in the Finns

OBJECTIVES

We studied the clinical characteristics and molecular background underlying a severe phenotype of long QT syndrome (LQTS).

BACKGROUND

Mutations of cardiac ion channel genes cause LQTS, manifesting as increased risk of ventricular tachycardia and sudden death.

METHODS

We studied two siblings showing prolonged QT intervals corrected for heart rate (QTc), their asymptomatic parents with only marginally prolonged QTc intervals and their family members. The potassium channel gene HERG was screened for mutations by deoxyribonucleic acid sequencing, and the electrophysiologic consequences of the mutation were studied in vitro using the whole-cell patch-clamp technique.

RESULTS

A novel missense mutation (L552S) in the HERG channel, present in the homozygous state in the affected siblings and in the heterozygous state in their parents, as well as in 38 additional subjects from six LQTS families, was identified. One of the homozygous siblings had 2:1 atrioventricular block immediately after birth, and died at the age of four years after experiencing unexplained hypoglycemia. The other sibling had an episode of torsade de pointes at the age of two years. The mean QTc interval differed significantly (p < 0.001) between heterozygous symptomatic mutation carriers (500 ± 59 ms), asymptomatic mutation carriers (452 ± 34 ms) and noncarriers (412 ± 23 ms). When expressed in vitro, the HERG-L552S formed functional channels with increased activation and deactivation rates.

CONCLUSIONS

Our data demonstrate that homozygosity for a HERG mutation can cause a severe cardiac repolarization disorder without other phenotypic abnormalities. Absence of functional HERG channels appears to be one cause for intrauterine and neonatal bradycardia and 2:1 atrioventricular block.     

Effects of methylprednisolone administration in acute myocardial infarction

Methylprednisolone sodium succinate, 50 mg/kg body weight, was given as an intravenous bolus injection to 15 dogs with acute myocardial infarction and the results were compared with data in control animals. Methylprednisolone was thought to improve the critical oxygen balance of the infarcted heart by two mechanisms: (1) by diminishing heart rate, afterload and preload in the initial 15 minutes after its administration and thereby decreasing the oxygen need of the heart, and (2) by increasing coronary arterial blood flow. Both mechanisms were believed to contribute to the increase in cardiac output, efficiency and ventricular performance. This improvement in performance was presumably not due to a positive inotropic effect, since studies in isolated heart muscle showed no effect of methylprednisolone on contractility. Regional circulations other than the coronary circulation seemed to be little affected by administration of methylprednisolone except for blood pressure-related increases in superior mesenteric and femoral arterial blood flow.     

Myocardial salvage by intracoronary thrombolysis in evolving acute myocardial infarction: evaluation using intracoronary injection of thallium-201

Immediate objective assessment of viabillty of reperfused myocardium following intracoronary (IC) thrombolysis by evaluation of ventricular function may be limited due to delay in restoration of function. Thus we assessed myocardial uptake of thallium-201 (TI-201) following IC injection postreperfusion as an index of myocardial salvage in 12 experimental dogs and in five patients with evolving acute myocardial infarction (AMI). In seven dogs with mean of 313 minutes of experimental coronary occlusion, immediate postreperfusion IC TI-201 images revealed absence of myocardial uptake in prevlously occluded zones. These TI-201 defects correlated with presence of necrosis as demonstrated by histochemical staining with triphenyl-tetrazolium chloride (TTC). In contrast, in five dogs with mean of 37 minutes of coronary occlusion, reperfused myocardium showed normal TI-201 uptake following its IC injection; this normal TI-201 uptake pattern correlated with absence of necrosis by TTC technique in all five dogs. In five patients with evolving AMI, control TI-201 images obtained following IV injection prior to IC thrombolysis showed myocardial perfusion defects corresponding to distribution of the occluded vessel. Following reperfusion, 30 to 50 mCi of TI-201 was injected into the reopened coronary artery. In two patients with mean symptom onset of reperfusion time of $\text{2}\text{1}\text{2}\text{hours}$, immediate postreperfusion IC TI-201 images demonstrated normal or improved TI-201 uptake in reperfused myocardium. By radionuclide ventriculography, segmental wall motion remained abnormal in the reperfused regions 6 hours postreperfusion and showed improvement by the time of 10-day study. In the remaining three patients with symptom onset to reperfusion time of 5 hours, immediate postreperfusion IC TI-201 images did not show improvement, correlating with persistent wall motion abnormalities 10 days postreperfusion. In all five patients, repeat 10-day IV TI-201 images were unchanged from the immediate postreperfusion IC TI-201 images. We conclude that (1) prereperfusion TI-201 imaging with repeat TI-201 injection into the reopened coronary artery appears to delineate the extent of myocardial salvage in both experimental and clinical studies and (2) this method of IC TI-201 imaging allows immediate assessment of myocardial viabillty which may facilltate decisions regarding the need for additional myocardial revascularization modalities.     

Granulocyte-macrophage colony-stimulating factor augmentation of T-cell receptor-dependent and T-cell receptor-independent thymocyte proliferation

The effects of granulocyte-macrophage colony-stimulating factor (GM- CSF) are not confined to cells of the myeloid lineage. GM-CSF has been shown to have effects on mature T cells and both mature and immature T- cell lines. We therefore examined the GM-CSF responsiveness of murine thymocytes to investigate whether GM-CSF also affected normal immature T lymphocytes. The studies presented here indicate that GM-CSF augments accessory cell (AC)-dependent T-cell receptor (TCR)-mediated proliferation of unseparated thymocyte populations. To identify the GM- CSF responsive cell type, thymic AC and T cells were examined for GM- CSF responsiveness. We found that GM-CSF augmentation of TCR-induced thymocyte proliferation appears to be mediated via augmentation of AC function, and not via direct effects on mature single-positive (SP) thymocytes. Enriched double-negative (DN) thymocytes were also tested for GM-CSF responsiveness. GM-CSF induced the proliferation of adult and fetal DN thymocytes in an AC-independent and TCR-independent single- cell assay. Thus, in contrast to the SP thymocytes, a DN thymocyte population was directly responsive to GM-CSF. GM-CSF therefore may play a direct role in the expansion of DN thymocytes and an indirect role in the expansion of SP thymocytes.