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We utilized reverse transcription polymerase chain reaction (RT-PCR) to amplify epsilon, G gamma and A gamma globin cDNAs from single red blood cells isolated from a day-10 transgenic fetus harboring a single copy of the human beta-YAC. A detailed structural analysis of the beta-YAC showed a single copy of each beta-like globin gene is present and linked to the locus control region (LCR). RNase protection analysis of RNA isolated from erythroid tissues from day-8 to day-16 of development and the adult stage showed proper developmental switching of the beta-like globin gene expression. Using epsilon / gamma and G gamma / A gamma primer sets in separate RT-PCR reactions on RNA from single day-10 red blood cells we observed an intercellular variation in the epsilon and gamma RT-PCR products that may be indicative of a change in the LCR preference from the epsilon gene promoter to the gamma gene promoter during switching. We also found that the majority of the red blood cells examined contain all three globin mRNA species. These observations suggest that either the LCR is capable of interacting simultaneously with more than one globin gene promoter or alternatively, the LCR may interact with only one promoter at any given time, but its interaction oscillates between promoters (flip-flop mechanism) resulting in expression of more than one gene from a single beta-globin locus.

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Transgenic mice were generated using a purified 248-kb yeast artificial chromosome (YAC) bearing an intact 82-kb human beta-globin locus and 148 kb of flanking sequence. Seventeen of 148 F0 pups were transgenic. RNase protection analysis of RNA isolated from the blood of 13 gamma- and beta-globin-positive founders showed that only the human beta-globin gene was expressed in the adult founders. Studies of F1 and F2 fetuses demonstrated that the genes of the beta-locus YAC displayed the proper developmental switches in beta-like globin gene expression. Expression of epsilon- and gamma-globin, but not beta-globin, was observed in the yolk sac, there was only minor gamma and mostly beta expression in the 14-day liver, and only beta mRNA in the blood of the adult animals. Structural data showed that the locus was intact. These results indicate that it is now possible to dissect regulatory mechanisms within the context of an entire locus in vivo by using the ability to perform mutagenesis efficiently in yeast via homologous recombination, followed by purification of the altered YAC and its introduction into mice.

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Concentration of iron in plasma, total iron-binding capacity (TIBC), and transferrin saturation are often determined by standard spectrophotometric methods, but iron concentration may be quantified by immunoprecipitation or, electrochemically, by controlled-potential coulometry. Because these iron assays do not all measure the same form(s) of iron, we studied subjects in various states of iron nutriture: normal adults, iron-deficient patients, thalassemia patients with unsaturated transferrin or oversaturated transferrin, and patients with idiopathic hemochromatosis. The spectrophotometric and coulometric methods detected essentially all non-heme iron in plasma; results correlated well but showed a negative bias toward the coulometric method. Results by an immunoprecipitation procedure, which measures only transferrin-bound iron, correlated well with those obtained coulometrically but were slightly higher than the latter. The characteristics of the various methods for iron must be understood by the clinical laboratory if diagnosis of iron disorders is to be accurate.

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We evaluated plasma iron (PI) and total iron-binding capacity (TIBC) or transferrin in normal individuals and in patients with iron imbalance. The standard colorimetric measurements of PI and TIBC and the standard isotope-dilution measurement of TIBC were compared with an immunoprecipitation method and also with immunoelectrophoresis of transferrin. PI concentrations as measured by the standard and immunoprecipitation methods agreed closely for all individuals except those with saturated transferrin, where nontransferrin iron increased the results in the standard assay. This excess iron in saturated plasma may be derived from either free iron or iron-bearing ferritin. There were also differences in TIBC between the two methods. Iron-deficient sera gave higher values for transferrin when measured by immunoelectrophoresis. Unsaturated iron-binding capacity was increased in the isotope-dilution method in some iron-saturated plasma, compounding errors when added to erroneously high PI values to compute TIBC. Perhaps some exchange of iron occurred between added iron and transferrin iron in the isotope-dilution method. These measurements confirm the accuracy of the standard colorimetric method of measuring PI and TIBC except in iron-saturated plasma. However, the greater specificity of a polyclonal immunoprecipitation method of measuring PI and TIBC makes it particularly useful in differentiating transferrin-bound iron from nontransferrin iron.

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Maternal and fetal iron balance through pregnancy was examined in the rat. The 20th day was selected for detailed study because of the peak iron requirements at that time. The standard diet provided a borderline iron supply to the fetus due to the limited availability of its iron for absorption. When a more available form of iron was used, normal fetal development occurred over a range of dietary iron content from 16 to 2500 mg/kg. At a level of 5 to 8 mg/kg, there was attrition of placental tissues with frequent fetal death and resorption. When the iron-deficient pregnancy was sustained, both maternal and fetal iron deficiency were present. At progressively higher levels of dietary iron, feto-placental iron content was constant despite a progressive increase in maternal iron stores. Fetal iron supply appeared to be determined primarily by plasma iron concentration, and, at normal levels, about 25% of transferrin iron passing through the uterine vasculature, was removed by the intact placentas. Low levels of plasma iron resulted in damage to fetal tissues and reduced the capacity of placental tissues to take up iron. At high levels of plasma iron, plasma iron turnover initially increased 5-fold over basal levels in nonpregnant animals due to increased placental uptake. However, with continued hyperferremia, uptake was regulated so as to maintain fetal iron at a normal level. A comparison of these data with human iron requirements explained the occurrence of both maternal and fetal iron deficiency in the rat, but only maternal iron deficiency in the human.

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The American Academy of Allergy sponsored a 2-yr "double blind" multicenter study of the effect of Rinkel injection therapy (RIT) compared with a histamine placebo in subjects with atopic rhinitis. Accumulated data included the symptom, medication, and physical examination scores and specific IgE antibody levels measured by the radioallergosorbent test (RAST). A total of 155 subjects (81 treated, 74 placebo) entered the project from six centers during their respective ragweed, grass, and mountain cedar pollen (from one center) seasons for a total of 11 pollen seasons. The total mean cumulative dose of extract was 18.6 PNU, which is much lower than recommended for standard immunotherapy. With one exception, none of the centers reported a consistent significant difference between the pollen extract-treated and placebo-treated groups in any of the weekly mean scores or the RAST before, during, and after the pollen seasons. For 4 wk after the height of the mountain cedar season the group treated with pollen extract showed a significant decrease in weekly mean symptom and medication scores as compared with the placebo group. The overall comparison of the mean seasonal scores for the entire study, however, showed no significant difference between the treated and placebo groups. We conclude the RIT is no more effective than a histamine placebo in influencing the weekly mean symptom, medication, and physical examination scores or IgE antibody levels.

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