CD44 as a Potential Screening Marker for Preliminary Differentiation Between Congenital Dyserythropoietic Anemia Type II and Hereditary Spherocytosis.

BACKGROUND: Bone marrow examination has been the confirmatory test for congenital dyserythropoietic anemia type II (CDAII). Occasional spherocytes on peripheral blood smear can confound the diagnosis. Since a screening test is still unavailable, we explored the feasibility of using flow cytometry as a preliminary screening method. METHODS: Thirteen monoclonal antibodies with specificities for eight erythrocyte membrane proteins were used in FACS analysis to probe the cellular features of red cells from CDAII, normal adults, hereditary spherocytosis (HS), and cord red cells. Confocal microscopy was performed on normal and CDAII to determine the overall distribution of CD44 and CD47. Their expression levels on cultured erythroblasts were also analyzed. RESULTS: The densely stained band 3 as seen in CDAII in gel electrophoresis was also obtained for Dantu phenotype. Likewise analysis of CDAII cases (n = 26) using the eosin-5'maleimide (EMA) binding test found 57% of patients giving results either positive or in the grey area for HS. Enhanced fluorescence of CD44 was detected in 96% of the CDAII patients, and anti-CD47 binding was also elevated to a lesser degree. Although RNA expressions of CD44 and CD47 in the cultured erythroblasts of normal controls and CDAII were similar, confocal microscopy revealed more CDAII red cells giving elevated fluorescence than normal red cells. CONCLUSIONS: A distinction between CDAII and HS can be made using the EMA Binding test and anti-CD44 binding. Confirmation of CDAII can subsequently be made based on clinical presentation together with either bone marrow examination or DNA sequencing of SEC23B. © 2016 International Clinical Cytometry Society.

Quantitation of monoclonal antibodies by ELISA. The use of purified mouse IgG and mouse IgM monoclonal antibodies as standards in a quantitative ELISA measuring monoclonal antibodies produced by cell culture.

Murine monoclonal antibodies (MAbs) of IgG1, IgG2a and IgG2b subclasses and IgM class in hybridoma culture supernatants were quantified using a sensitive, reliable, optimized indirect double antibody sandwich ELISA. In the ELISA, the MAb in the culture supernatants was sandwiched between affinity isolated heavy chain specific polyclonal antibodies used for capture and detection. Quantitation was achieved by comparison with a standard curve produced by a purified MAb of the same class, subclass or ideally the same clone as the MAb to be quantified. These quantitative results were compared with those obtained using purified IgG and IgM polyclonal serum samples as standards and those obtained by total protein estimation using measurement at OD280nm. The IgG subclass MAbs used as standards were purified using protein G and the IgM class MAb was purified by ion exchange followed by gel filtration chromatography. Bovine IgG contamination of the MAb supernatants and the purified MAbs was also measured by a double antibody sandwich ELISA.

Effects of phenobarbitone and phenytoin on folate catabolism in the rat.

The effects of phenobarbitone and phenytoin on the catabolism of oral [2-14C] and [3',5',7,9-3H] folic acid were investigated. Normal rats were found to excrete an excess of 3H-labelled compounds into the urine and 14C-labelled compounds into the faeces. Phenytoin abolished this urinary 3H imbalance and also delayed and prolonged the overall excretion of radioactive material. Phenobarbitone appeared to increase the amounts of urinary scission products in the first 24 hr but over the 0-72 hr period both anticonvulsants decreased folate polyglutamate catabolism. As the anticonvulsants used in these experiments decreased folate catabolism in the rat it is unlikely that the megaloblastic anaemia caused by chronic anticonvulsant therapy is due to induction of the enzymes responsible for folate breakdown in vivo.

Toxicity of benzyl cyanide in the rat.

Lethal doses of benzyl cyanide in the rat were 0.64 mmol/kg (i.p.), 1.8 and 2.6 mmol/kg (p.o.) for female and male rats, respectively. Sublethal oral doses of benzyl cyanide were nephrotoxic causing increased excretion of protein, amino acids and glucose. Cyanide was slowly liberated from the dosed nitrile and excreted as cyanide and thiocyanate, the proportion of the former increasing with increasing i.p. dose and with the highest oral dose. The oral cyanide antidote was ineffective against benzyl cyanide and the intravenous antidote, Kelocyanor (cobalt edetate), had little beneficial effect.

Limitations of student evaluations of curriculum.

RATIONALE AND OBJECTIVES: Medical student surveys are used extensively in the development and modification of curriculum. The purpose of this study was to look at medical student surveys of a radiology lecture series, evaluating the accuracy of student perceptions of learning and factors affecting them. MATERIALS AND METHODS: After a "Case of the Week" lecture series, 156 3rd-year medical students returned a survey evaluating the experience with 10 questions on a four-point scale (1 = disagree, 4 = agree very much) and took a clinical competency assessment (CCA) examination with a radiology substation. Survey responses were compared with actual examination performance, analyzed for how overall learning was characterized in specific educational objectives, and evaluated for factors affecting perceived learning. RESULTS: The mean response for perceived CCA examination preparedness was 1.83. The mean radiology station test score was 90.43%. Correlations between student perception of learning and the scoring of focused learning objectives ranged from 0.33 to 0.48 (P < .01). Students responding 1 to items assessing perceived lecture organization, stimulation to read, and interest in the field of radiology had mean scores for perception of overall learning of 2.09-2.44 and mean scores for recommendation of course continuation of 1.68-2.46. Students responding 4 had means of 3.25-3.81 and 3.06-4.0, respectively. CONCLUSION: Student perceptions of the value of curriculum were inaccurate compared with external measures of performance, and students poorly related their general impressions to specific learning objectives. Perceived lecture organization, stimulation to read, and interest in radiology as a specialty affected perceived overall learning and perceived value of the lecture series.

Synthesis and biological properties of some 3-[(N-substituted-amino)pyridinium-4-thiomethyl]-7-[2-(2-amino-thiazol- 4-yl)-2-(Z)-(methoxyimino)acetamido]ceph-3-em-4-carboxylates.

The synthesis and antibacterial activity of a series of beta-lactamase stable, broad spectrum 7-[2-(2-amino-thiazol-4-yl)-2-(Z)-(methoxyimino)acetamido]-cephalo sporins, characterised by a C-3-[N-(substituted-amino)pyridinium-4-thiomethyl] group, is described. Gram-positive and Gram-negative bacteria including extended spectrum beta-lactamase-producing strains were most susceptible to the N-amino- and N-methylamino derivatives (3a) and (3b); with the exception of Pseudomonas aeruginosa, (3b) was more active in vitro and in vivo than cefpirome or ceftazidime.

Novel C-2 substituted carbapenem derivatives. Part I. Synthesis and biological activity of non-aromatic heterocyclic derivatives.

A new series of carbapenems, having a saturated or partially unsaturated heterocycle at C-2, has been synthesised. The in vitro antibacterial activity of these compounds and their stability to human dehydropeptidase-1 (DHP-1) are described. The stereochemistry of the C-2 side-chain and the presence of a double bond in the heterocycle were shown to have significant effects on the stabilities of the compounds to DHP-1.

Structure-activity relationships within a series of C(7)-substitutedoxyiminocephalosporins containing the C(3)-methylaminopyridiniumthiomethyl substituent. Synthesis and biological properties of BRL 57342 and some close analogues.

(6R,7R)-7-[2-(2-Amino-4-thiazolyl)-2-[(Z)-[(S)-carboxy(3,4- dihydroxyphenyl)methyl]oxyimino]acetamido]-3-(1-methylaminopyri dinium-4-thiomethyl)ceph-3-em-4-carboxylate sodium salt (BRL 57342, 1f) combines excellent in vitro antibacterial potency against Gram-positive and Gram-negative bacteria, including P. aeruginosa and Acinetobacter spp., with excellent stability to extended spectrum beta-lactamases. This potency is reflected in in vivo efficacy studies.

Studies on semi-synthetic 7 alpha-formamidocephalosporins. III. Synthesis and antibacterial activity of some 7 beta-[D-2-(aryl)-2-[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl amino] acetamido]-7 alpha-formamidoceph-3-em-4-carboxylate derivatives.

The synthesis and antibacterial activity of 7 beta-[D-2-(aryl)-2-[(4-ethyl-2,3-dioxopiperazin-1-yl) carbonylamino] acetamido]-7 alpha-formamidocephalosporins with various substituents at the C-3 position of the cephalosporin nucleus is described. Inhibition of Gram-positive and Gram-negative bacteria including beta-lactamase producing strains was observed with phenyl as the aryl residue. The 3,4-dihydroxyphenyl group further enhanced the activity against Gram-negative organisms; in this series, the 3-[(1-methyl-1H-tetrazol-5-yl)thiomethyl] and 3-[(1-carboxymethyl-1H-tetrazol-5-yl)thiomethyl] analogues (2 and 12b) exhibited exceptional activity against Gram-negative bacteria, including Pseudomonas aeruginosa.

Synthesis and biological activity of 3-(N-substituted pyridinium-4-thiomethyl)-7 alpha-formamido cephalosporins.

The synthesis and antibacterial activity of a series of 3-(1-substituted pyridinium-4-thiomethyl)-7 alpha-formamido cephalosporins is described. All the derivatives showed good potency and stability to bacterial beta-lactamases. The antibacterial efficacy seen with the N-alkyl pyridinium substituents was enhanced by the introduction of a catecholic side chain at C-7 and by preparation of N-(substituted amino)pyridinium derivatives.

Novel C-2 substituted carbapenem derivatives. Part IV. Synthesis and biological activity of five membered heteroaromatic derivatives.

The synthesis, antibacterial activity, and stability to human dehydropeptidase-1 (DHP-1) of a novel series of (5R,6S)-6-[(1R)-1-hydroxyethyl]-2-heterocyclylcarbapen-2-em-3-carb oxylates are described. Of the compounds investigated 1,5-disubstituted pyrazol-3-yl and 3-substituted isoxazol-5-yl derivatives have the best combination of antibacterial activity and stability to DHP-1. They are particularly active against community-acquired respiratory tract pathogens and have stabilities to DHP-1 superior to that of meropenem.

The old-new distinction and naturalization: 1900.

PIP: The author analyzes differences in naturalization baetween "old" groups of migrants coming primarily from Northern and Western Europe and "new" migrants from Southern and Eastern Europe. The primary focus is on naturalization as a measure of assimilation into the United States. Reasons for differences between the two groups are discussed^ieng

Fertility variation among the U.S. foreign stock population in 1900

PIP: In the late 19th century, the US population included a large number of 1st and 2nd generation immigrants from European countries, often with relatively high fertility levels. This article investigates the degree to which fertility behavior changed as a result of social structural characteristics of the environment such as urbanization and children's role, the diffusion of fertility values and information from the native population, and the role of cultural values or resistance to change. By 1900 nationality groups within urban and rural environments in the US were showing amazingly similar degrees of adaptation in fertility patterns. Distinctive fertility patterns were being maintained over generations, but there appeared to be little inherence in the national culture or mentalities of the various European populations which retarded the speed of reproductive change, or even the legitimacy of smaller families. The clear continuing variations by nationality in fertility were geared to reflect social structural differences such as agricultural background and the functional role of children, rather than unique cultural outlooks. Even within the 1st generation, fertility variation across states responded in a regular manner to social structural factors, such as the agricultural orientation of the population and the importance of child labor. Other evidence suggests that some diffusion of fertility values must have been occurring from the native white to the foreign born population, especially within the urban parts of the US where greater contact was probably facilitated. The foreign born seemed particularly resistant to adoption of low fertility behavior in rural states where they comprised a significant segment of the population, and thus were probably able to isolate themselves more easily. These results do not support the notion of relative unique cultural adaptation. It may be that studies of the European transition have placed too much emphasis on cultural mentalities within various populations as a cause of different reproductive trends.^ieng

Own-child estimates of U. S. white fertility, 1886-1899.

PIP: Uses data from a 1/750 household sample of the 1900 census manuscripts to make own-child estimates of fertility behavior among American white women in the years between 1886 and 1899. Fertility behavior is estimated by linking children's records with those of their mothers. New estimates of American total fertility rates for each year are provided separately for native and foreign-born whites. The degree of conscious control of fertility at this important period in American demographic history is estimated on the basis of age-specific patterns of reproduction. Variations in fertility patterns within major regional and urban-rural residence categories are analyzed, especially for the last 5 years of the century. Data on trends in fertility in the last 14 years of the century suggest the close correspondence between the fertility decline and the development of a mass educational system, particularly for native whites. Patterns for the foreign born during this time period are less easily interpretable within a modernization framework. Evidence on patterns of family limitation indicates the degree to which this period represented a transition from the minimal degree of conscious control over fertility at earlier points in the 19th century to the extremely rationalized system of reproduction which developed after 1940. The significant amount of fertility control at this time is not surprising, given other related studies, particularly since highly effective methods of birth control were probably not generally practiced. Abortion, however, may have been a very important means of family limitation. Patterns of reproduction in the late 1880s were highly varied within the United States. Native white women in the urban North Atlantic region had essentially achieved modern patterns of fertility limitation by the turn of the century, while Southern farm women were only a few steps removed from natural fertility. Important questions are raised concerning how populations with relatively similar cultural and ethnic heritages could achieve such different reproduction patterns. Charts and graphs illustrating birth rates, fertility rates and patterns are given.^ieng

Detection of hereditary pyropoikilocytosis by the eosin-5-maleimide (EMA)-binding test is attributable to a marked reduction in EMA-reactive transmembrane proteins.

INTRODUCTION: Hereditary spherocytosis (HS) and hereditary pyropoikilocytosis (HPP, severe form of hereditary elliptocytosis) are unrelated red cell disorders caused by defects in distinct regions of the red cell cytoskeleton. The high predictive value of the eosin-5-maleimide (EMA)-binding test for the diagnosis of HS is because of its interaction with transmembrane proteins band 3, Rh protein, Rh glycoprotein and CD47, which are reduced on HS red cells. Our study was undertaken to determine why EMA-labelled HPP red cells were previously found to give much lower fluorescence readings than HS. METHODS: Flow cytometry was used to determine the relative amounts of monoclonal antibodies bound to red cells from normal adults, HS and HPP groups. Confocal microscopy was used to visualise the overall staining pattern of the red cells with selected antibodies. RESULTS: In flow cytometry, HPP red cells gave lower antibody binding to the four EMA-reactive membrane proteins than HS red cells and bound less antibody to glycophorins A and C, and CD59. Confocal images of Rh protein and band 3 immunostaining revealed a greater number of HPP red cells having partial or no fluorescence than in HS and normal controls. CONCLUSION: Lesser amounts of EMA-reactive membrane proteins were detected in HPP than HS red cells, thus confirming their lower fluorescence readings in the EMA-binding test. The concomitant reduction in glycophorins A and C, and CD59 in HPP could have caused cellular contraction, resulting in poikilocytosis.