Anders Elmgren

Treatment of Growth Hormone-Deficient Adults with Recombinant Human Growth Hormone Increases the Concentration of Growth Hormone in the Cerebrospinal Fluid and Affects Neurotransmitters

In a double-blind, placebo-controlled trial, the effects of recombinant human growth hormone were studied on cerebrospinal fluid concentrations of growth hormone, insulin-like growth factor 1 (IGF-1), insulin-like growth factor binding protein-3 (IGFBP-3), monoamine metabolites, neuropeptides and endogenous opioid peptides. Twenty patients, 10 patients in each of 2 groups, with adult-onset, growth hormone deficiency were treated for 1 month with recombinant human growth hormone (0.25 U/kg/week) or placebo. All the patients received the appropriate thyroid, adrenal and gonadal hormone replacement. In cerebrospinal fluid, the mean concentration of growth hormone increased from 13.3 +/- 4.4 to 149.3 +/- 22.2 muU/l (p = 0.002), during recombinant human growth hormone treatment. The cerebrospinal fluid IGF-I concentration increased from 0.67 +/- 0.04 to 0.99 +/- 0.10 micrograms/l (p = 0.005) and the IGFBP-3 concentration rose from 13.4 +/- 1.25 to 17.5 +/- 1.83 micrograms/l (p = 0.002). The dopamine metabolite homovanillic acid decreased from 282.1 +/- 36.0 to 234.3 +/- 26.5 nmol/l (p = 0.02) and the vasoactive intestinal peptide decreased from 4.1 +/- 0.6 to 3.7 +/- 0.4 pmol/l (p = 0.03). Cerebrospinal fluid immunoreactive beta-endorphin increased from 24.4 +/- 1.8 to 29.9 +/- 2.1 pmol/l (p = 0.002). There were no significant changes compared to baseline in the cerebrospinal fluid concentrations of enkephalins, dynorphin A, the norepinephrine metabolite 3-methoxy-4-hydroxyphenyl-ethyleneglycol, the serotonin metabolite 5-hydroxyindoleacetic acid, gamma-aminobutyric acid, somatostatin or corticotropin-releasing factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Evolution of Fucosyltransferase Genes in Vertebrates

Cloning and expression of chimpanzee FUT3, FUT5, and FUT6 genes confirmed the hypothesis that the gene duplications at the origin of the present human cluster of genes occurred between: (i) the great mammalian radiation 80 million years ago and (ii) the separation of man and chimpanzee 10 million years ago. The phylogeny of fucosyltransferase genes was completed by the addition of the FUT8 family of α(1,6)fucosyltransferase genes, which are the oldest genes of the fucosyltransferase family. By analysis of data banks, a newFUT8 alternative splice expressed in human retina was identified, which allowed mapping the human FUT8 gene to 14q23. The results suggest that the fucosyltransferase genes have evolved by successive duplications, followed by translocations, and divergent evolution from a single ancestral gene.

Significance of Individual Point Mutations, T202C and C314T, in the Human Lewis (FUT3) Gene for Expression of Lewis Antigens by the Human α(1,3/1,4)-Fucosyltransferase, Fuc-TIII

The Lewis α(1,3/1,4)-fucosyltransferase, Fuc-TIII, encoded by theFUT3 gene is responsible for the final synthesis of Lea and Leb antigens. Various point mutations have been described explaining the Lewis negative phenotype, Le(a−b−), on erythrocytes and secretions. Two of these, T202C and C314T originally described in a Swedish population, have not been found as single isolated point mutations so far. To define the relative contribution of each of these two mutations to the Lewis negative phenotype, we cloned and made chimeric FUT3 constructs separating the T202C mutation responsible for the amino acid change Trp68 → Arg, from the C314T mutation leading to the Thr105 → Met shift. COS-7 cells were transfected and the expression of Fuc-TIII enzyme activity and the presence of Lewis antigens were determined. There was no decrease in enzyme activity nor of immunofluorescence staining on cells transfected with the construct containing the isolated C314T mutation compared with cells transfected with a wild type FUT3 allele control. No enzyme activity nor immunoreactivity for Lewis antigens was detected inFUT3 constructs containing both mutations in combination. The T202C mutation alone decreased the enzyme activity to less than 1% of the activity of the wild type FUT3 allele. These results demonstrate, that the Trp68 → Arg substitution in human Fuc-TIII is the capital amino acid change responsible for the appearance of the Le(a−b−) phenotype on human erythrocytes in individuals homozygous for both the T202C and C314T mutations.

Typing for the Human Lewis Blood Group System by Quantitative Fluorescence–Activated Flow Cytometry: Large Differences in Antigen Presentation on Erythrocytes between A1, A2, B, O Phenotypes

Background: Lewis phenotyping by hemagglutination is an unreliable routine method for Lewis antigen designation. Now genomic typing of the Lewis gene is available. Additionally, flow cytometry has been used for typing. We wanted to compare the results of Lewis typing in healthy individuals using the three methods. Materials and Methods: Ninety–three randomly selected plasma donors were genotyped for inactivating Secretor (FUT2) G428A and Lewis (FUT3) T59G, T202C, C314T, G508A and T1067A point mutations. All Le(a+b–) individuals (nonsecretors) were homozygous for the FUT2 G428A mutation and all Le(a–b–) individuals had inactivating mutations on both FUT3 alleles. Fixed erythrocytes were analyzed by fluorescence–activated flow cytometry and the results were compared with hem– agglutination and genotypic data. Antigen availability was expressed as median fluorescence intensity and as percentage positive cells with fluorescence intensities ≥102. Results: Using an anti–Lea reagent a mean of 99% of erythrocytes from Le(a+b–) individuals and 1% of erythrocytes from Le(a–b–) or Le(a–b+) individuals were stained positive. Using an anti–Leb reagent, a mean of 71% of erythrocytes from A1, 95% from B and 99% from O and A2 Le(a–b+) individuals and less than 10% of erythrocytes from Le(a–b–) or Le(a+b–) individuals were stained positive. After papain treatment 100% of the erythrocytes from A1 and A1B Le(a–b+) individuals stained positive without increase in background staining. The flow cytometric technique revealed large differences in staining intensities, within each ABO Le(a–b+) subgroup which was not directly correlated to plasma donation frequencies nor to Secretor or Lewis genotypes. Conclusion: Flow cytometry may prove valuable as a Lewis blood group typing technique but also as a research tool when investigating Lewis phenotypes of human erythrocytes.

Nomenclature of Granulocyte Alloantigens

and the detection of new ones has made a new nomenclature for these antigens necessary. Neutrophil alloantibodies are known to cause neonatal alloimmune neutropenia, immune neutropenia after bone marrow transplantation, refractoriness to granulocyte transfusions, and severe transfusion-related acute lung injury. With respect to the increasing use of granulocyte transfusions this is of special importance, since alloimmunization to granulocyte antigens can decrease the recovery and half-life of transfused granulocytes, can prevent their migration to sites of infection and can cause severe pulmonary transfusion reactions. The Granulocyte Antigen Working Party of the ISBT agreed at their meeting in S’Agaró, Spain, in 1998, to establish a new nomenclature system for well-defined neutrophil alloantigens, which is based on the following rules: (1) Neutrophil antigens are to be called HNA for ‘human neutrophil antigen(s)’. (2) MemISBT Working Party

Identification of two functionally deficient plasma ?3-fucosyltransferase (FUT6) alleles

One Indonesian individual without detectable plasma α3‐fucosyltransferase activity was identified with three point mutations, 730C>G (L244V), 907C>G (R303G), and 370C>T (P124S), in the coding region of one FUT6 allele. Another individual, expressing weak plasma α3‐fucosyltransferase activity, had the 907C>G together with the 370C>T mutation, but did not have the 730C>G mutation. PCR‐RFLP analyses of complete families confirmed the segregation of these alleles and illustrated the existence and inheritance of the [370C>T; 907C>G] mutated allele in three additional families. Altogether, this allele was found heterozygously in nine Indonesian and two Swedish individuals, all with detectable plasma α3‐fucosyltransferase activities. The FUT6 allele with the three mutations (370C>T; 730C>G; 907C>G) was identified heterozygously in only two Indonesian individuals, both having the inactivating 739G>A mutation in the other allele and both lacking plasma α3‐fucosyltransferase activity. Enzyme studies made on transiently transfected COS‐7 cells demonstrated that the combination of the 370C>T, 730C>G and 907C>G mutations decreased the Vmax by more than 80%, but caused no obvious change of the apparent Km values for GDP‐fucose and Gal‐N‐acetyllactosamine. In comparison, chimeric constructs with the isolated 730C>G or 907C>G mutations decreased the Vmax values by about two thirds and one third, respectively. Hum Mutat 16:473–481, 2000.