Genotype-phenotype correlation and frequency of the 3199del6 cystic fibrosis mutation among I148T carriers: results from a collaborative study.

PURPOSE: We expect that the mutation panel currently recommended for preconception/prenatal CF carrier screening will be modified as new information is learned regarding the phenotype associated with specific mutations and allele frequencies in various populations. One such example is the I148T mutation, originally described as a severe CF mutation. After implementation of CF population-based carrier screening, we learned that I148T exists as a complex allele with 3199del6 in patients with clinical CF, whereas asymptomatic compound heterozygotes for I148T and a second severe CF mutation were negative for 3199del6. METHODS: We performed reflex testing for 3199del6 on 663 unrelated specimens, including I148T heterozygotes, compound heterozygotes, and a homozygous individual. RESULTS: Less than 1% of I148T carriers were also positive for 3199del6. Excluding subjects tested because of a suspected or known CF diagnosis or positive family history, 0.6% of I148T-positive individuals were also positive for 3199del6. We identified 1 I148T homozygote and 6 unrelated compound heterozygous individuals with I148T and a second CF variant (2 of whom also carried 3199del6). In addition, one fetus with echogenic bowel and one infertile male were heterozygous for I148T (3199del6 negative). CONCLUSIONS: Reflex testing for 3199del6 should be considered whenever I148T is identified. Reflex testing is of particular importance for any symptomatic patient or whenever one member of a couple carries a deleterious CF mutation and the other member is an I148T heterozygote. Further population data are required to determine if I148T, in the absence of 3199del6, is associated with mild or atypical CF or male infertility.

Relationship of nematode cholinesterase activity and nematode burdens to the development of resistance to trichostrongyle infections in sheep.

The changes in nematode cholinesterase (ChE) activities were examined in relation to the development of resistance in (1) a flock of young grazing sheep, (2) grazing and penned sheep treated with dexamethasone and (3) penned sheep receiving a single mixed infection. Nematodes from grazing sheep with high faecal egg counts (FECs) had higher ChE activities than those from sheep with low FECs. Female nematodes tended to have higher ChE activities than males, and ChE activities in both tended to decline with increasing age of the sheep. The decline in female Trichostrongylus colubriformis ChE activity was associated with a decline in both worm length and in utero egg count. No decline in nematode ChE activity was observed when grazing sheep were treated with dexamethasone. ChE activity of T. colubriformis established in immunosuppressed penned sheep declined 10-20 fold 8 weeks after cessation of treatment. Nematode burdens in the small intestine and abomasum of grazing sheep were significantly correlated, and in individual species they were also correlated with ChE activities. The development of resistance in sheep and the elimination of adult nematode burdens is discussed in relation to gastrointestinal mucosal globule leucocyte numbers, mucus antiparasite activity and the impairment of nematode metabolic function.

Relationship of gastrointestinal histology and mucus antiparasite activity with the development of resistance to trichostrongyle infections in sheep.

Changes in the numbers of globule leucocytes, mast cells, eosinophils and goblet cells in the gastrointestinal mucosa were examined in relation to the development of resistance and elimination of nematodes in grazing sheep in their first year of life. Sheep immunised against Trichostrongylus colubriformis, and sheep treated with dexamethasone were also examined. A strong association between resistance to infection and the presence of globule leucocytes was found. In contrast, the numbers of mast cells or goblet cells were not correlated with resistance. Globule leucocyte and eosinophil numbers were also correlated with antiparasite activity in mucus. Immunising infections of T. colubriformis given to 10-month-old sheep, their duration limited by thiabendazole treatment, gave rise to considerable immunity to homologous challenge infections. Larvae that developed to the 4th stage were as effective at stimulating immunity as those that developed to the 5th stage. Dexamethasone treatment abrogated resistance to trickle challenge infection with T. colubriformis and reduced mucosal globule leucocyte and mast cell numbers. After cessation of drug treatment, the re-establishment of resistance and adult worm elimination were associated with repopulation of the mucosa with large numbers of globule leucocytes and high antiparasite activities in mucus.

Relationship of histamine in tissues and antiparasitic substances in gastrointestinal mucus to the development of resistance to trichostrongyle infections in young sheep.

The development of resistance to nematode infection and self-cure in a flock of young grazing sheep were examined in relation to changes in the levels of histamine in tissues and levels of antiparasitic substances in gastrointestinal mucus. Analysis of faecal egg counts showed that when the sheep were ranked according to individual mean monthly egg counts there was a significant trend to similar rankings in successive months. Sheep with high and low egg counts were slaughtered at monthly intervals for examination and comparison. Histamine levels in blood and intestinal content fluids were similar in both groups of sheep and were highest during maximum challenge by larval nematodes. Antiparasite activity of the intestinal mucus was significantly higher in sheep with low egg counts than those with high counts, between January and May, and was associated with significantly lower burdens of fourth stage larvae.

Some properties of the sulphoxidases and sulphoxide reductases of the cestode Moniezia expansa, the nematode Ascaris suum and mouse liver.

1. The anthelmintics bithionol, phenothiazine, albendazole and fenbendazole were oxidized to sulphoxides by enzymes in the cytosol of the proglottids of the cestode Moniezia expansa and the cytosol of the intestinal epithelial cells of the nematode Ascaris suum. Enzymes in these tissues were also able to reduce these sulphoxides to the thioethers in the absence of oxygen. 2. Sulphoxidation and sulphoxide reduction also occurred in mouse liver enzyme preparations. About 20% of the sulphoxidation activity was not associated with microsomes and was not inhibited by CO; about 50% of the reductase activity was found in the microsomes. 3. The pH optima for sulphoxidases from both helminths were in the range 7.0--7.2, and both required NADH or NADPH for activity. Low molecular weight thiols and flavins did not affect sulphoxidation. Enzyme activity was inhibited by 0.1 mM Cu2+, Hg2+, Cd2+ or Zn2+ and by p-chloromercuribenzoate or N-ethylmaleimide. 4. Both helminth sulphoxide reductases displayed pH optima in the range 1.2--7.4, and required NADH or NADPH for activity. Oxygen inhibited the reductases.

The metabolism of nitrophenolic and 5-arylazorhodanine anthelmintics by Ascaris suum, Moniezia expansa and by mouse- and sheep-liver enzymes.

1. The anthelmintics disophenol (2,6-diiodo-4-nitrophenol), nitroxynil (3-iodo-4-hydroxy-5-nitrobenzonitrile) and nitrodan (3-methyl-5-(4-nitrophenylazo)rhodanine) were reduced in vitro to the corresponding amines by intact Ascaris suum, Moniezia expansa, by enzymes prepared from these helminths, and by mouse- and sheep-liver homogenates. Helminth reductases required NADH2 and glutathione as cofactors and were inhibited about 50% by 2.0 x 10(-7) M allopurinol. Azo bonds of nitrodan and its analogues were not reduced by the helminths but were reduced by mouse- and sheep-liver enzymes. 2. Mouse- and sheep-liver enzymes, in addition to effecting nitro reduction, metabolized nitroxynil by hydrolysis to 3-iodo-4-hydroxy-5-nitrobenzamide and 3-iodo-4-hydroxy-5-nitrobenzoic acid. No hydroxylation products were found. Nitrodan was oxidized by the mammalian microsomal oxidation enzyme system to the thiazolidinedione derivative, but not by helminth enzymes.

Glutathione conjugation of some xenobiotics by Ascaris suum and Moniezia expansa.

1. The cestode Moniezia expansa and the nematode Ascaris suum both possess enzymes catalysing the conjugation of glutathione (GSH) with 1-chloro-2,4-dinitrobenzene. 2. The GSH-S-aryl transferase (GSH-S-transferase A) was present in the cytosol of the cestode proglottids and of the nematode intestinal epithelial cells. Other tissues did not contain measurable activity. The enzymes from both species had mol. wt. of about 37 000 and broad pH optima around pH 8.3. Both enzymes were inhibited by Cu2+, Fe3+ and Hg2+ at 1 mM and stimulated by Co2+. 3. Neither M. expansa nor A. suum possessed measurable DDT dehydrochlorinase activity. GSH-S-epoxide transferase (GSH-S-transferase E) activity was indicated in both species; neither species effected the conjugation of bromo- or chlorobenzene. 4. Halogenated anthelmintics were not metabolized to GSH conjugates in the helminths studied and did not inhibit GSH-S-aryltransferase activity towards chlorodinitrobenzene.