D.R. Turner

Mutations in human lymphocytes studied by an HLA selection system

Human lymphocytes mutated at the HLA-A2 or HLA-A3 alleles were enumerated and studied by primary selection using antibody and complement, followed by limiting dilution cloning and secondary selection using immunofluorescence or antibody and complement. The geometric mean frequency of in vivo mutant lymphocytes was 3.08 X 10(-5) for the HLA-A2 allele and 4.68 X 10(-6) for the HLA-A3 allele. Mutagenesis by X-radiation or mitomycin produced a dose-related increase in mutant frequency. HLA-B phenotyping and Southern Analysis of the HLA-A gene suggested that mutation was frequently due to gene deletion, which was often substantial.

Effect of age on lymphocyte proliferation

The relationship of lymphocyte proliferative capacity to age was studied using lymphocytes from neonates, from young adults aged 20-30 and from healthy individuals aged 70-90. Mass cultures expanded exponentially and eventually died after a final expansion of 10(19)-10(52). They therefore showed a Hayflick effect, but in contrast to reported findings for other cell types there was no relationship between age and the magnitude of the final expansion. Cytogenetic and molecular studies showed that monoclonality developed in all mass cultures. Study of individual clones also showed exponential growth followed by cessation. The magnitude of the expansion, 10(4)-10(35), was substantially less than that observed for mass cultures, but was related to age. We conclude that lymphocytes have a heterogeneous proliferative potential, that the overall proliferative potential declines with age but that rare cells having extended proliferative potential continue to be present into old age. The development of monoclonality during the course of mass cultures has implications for the interpretation of findings from such cultures since observations drawn from the later stages of culture reflect the properties of rare cells having high proliferative potential and do not necessarily reflect the properties of the overall population.

Reappraisal of the causes of the “hook effect” in two-site immunoradiometric assays

Abstract A simulation of two-site immunoradiometric assays (2-site IRMA) based on calculations of chemical equilibria was derived and used to investigate assay conditions under which aberrant results may occur. In simulated 2-site IRMA, the assay response was governed in a complicated but predictable way by (i) the amount of analyte immobilized by the solid-phase antibody in the first step of the assay, (ii) the extent of competing side reactions in the second step of the assay, and (iii) the extent of complexing of immobilized analyte by labeled antibody in the second step of the assay. This last determinant of assay response was particularly important when inadequate concentrations of labeled antibody were present in the second step of the assay. Under these conditions a fall in the assay response occurred at high sample concentrations. This previously reported artefact, known as the “hook effect”, could thus be produced in simulated assays without assuming that the solid-phase antibody was contaminated with low-affinity binding sites or that incomplete washing of the solid phase occurred after the first assay step.

Ageing in vivo does not alter the kinetics of DNA strand break repair.

The alkaline elution technique has been used to measure the apparent rate constant for repair of DNA strand breaks induced by X-radiation in human transformed lymphocyte DNA. Repair follows first-order kinetics and is essentially complete within 60 min. Although biological variation is observed there is no significant change in rate of repair with age of lymphocyte donor. In light of this it is unlikely that age-associated changes in DNA which have been widely observed arise from a changed persistence of strand breaks.

Age-related variations in human lymphocyte DNA

The molecular weight of DNA from human peripheral lymphocytes has been measured on alkaline sucrose density gradients. The number average molecular weight (MN) of DNA is found to decrease as the age of the donor increases. This result is discussed with reference to lesions in DNA, both repairable and non-repairable, which may accumulate with age.

Karyotypic abnormality of the X chromosome is rare in mutant HPRT− lymphocyte clones

Lymphocyte clones mutated at the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) locus on the X chromosome were studied by synchronization and G banding to determine the proportion of mutant clones having visible karyotypic change. 47 spontaneously mutant clones, 17 mutant clones induced by X-irradiation and 33 wild-type clones were studied. All clones were karyotypically normal except for 1 clone induced by X-irradiation in which an interstitial deletion of the short arm of the X chromosome had been inserted into the long arm of the same chromosome between q23 and q24; this change may have been coincidental or may have resulted in a position effect mutation. It was concluded that the great majority of mutations were not associated with a visible chromosome abnormality. This conclusion complements molecular studies which suggest that gene changes at the HPRT locus in HPRT- mutants generally extend over segments of DNA too small to be resolved by karyotypic analysis.

Lymphocyte DNA in Aplastic Anaemia

Summary. Using ultracentrifugation on alkaline sucrose density gradients the DNA of lymphocytes from 14 patients with aplastic anaemia and 23 controls was studied before and after exposure to bleomycin, an agent known to cause strand breaks in DNA. Before exposure to bleomycin the DNA from aplastic patients had more strand breaks than the DNA from the controls of similar ages. Following exposure to bleomycin an abnormal number of DNA strand breaks was produced in 10 of 14 patients and this molecular evidence of drug sensitivity correlated well with the sensitivity of the proliferating lymphocytes to bleomycin in tissue culture. Furthermore, two relatives of patients with aplastic anaemia showed similar abnormality of DNA before and after exposure to bleomycin and increased sensitivity to bleomycin in tissue culture. These results suggest that an abnormality of DNA structure and/or repair may be present in some patients with aplastic anaemia.

An analysis of the behaviour of the solid phase radioimmunometric assay of serum ferritin

Two artefacts of solid phase radioimmunometric analysis of serum ferritin confuse the interpretation of results: (1) Samples with high ferritin content may give apparently normal results. (2) Progressive dilution of samples gives increasing final results. The behaviour of this analytical system was investigated using a theoretical model based on chemical equilibria. It was shown that artefactually low results are obtained if low affinity binding of ferritin to the solid phase antibody occurs in the first assay step. This loosely bound ferritin dissociates in the second assay step and complexes with radio-labelled antibody, preventing the latter binding to immobilized ferritin. Furthermore, competition between isoferritins for antibody binding sites results in different populations of ferritin molecules being bound to the solid phase antibody, depending on the dilution of the sample. This results in a non-linear dilution curve. If the serum ferritin is grossly heterogeneous, this methodology cannot give a valid result.