Quantitative and spatial information on the composition of chimaeric fetal mouse eyes from single histological sections.

The spatial distribution of cells in chimaeric tissues, composed of two genotypes, provides insights into the extent of cell mixing during development and growth. However, direct measurement of patch sizes is not usually meaningful because, when the proportion of one genotype is high, a single patch may encompass several adjacent coherent clones of like genotype (clone aggregation). Two previously used methods of comparing patch lengths were evaluated to overcome this problem. The corrected mean patch length (corrected for the predicted effects of random clone aggregation) is a more useful summary statistic than the median patch length of the minor genotype, because its use is not restricted to grossly unbalanced chimaeras, but its validity has been questioned. The two methods gave almost identical numerical summaries of patch sizes in the retinal pigment epithelium of fetal chimaeras, thereby validating the use of the corrected mean patch length for this tissue. The present study also showed that the corrected patch length was unaffected by the presence of cells hemizygous for the TgN(Hbb-b1)83Clo transgene and that the proportion of pigmented cells in a single histological section was representative of the overall composition of the chimaeric fetus.

Novel affinity chromatographic system for the single-step purification of glycosaminoglycans from complex systems using volatile buffers.

A new system for the isolation and purification of glycosaminoglycans (GAGs) and related molecules from complex systems such as plasma is described. Affinity chromatography which exploits the very high affinity between the polymeric base Polybrene and sulphated polysaccharides was used. A novel volatile buffer system composed of ammonium formate and formic acid, which allows the complete recovery of samples, was developed, and elution conditions were optimised for the separation and purification of GAGs of different charge densities. Using this system the losses associated with dialysis and desalting, frequently necessary preliminaries to further analysis, are avoided.

Plasma exchange as a treatment for endogenous glycosaminoglycan anticoagulant induced haemorrhage in a patient with myeloma kidney.

A 57-year-old man with end-stage renal failure secondary to myeloma kidney developed haemorrhagic complications due to endogenous glycosaminoglycan anticoagulant production. Glycosaminoglycan levels and anticoagulant effect were reduced by plasma exchange and this contributed to control of the haemorrhagic manifestations.

The absorption, clearance and metabolic fate of dermatan sulphate administered to man--studies using a radioiodinated derivative.

An iodinated derivative of dermatan sulphate was administered by the intravenous, subcutaneous and oral routes to healthy human volunteers in conjunction with unlabelled dermatan sulphate. Following intravenous injection clearance of radiolabel and concentration as measured by competitive binding assay were highly correlated and displayed complex kinetics which were not dose-dependent. Intact 125I-dermatan sulphate was absorbed following both subcutaneous and oral administration, though there appeared to be selective uptake by the gut of a subfraction comprising the smaller or less sulphated molecules. The intact material was subsequently excreted unchanged in the urine. Degradation products of dermatan sulphate were not detected by either gel filtration or affinity chromatography on Polybrene-Sepharose at any time in either plasma or urine, indicating that administered dermatan sulphate is not catabolised by man.

Oligoclonal tumours.

Tumours are commonly classified as monoclonal or polyclonal. The question of how many clones are present in a polyclonal tumour is seldom asked; it is important, however, because the answer may show whether or not clones arise and develop independently, and whether the number of clones in tumours of a particular kind tends to increase or decrease with time. We have used two procedures to assess the clonality of chemically-induced murine fibrosarcomas, one based on the heterozygosity of the tumour hosts for an X-linked marker, the other on the expression of tumour-associated transplantation antigens (TATA) by the tumours. As we have reported previously, many of these tumours are pleoclonal. Evidence now presented suggests that the clones do not develop independently and that many of the tumours are biclonal.

The effect of passage in vivo and in vitro on the properties of murine fibrosarcomas: III. Cell surface molecules and production of growth factors.

Three factors may be responsible for the sharp difference in tumourigenicity between cloned murine fibrosarcoma lines maintained in vitro, and cells of the same lines after in vivo passage, initially in a T cell deficient mouse and subsequently in normal mice: acquisition during passage of resistance to NC cells; acquisition during passage of a surface molecule, probably a sialic acid, which protects the cell against T cell-mediated lysis; and ability of the passaged cells, but not the non-passaged cells, to produce sufficient amounts of autocrine growth factors necessary for growth in vivo. The tumourigenicity of the passaged cells cannot be attributed to failure to express TATA or MHC class I molecules.

The effect of passage in vitro and in vivo on the properties of murine fibrosarcomas. II. Sensitivity to cell-mediated cytotoxicity in vitro.

The sensitivity of cultured and mouse-passaged cloned lines of chemically-induced murine fibrosarcomas to killing by NK and NC cells, and to cell-mediated immunity, has been studied in in vitro assays, using target cells labelled with 51Cr or 125IUDR. None of the lines tested proved sensitive to NK cells. Three cultured lines were, at most, only slightly sensitive to NC cells; a fourth cultured line was moderately sensitive and became less so, but not completely insensitive, after passage in susceptible hosts. The primary object of these experiments was to test the hypothesis that cultured cell lines which ordinarily fail to grow in normal mice are able to grow after being passaged in a susceptible immunodeficient host because, during this passage, they become resistant to NK or NC cells. This has been shown to occur with one clone, but will not serve as a general explanation because, with other clones, both cultured and mouse-passaged lines were NC-insensitive. The cell-mediated immunity assays confirm our previous conclusion that cultured and mouse-passaged lines of the same clone differ little, if it all, in immunogenicity.

The effect of passage in vitro and in vivo on the properties of murine fibrosarcomas I. Tumorigenicity and immunogenicity.

Cloned cell lines of chemically-induced murine fibrosarcomas maintained in tissue culture usually fail to grow when transplanted to normal syngeneic mice. They grow, however, in various categories of T cell deficient mice and after such passage grow readily in normal mice. Both cultured and mouse-passaged lines possess strong TATA. Three alternative explanations are suggested which might account for these findings. Emergence during the initial passage of a population of tumour cells resistant to NC cells. Acquisition during the initial passage of a protective surface molecule that interferes with the efferent side of the immune response when the tumour cells are subsequently transplanted to a normal host. Loss during the initial passage of a Class I MHC molecule which prevents dual recognition of the tumour cells by T cells when they are transplanted to a normal host. New experiments are proposed to distinguish between these possibilities.

Specificity of tumour associated transplantation antigens (TATA) of different clones from the same tumour.

The TATA of two clones from the same murine methylcholanthrene-induced fibrosarcoma have been investigated by immunizing syngeneic mice with irradiated cells of one or both clones and challenging them 14 days later with viable cells. The tumour had been induced in a female backcross CBA mouse heterozygous for the A and B alloenzymes of phosphoglycerate kinase-1 (PGK-1). One clone expressed A and the other B, and both A and B hosts were used in the experiments. Each clone was found to possess strong TATA but there was no demonstrable cross reactivity. The clonal composition of tumours produced by inoculating mice with a mixture of the two clones was profoundly altered by prior immunization with one of them. A second experiment was performed with 3 clones from another tumour; these expressed PGK-1 A, B and AB respectively. Again, there was no evidence of immunological cross reactivity between the A and B clones, but there was some cross reactivity between the A clone and AB clone. These results, coupled with previous observations of changes in the clonal composition of pleoclonal murine fibrosarcomas in culture and on transplantation, suggest that the antigenic specificity of these tumours is less stable than is commonly supposed.

Accumulation of UDP-glucose pyrophosphorylase by axenically grown amoebae of Dictyostelium discoideum.

Amoebae of the slime mould Dictyostelium discoideum AX2 possess only low UDP-glucose pyrophosphorylase activity when grown on autoclaved Klebsiella aerogenes (approx. 30 units/mg of protein), but accumulate the enzyme to approx. 150-200 units/mg of protein during vegetative growth in axenic medium. The vegetative accumulation of UDP-glucose pyrophosphorylase by axenically grown cells is prevented if autoclaved K. aerogenes are included in the axenic medium, suggesting the absence of a specific inducer. Affinity chromatography using anti-(UDP-glucose pyrophosphorylase) antibody and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicate that the enzyme accumulated during axenic growth and that normally accumulated during development are immunologically cross-reactive and that both are composed of two subunits with mol.wts. 55,600 and 57,500 present in approximately equal amounts in the active enzyme.