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The authors report two cases presenting to a rheumatologist, one with palindromic rheumatism and previously undiagnosed ulcerative colitis, and one with rheumatoid arthritis. Both were subsequently found to have early sclerosing cholangitis with some response treatment.

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We have used the Health Assessment Questionnaire (HAQ) to follow changes in disability in an unselected group of 245 patients with RA. The HAQ has been widely used in cross-sectional studies of disability in RA, but little is known about the dynamics of the change in HAQ score with long term follow-up. If it is to prove useful as a measure of health outcome it must not only be able to accommodate a wide range of disability but also show adequate sensitivity to change in disability. We administered the HAQ to 245 RA inpatients and outpatients at the beginning and end of a 5-yr period to address this important question. The mean change in individual HAQ score in the 175 patients for whom complete data was available was +0.18 (SD 0.66) over 5 yr, i.e. 0.03 units per year. It is likely that the observed rate of change in HAQ score is an under-estimate of the true rate of progression of disability, as the scale failed to accommodate change in disability toward its upper limit. The inherent design of the HAQ creates several 'ceilings' in functional subcategories (such as lower limb function) which may be masked by the overall HAQ score. Longitudinal studies of disability using the HAQ as outcome measure should therefore be interpreted with caution, and close attention paid to the baseline HAQ score.

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Flow cytometry and laser scanning confocal imaging have been used to analyze the uptake of the anticancer topoisomerase II poison mitoxantrone by intact mammalian cells and the results correlated with the induction of DNA damage. Unlike Adriamycin, mitoxantrone displays only minimal levels of red fluorescence when excited at 514 wavelength. However, using these excitation and emission conditions, flow cytometry could detect low levels of fluorescence in human transformed fibroblasts exposed to high concentrations (5-20 microM) of mitoxantrone for 1 h. Over this dose range whole cell fluorescence was a function of cell size and increased with drug concentration while drug-induced DNA-protein cross-linking showed saturation. Confocal microscopy revealed the time- and dose-dependent appearance of fluorescence, interpreted here as reflecting the disposition of drug molecules, preferentially within the cytoplasm, nuclear membrane, and nucleoli. This pattern contrasted with the intense intranuclear fluorescence observed in Adriamycin-treated human cells. Loss of the nuclear membrane during mitosis resulted in an apparent increase in chromatin-associated fluorescence. Photon counting procedures revealed a predominantly cytoplasmic, possibly lysosomal, location for fluorescence from human cells exposed for 1 h to a low but cytotoxic concentration (0.1 microM, yielding approximately 90% cell kill) of mitoxantrone. At this low concentration, human cells displayed minimal levels of DNA strand cleavage or DNA-protein cross-linking. Murine cells, displaying mitoxantrone resistance as part of the P-glycoprotein-mediated multidrug resistance phenotype, showed specific extinction of mitoxantrone-associated fluorescence from inside nuclei but not from within extranuclear compartments. The study demonstrates the feasibility of high resolution studies on the intracellular distribution of mitoxantrone in intact living cells. We suggest a mechanism by which cytoplasmic sequestration of mitoxantrone may be important in determining the response of normal and multidrug-resistant cells as they attempt to progress through mitosis.

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Techniques for the assessment of DNA damage and repair in individual cells are pertinent to several areas of research, in particular the study of the heterogeneity of tumour cell populations in response to anticancer agents. We describe an adaptation of an in situ alkaline denaturation assay performed on individual nuclei of lysed cells, termed nucleoids, trapped within an agarose film. A novel aspect of the technique described in the application of confocal laser scanning fluorescence microscopy for the measurement of nucleoid relaxation in response to DNA damage. The volumes of spherical nucleoids and their relative DNA contents were determined by ethidium bromide staining and the analysis of confocal sections through the equatorial planes of the nucleoids. Mean nucleoid volume increased as a linear function of X-ray dose (0.5-8 Gy) administered to intact cells prior to lysis. We provide evidence of heterogeneity, in asynchronous cultures, in the DNA unfolding/unwinding characteristics of cells irrespective of cell cycle age. Bivariate plots of relative DNA content versus nucleoid volume allowed the direct assessment of cellular repair capacity with respect to cell cycle position.

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The ability of SV40-transformed human (ataxia-telangiectasia) fibroblasts to maintain Epstein-Barr virus (EBV)-based plasmids and cosmids extrachromosomally has been investigated. Transfection of a culture of cells with two different plasmids gave rise to cell clones which were able to maintain both plasmids extrachromosomally. When an EBV-based cosmid library was transfected into the cells and an individual cell clone was isolated, the extrachromosomal DNA derived from the cosmid contained numerous deletions and rearrangements. When individual cosmids were transfected into the culture, and several cell clones were isolated, the intracellular cosmid-derived DNA again showed the presence of multiple deletions and rearrangements. We conclude that although SV40-transformed cells are able to maintain more than one different EBV-based plasmid extrachromosomally, large EBV-derived molecules are extensively rearranged. SV40-transformed human fibroblasts cannot therefore be usefully used in attempting to clone genes from EBV-based cosmid libraries.

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An SV40-transformed Fanconi's anaemia (FA) cell line, GM6914, exhibits approximately 2.4-fold increased sensitivity to the cytotoxic effects of nitrogen mustard (NM) when compared with the normal line, MRC5-V1. Host cell reactivation of NM-treated plasmid has been investigated using transient expression vectors which contain the chloramphenicol acetyltransferase (CAT) gene. In both cell types there is a similar, dose-dependent reduction in CAT expression which correlates with an increase in NM-induced DNA-interstrand crosslinking. The data are consistent with two possible mechanisms for inactivation of the plasmid. Either a single crosslink anywhere within the plasmid is sufficient to prevent transcription of the cat gene. Alternatively, inactivation may result from some other more prevalent NM-induced lesions within the cat coding sequence.

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