A comparison of immunohistochemical markers of cell proliferation with experimentally determined growth fraction.

The relationship between immunoreactivity for cell proliferation markers (Ki67 and PCNA) and the growth fraction as determined by the fraction of labelled mitoses method was assessed in xenograft tumours grown from the LoVo cell line in nude mice. FLM curves were constructed by injecting tritiated thymidine and then preparing autoradiographs from the tumours. From this data an estimate of growth fraction and cell cycle time were made. Using frozen material from the same tumours, the Ki67 index was determined by immunostaining. PCNA staining was determined in the fixed material which had been used for the autoradiographs. The results show that Ki67 staining follows the same trend as the FLM-determined growth fraction as the tumour increases in size and the rate of growth decreases. However the Ki67 index does produce a consistent overestimate of the growth fraction in this in-vivo system, as compared to observations in cell culture. PCNA staining showed virtually 100 per cent positive staining in all the tumours, which is likely to reflect the long half-life of the antigen, compared to the very fast cell-cycle time of the xenograft tumours. These results show that staining with proliferation markers is not a precise determinant of growth fraction. Ki67 staining is a method that can be usefully applied as an operational marker of cell proliferation, but should not be used uncritically. Further caution is necessary in the use of PCNA. The findings also demonstrate the need to use a range of methods when assessing a new proliferation marker.

Proliferating cell nuclear antigen immunolocalization in gastro-intestinal epithelia.

Proliferating cell nuclear antigen (PCNA), also called DNA polymerase delta-associated protein, is found in the cells of the proliferative compartment of normal tissues and is essential for DNA replication. It can be recognized by many monoclonal antibodies to various epitopes on the molecule. In this investigation one of these, PC10, has been used on formalin-fixed, paraffin-embedded, human and rodent gastro-intestinal epithelial tissues to assess numerically the labelling index of PC10 and to compare it, in the rat liver and gastrointestinal tract, with the S-phase fraction as determined by bromodeoxyuridine (BrdUrd) labelling. The distribution of PC10-labelled cells was recorded with respect to cell position in the intestinal crypts of man. In tissues where both modes of assessment were used, PC10 staining in the well-established proliferative compartments was found to be more extensive than that of BrdUrd. The higher labelling index with PC10 can be explained by its identification of PCNA outside the S phase of the cell cycle and also by the long half-life of PCNA protein in post-proliferative intestinal epithelial cells as they migrate towards the villus. Nevertheless the data suggest PC10 immunostaining in gastro-intestinal epithelia is an operational marker of cell proliferation which is reproducible, quantifiable and can be performed on routinely processed tissues.

Kinetics of enteroendocrine cells with implications for their origin: a study of the cholecystokinin and gastrin subpopulations combining tritiated thymidine labelling with immunocytochemistry in the mouse.

Evidence for a common endodermal stem cell has been derived from kinetic studies in mouse small intestine which indicate that the turnover characteristics of endocrine cells are similar to those of other cell lineages (columnar and goblet cells). We have used continuous tritiated thymidine labelling and peptide immunocytochemistry on resin embedded semithin sections, a combination of techniques which have not been used before in the small intestine. Our data show that the turnover time for endocrine cells in the small intestine is 10 days, considerably longer than the four days suggested by previous studies, although for columnar and mucous cell lineages, turnover rates are similar to the published literature. In the stomach, the turnover time was very slow indeed (of the order of 45-60 days). These results show that endocrine cells do not share turnover characteristics with the other cell types and suggest that they constitute a kinetically distinct cell population independent of the other cell lineages. These data are not consistent with a common stem cell origin for gut endocrine cells.

Transplanted isolated hepatocytes: effect of partial hepatectomy on proliferation of long-term syngeneic implants in rat spleen.

Proliferation of isolated hepatocytes in long-term splenic implants was assessed by flash labelling with a 1 pulse of tritiated thymidine (3H TdR). Cell kinetics showed that the basal labelling index was 0.9% which was greater than normal non-regenerating liver. Twenty four hours following partial hepatectomy the labelling index was 2.0%, a significant rise. These results suggest that hepatocytes transplanted to the spleen constitute a suitable model for screening putative hepatotrophic factors, and are of relevance in establishing clinically useful models of hepatocyte transplantation.

Specificity of indium-111 granulocyte scanning and fecal excretion measurement in inflammatory bowel disease--an autoradiographic study.

The validity of 111In granulocyte scanning and fecal excretion measurement, as a reflection of loss of cells into the gastrointestinal tract, was studied using an autoradiographic technique in 11 patients in whom 111In granulocyte scan and colonoscopy were carried out simultaneously. 111In granulocytes were injected 1.5-4 hr prior to colonoscopy, and intraluminal fluid, mucosal brushings, and colonic biopsies were collected during the colonoscopy. In two patients with no histological evidence of inflammatory bowel disease, and four patients with clinically and histologically inactive inflammatory bowel disease, no 111Indium was detected in fluid, brushing, or biopsies. In five patients with active disease, 85% of the 111In activity in colonic fluid was precipitated by low-speed centrifugation. Autoradiography confirmed that the label remained attached to whole granulocytes in colonic fluid and mucosal brushings. Studies on biopsies, at intervals up to 4 1/2 hr following labeled granulocyte injection, demonstrated labeled polymorphonuclear neutrophils (PMNs) on the inflamed epithelial surface, with occasional cells in crypt abscesses by 110 min. We conclude that the techniques of 111In granulocyte scanning and fecal counting in patients with IBD are specifically measuring cell loss; labeled PMNs are capable of migrating through the gastrointestinal mucosa, in active disease, within 2 hr of administration.

A medical team approach to children who fail to progress in school.

A multidisciplinary team composed of a psychologist, a speech therapist, an occupational therapist, a remedial teacher and a social worker and headed by a paediatrician examined 502 children referred for failure at school. The advantages and disadvantages of the team approach to such children are discussed. The establishment of multidisciplinary teams comprising medical, paramedical and educational members to diagnose and treat these children's problems and to monitor their progress, together with close liaison with the educational and welfare authorities, is recommended as being in the best interests of the child.