Summary. A low rate of spontaneous cell death by apoptosis was found in a L>HA/2 mouse mastocytoma growinj? in culture. It was not significantly altered by additun. of .splenic lymphocytes horn normal G57BL miee. but was massively enhanced by lymphocytes from mice previously immunized against tbe tumour Ge Is sbow-ing ultrastrnctural changes of early apoptosis in tbe latter experiments had Knipbootes firmly attached to tbeir surfaces, suggesting that cellular iimnnne attack induces apoptosis directly: the cell budding to produce niembiane-bonnded fragments with well preserved organelles characteristic of tbe later stages of the process was assoeiated with lymphocytic detachment. lhe.se cdl fragments or apoptotic bodies were not pbagocyto.sed by vhvUc cells as they are uhen apoptosis takes place in tissues, and underwent secondary degeneration with ballooning of organelles and rnptuie of membranes while still suspended in the medium.The niajor wave of lymphocyte-induced apoptosis occurred within the hrst .3 h of ineubation. The number of well preserved apoptotic bodies reached a peak at Hi h and thereafter declined, whereas ceil debris increased progressively. The n.aximum rate of release of alxsorbed .'"Cr from tumour cells coincided m time witb tbe wave of apoptosis, but difficulty in asse.ssing the rel'Uive rates of formation and secondary degeneration of apoptotic bodies made it in.possible to determine whether the release was particularly associated with one or other of these processes.That cellular i.nmune attack shonJd induce a type of cell death known to occur m normal tissues has implications for tbe hypothesis that tb-, immune system eontniuousiy motn'tors cells and eliminates tbose tbat undergo significant antigcnic change. * 'Present address: SC1K,O1

SUMMARY A technique for the extraction of cells from bone marrow trephine core biopsy specimens using collagenase digestion was assessed in 39 cases (33 diagnostic and six normal). Diagnostically useful numbers ofcells were extracted from all marrows. Morphological assessment ofcytocentrifuge preparations of these cells gave a correct diagnosis in 23 (60%) of cases compared with 27 (70%) for the corresponding aspirated marrow smears. Phenotypic analysis using flow cytometry showed persistence of a range of surface membrane antigens following collagenase digestion. Increased autofluorescence was a problem in some cases.Cytochemistry, bone marrow culture, and cytogenetic analysis could also be carried out on these cells. It is concluded that this technique has useful diagnostic applications in cases of dry taps.Dry bone marrow taps may occur in many haematological disorders. These include the densely cell packed marrow of acute leukaemia and some malignant lymphomas, the fibrotic marrow of myeloproliferative disorders and some lymphoproliferative disorders such as hairy cell leukaemia, and the hypocellular marrow of aplastic anaemia. When this occurs diagnosis is impaired as marrow aspirate smears are not available for cell morphology and cytochemistry. Furthermore, karyotyping, bone marrow culture, and immunological phenotyping techniques using cell suspensions cannot be done.In these circumstances attempts to assess cell morphology by "roll imprints" are made but cell numbers and quality are poor. Alternatively, plastic embedded ultra thin histological section ofbone marrow trephine biopsy specimens may be used,' but this technique is difficult, labour intensive, and not available in every laboratory. Frozen section immunohistochemistry has been advocated for immunological phenotying of marrow in cases of dry taps,2 but this technique is also demanding and so not widely used.We present and assess a simple alternative technique for use in cases of dry taps in which cell suspensions suitable for morphological assessment, cytogenetics and immunological phenotyping are prepared by collagenase digestion of trephine bone marrow core biopsy specimens.

Wilms' tumour (WT) is a paediatric kidney tumour arising from the embryonal metanephrogenic blastema. Recent reports suggest that the expression of messenger RNA (mRNA) for insulin-like growth factor II (IGFII) is elevated in WT. Total cytoplasmic RNA was extracted from 11 sporadic WTs and analysed for IGFII mRNA using dot-blot hybridization. The level of IGFII mRNA expression varied greatly and not all tumours displayed enhanced IGFII expression. Two successive WT xenografts were established in nude mice. The original WT and first passage xenograft showed a blastematous histology, while the second passage xenograft showed epithelioid differentiation and tubule formation. Analysis of the expression patterns of these xenografts showed elevated IGFII expression in the primary undifferentiated tumour and the second differentiated xenograft, while the first undifferentiated xenograft failed to exhibit enhanced IGFII expression. These data show that elevated IGFII mRNA is not an essential component of the progression of WT and that WT tumourigenicity is independent of the level of IGFII expression. Therefore, IGFII overexpression in WT is most likely a tumour epiphenomenon.

Summao'. Dietary restriction, adretiiiline. hydrocortisone or surgery reduced the rate at which pulmonarily arresied '-''IUdR-labelled murine lumour cells were lost within 7 h of intravenous (i.v.) injection. Mice that had been adrenalectomised 10 days previously showed a normal intrapiilmonary tumour cell loss rate wilh furiher surjjery reducing this rate to approximately half that observed in normal mice thai had been subjected to surgery. Thus, although it is likely that adrenal hormones play an important role in decreasing the rate of early intrapulmonary tumour cell loss, additional factors must be implicated.Mice subject to dietary restriction, adrenaline, hydrocortisone or surgery had reduced levels of in viirn growth inhibitor(s) In their sera. Despite this, individual surgically treated animals showed no correlation between serum in vitro growth inhibitor levels and rate of loss of i.v. injected tumour cells from the lungs. Furthermore, [he 24 h pre-incubation of tumour cells in inhibitor-rich serum did not influence the subsequent loss rate of such cells following i.v. injection into mice.Electron microscopic studies indicated that dietary restriction, adrenaline and surgery reduced the rale of intravascular tumour cell death. The decreased tumour cell death rate in mice receiving these treatments could not be related, however, to any consistent morphological change in the pulmonary vasculaturc.The decreased rate of intravascular lumour cell death in treated mice was followed by an increased number of lung tumours with only one of the tumour lines studied, indicating that the intravascular death rate need not be a major determinant of pulmonary tumour incidence.

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