(Osborne, 1992) and different studies of prognosis have yielded widely differing results (Hawkins, 1993). We have selected four putative biochemical indices of prognosis and evaluated them in a prospective study. The putative markers chosen were the oestrogen receptor (ER), the epidermal growth factor receptor (EGFR), cathepsin D (cath D) and cyclic AMP-binding protein(s) (c-AMP-b).The oestrogen receptor, a nuclear protein (Mr 66 kDa) has been shown to relate to both endocrine sensitivity (Jensen, 1975) and prognosis in multiple studies, including our own previous work (Humeniuk et al., 1982;Hawkins et al., 1987aHawkins et al., , 1991, although some consider that it is only of prognostic significance in the early years after treatment (e.g. Saez et al., 1983). The receptor for epidermal growth factor is a membrane protein (Mr 180 kDa), which has been reported to be a sign of bad prognosis, again in multiple studies (Sainsbury et al., 1987;Gasparini et al., 1992). The proteolytic enzyme, cathepsin D (Mr 52 kDa), has been suggested to be involved in metastasis formation and reported in several studies also to be a sign of bad prognosis when present in high levels (for a review see Rochefort, 1990). Lastly, work in our own laboratories and elsewhere has demonstrated that high levels of cyclic AMP-binding protein(s), the regulatory subunits of the enzyme protein kinase A, represent another sign of poor prognosis, in both retrospective (Miller et al., 1990) and prospective (Miller et al., 1993)

The response of granulocyte-macrophage progenitor cells (in vitro colonyforming cells) and of colony-stimulating (CS) factor in serum were studied in mice infected intraperitoneally with 103 viable Salmonella typhimurium. Increases in the number of colony-forming cells in marrow and spleen and increases in the serum level of CS factor occurred during the infection. There was no evidence to suggest that progressive infection was associated with failure of macrophage production. Medium rich in CS factor increased the bactericidal activity of macrophages in vitro and it was suggested that CS factor could be involved in macrophage activation.

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The antiviral drug methisazone (N-methylisatin ,B-thiosemicarbazone) was tested for its effect on immune responses to sheep erythrocytes and on hemopoietic colony-forming cell (granulocyte-macrophage progenitor cell) responses to complete Freund's adjuvant in mice. Suppressive activity was demonstrated in both systems, the immune system being more readily and more consistently susceptible. Evidence is presented which suggests that the insoluble particulate form of the drug has both stimulatory and suppressive effects on the colony-forming cell system, whereas the soluble form is only suppressive. Methisazone increased the mortality from ectromelia in adjuvant-treated animals.

The responses of in vitro colony-forming cells (granulocyte-macrophage progenitor cells) were studied in the bone marrow, spleen, and blood of mice after intraperitoneal injection of 103 plaque-forming units of ectromelia virus. This study showed that a colony-forming cell response occurred during infection and was accompanied by an increase in the serum level of colony-stimulating factor and, at a later stage, colony-inhibiting factor. Changes in the proportions of colonyforming cells were not due to relative changes in other cell populations. The relationships between colony-forming cell responses and levels of infection were complex, higher levels of infection being associated with good colony-forming cell responses in bone marrow and apparently poor colony-forming cell responses in blood and spleen.

In 1990, 215 patients with operable breast cancer were entered into a prospective study of the prognostic significance of five biochemical markers and 15 other factors (pathological/chronological/patient). After a median follow-up of 6.6 years, there were 77 recurrences and 77 deaths (59 breast cancer-related). By univariate analysis, patient outcome related significantly to 13 factors. By multivariate analysis, the most important of nine independent factors were: number of nodes involved, steroid receptors (for oestrogen or progestogen), age, clinical or pathological tumour size and grade. Receptors and grade exerted their influence only in the first 3 years. Progestogen receptors (immunohistochemical) and oestrogen receptors (biochemical) were of similar prognostic significance. The two receptors were correlated (r =+0.50, P=0.001) and displaced each other from the analytical model but some evidence for the additivity of their prognostic values was seen when their levels were discordant.

Intraperitoneal injection of complete Freund's adjuvant which caused an extensive cellular response in the reticuloendothelial system greatly increased the severity of infection which resulted from footpad inoculation of ectromelia virus in mice. The mortality was greatest during the proliferative phase of the cellular response, indicated by the colony-forming cell response. Exposure of colony-forming cells to virus in vitro showed that those from normal bone marrow were much less susceptible to virus than those from the bone marrow of adjuvant-treated mice and more particularly those from the spleen of adjuvant-treated mice. Severe bleeding to induce anemia and a consequent erythropoietic response did not increase the mortality from ectromelia. Previous results were interpreted on the basis of these findings and a more general application of them to the pathogenesis of poxvirus infection was discussed.It was reported in the previous paper (13) that during infection with ectromelia virus mice can develop an increase in the number of hemopoietic colony-forming cells (CFC) in bone marrow, blood, and spleen. However, it was also shown that the relationship between the level of infection and the CFC response in the different tissues was complex, and that this relationship in bone marrow was different from that in spleen and blood (13). No clear-cut pattern emerged regarding the possible role of a CFC response in the pathogenesis of the disease since the results could be interpreted in various ways. The experiments reported in this paper were carried out in an attempt to resolve these problems by (i) studying the infection in mice whose CFC system was stimulated before infection and (ii) by studying the susceptibility of CFC to virus infection in vitro. MATERIALS AND METHODSThe mice, the strain of virus, and the technique of virus and infectious cell assay were described or referred to in a previous paper (13).Hemopoietic stimuli. Intraperitoneal injection of complete Freund's adjuvant has been shown to give an intensive stimulus to the CFC system (12). The complete adjuvant (Difco) was used as supplied, without emulsification, but ensuring complete suspension of the mycobacterial component. Each mouse was given 0.2 ml by intraperitoneal inoculation.A strong erythropoietic stimulus was given by making mice anemic. This was achieved by bleeding 0.3 ml from the tail on 3 successive days, a procedure which resulted in the peripheral blood hematocrit falling from 45% to 20 to 25%7. Virus was given the day after the third bleeding. Infection of CFC in vitro. Bone marrow and spleen cells were collected from either normal mice or from mice which had been given adjuvant 7 days previously. Cell suspensions were made as described in the previous paper (13) and were diluted to a concentration of 4 X 106 per ml. A 1-ml amount of this suspension was mixed with an equal volume of Eagle's medium containing 4 X 107 plaque-forming-units (PFU) of ectromelia virus. The mixture was incubated at 37 C in a water bath for 2 hr with gentle s...

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