The Wellcome Foundation lecture, 1986. The molecular regulators of normal and leukaemic blood cells

The development of a cell-culture system for the cloning and clonal differentiation of different types of blood cell has made it possible to identify: (i), the proteins that regulate growth and differentiation of different cell lineages in normal and leukaemic blood cells; (ii), the molecular basis of normal and abnormal control of cell development in blood-forming tissue; and (iii), how to suppress malignancy in leukaemic cells. By using myeloid blood cells as a model system, it has been shown that normal blood cells require different proteins to induce cell viability and multiplication (growth-inducers) and differentiation (differentiation-inducers), that there is a hierarchy of growth-inducers which act at various stages of cell development, and that a growth-inducer can switch on production of a differentiation-inducer. Gene cloning has established a multigene family for these proteins. Identification of these proteins and their interaction has shown how growth and differentiation are regulated in normal development and demonstrated the mechanisms that uncouple growth and differentiation so as to produce malignant cells. Normal cells require an external source of growth-inducing protein for cell viability and multiplication. Cells can become leukaemic by genetically changing this normal requirement for growth without blocking response to normal differentiation-inducers. The mature cells induced by adding these normal protein-inducers are then no longer malignant. Other genetic changes which inhibit differentiation by the normal blood-cell regulatory proteins can occur in the evolution of leukaemia. But even these leukaemic cells may still be induced to differentiate by other compounds that can induce differentiation by alternative pathways. The differentiation of leukaemic to mature cells, which stops the cells from multiplying, results in the suppression of malignancy by bypassing genetic changes that produce the malignant phenotype. The activity of blood-cell growth- and differentiation-inducing proteins has been shown in culture and in the body. They can, therefore, be clinically useful to correct defects in the development of normal and leukaemic blood cells.     

The Wellcome Foundation lecture, 1982. Opioid peptides and their receptors

The remarkable feature of the opioid system is the complexity of its ligands and their interactions with the mu-, delta- and kappa-binding sites. The three endogenous opioid precursors give rise to more than ten opioid fragments. The fragments of pro-opiocortin and pro-enkephalin have affinities mainly to the mu- and delta-binding sites and those of pro-dynorphin have a preference for the kappa-binding site. It is important to realize that some of the larger fragments may have pharmacological actions that are of a non-opioid character. As the endogenous opioid peptides bind to more than one of the types of binding sites, it was necessary to obtain synthetic compounds that bind almost exclusively at one site. There are now agonists for which this aim has been achieved but we still require antagonists that are exclusively selective for only one opioid site. The results obtained with opioid peptides or non-peptides having such qualities would be the physiological basis for a correlation of the binding at mu-, delta- and kappa-receptors with their pharmacological effects. Furthermore, since almost all endogenous opioid ligands are degraded by peptidases, it is necessary to synthesize non-toxic inhibitors of those peptidases that play a role in opioid transmission. Related to this problem is the need to develop methods for the study of the release of various endogenous opioid peptides under physiological conditions.     

The molecular pathology of haemophilia B. Fourth Wellcome Trust lecture

Haemophilia is a rare inherited disease of blood clotting known since biblical times. The rarer form (haemophilia B) occurs in about 1 in 30,000 males and there are about 900 patients in the U.K. at present. Biochemically, patients either lack or have a defective protein (called factor IX) which is needed for the clotting of blood in response to injury. Only males get the disease. However, females can carry the trait in a latent form and transmit the disease to their offspring. Untreated, the disease leads to internal bleeding into muscles and joints and is life-threatening. In the U.K. and in countries with effective health care programmes, patients are treated by periodic injection of factor IX concentrate, a drug isolated from the pooled plasma derived from many blood donors. This drug replaces their own absent or defective factor IX and allow them to enjoy a relatively normal lifestyle. I have reviewed recent studies on the molecular genetics of haemophilia B which started with the isolation of the gene coding the factor IX protein from normal individuals in 1984. Following this, it has been possible firstly to produce factor IX artificially in the laboratory from cloned copies of the messenger RNA of the factor IX gene. Secondly, it has been possible to improve the diagnosis of 'carriers'. Carrier females often wish to know whether they are carriers or not before they have children. If they are positively identified as carriers, the risk and implications of having a haemophiliac son can be discussed and therapeutic abortion considered.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Wellcome Foundation lecture, 1981. Nuclear magnetic resonance imaging in medicine: physical principles

In recent years nuclear magnetic resonance (n.m.r.) has become a means of providing excellent images of the interior of the human body which are proving useful in medical practice. The development of n.m.r. imaging, much of which was pioneered in Britain, is outlined. Proton image resolution of human anatomy is comparable with X-ray computed tomography images, but without the hazard of ionizing radiation. There is improved soft tissue discrimination and pathological contrast through the basic imaging parameters of the proton density and the relaxation times T1 and T2, whose differences from one tissue to another are exploited by use of appropriate radiofrequency pulse sequences. Images may be obtained directly of transverse, coronal and sagittal sections of the head and body. Single slices or multiple slices may be imaged and imaging may be done in three dimensions. The lecture describes the more important imaging techniques and gives illustrative examples of images obtained. The efficient use of time in n.m.r. imaging is discussed, particularly mentioning the multiecho-multislice procedure and the development of real-time n.m.r. imaging. Magnetic field strengths in current use for proton n.m.r. imaging range from 0.02 to 2 T. At the lower end of the range resistive magnets are used, while for higher fields superconducting magnets are needed. A considerable improvement in image quality is obtained by use of special receiver coils.     

The Wellcome Foundation lecture, 1984. Nuclear magnetic resonance imaging in medicine: medical and biological applications and problems

From early biological work and the first T1 nuclear magnetic resonance (n.m.r.) animal image in 1974, whole-body patient images, by using a two-dimensional Fourier transform method were achieved in Aberdeen in 1980 with a 0.04 T vertical resistive magnet. Different pulse sequences produce images dependent by different amounts on proton density, T1 and T2, and for clinical work it is advantageous to use more than one pulse sequence to image pathology. The slow improvement of spatial resolution with increasing standing magnetic field strength is discussed and information on the T1 and T2 contrast dependence is reviewed: it suggests that the gains from high fields may be less than believed hitherto. Electrocardiogram gating can be used to produce moving images of the beating heart; blood flow can be imaged and surface radiofrequency coils are used for improved detail. N.m.r. imaging has considerable potential for studying response to therapy; mental states and dementia; tissue generation; discriminating body fat and body fluids. Other nuclei such as 23Na can be imaged and the potential to image fluorine-labelled pharmaceuticals could be very exciting; n.m.r. contrast agents are now being developed. Images formed from T1 values measured for each pixel are very useful for diagnosis, but the numerical values themselves are less valuable for distinctive pathological identification. With 15 companies manufacturing n.m.r. imagers and over 200 in use in hospitals, the technique is rapidly becoming established in diagnostic clinical practice and some typical uses are presented.     

The Florey lecture, 1986. The regulatory biology of antibody formation

The regulatory biology of antibody formation entered a new phase of study with the development of selective theories of immunity. The discovery of the 'one cell - one antibody' dogma and the demonstration that only a small minority of B cells possessed receptors specific for a given antigen were consistent with Burnet's clonal selection hypothesis, which was later formally proven by preparing antigen-specific lymphocytes and inducing clonal activation in vitro. Clonal analysis has aided precise study of immunoregulation for both B and T lymphocytes. Clonal activation of B cells in the absence of T cells is now possible with high cloning efficiency. It requires the combined action of certain antigens and growth factors, collectively termed B-cell stimulatory factors (BSFS). Single cell analysis has shown that most BSFS so far tested, in contrast to most claims in the literature, possess the capacity (in synergy with antigen) to: stimulate B cells out of the G0 phase into active cell cycle; promote sequential mitotic divisions; and induce differentiation to active secretory status. This is clearly true for IL-1, IL-2, and BSF-p2. These multiple actions resemble those of the colony-stimulating factors in haemopoiesis. Regulation of antibody production by T lymphocytes can also be profitably analysed in clonal systems. The immunoregulatory problem of tolerance can also be analysed by means of clonal techniques. Studies are summarized which indicate that T-cell-mediated suppression and functional silencing of toleragen-specific lymphocytes are both cooperatively involved in many tolerance models. For the B lymphocyte, tolerance can be induced without an actual deletion of the cell involved; rather, the tolerant cell appears to have received and stored a negative signal, rendering it unresponsive to normally immunogenic stimuli. Thus, a state termed 'clonal anergy' has been induced within the cell. Functional clonal deletion has also been noted in several models to T-lymphocyte tolerance, but here it is not known whether clonal anergy or actual death of the relevant cell is at work. Self-tolerance sufficient to be consistent with good health need not mean a total absence of cells with any degree of self-reactivity. Indeed, it is clear that some B cells capable of forming antibody with some degree of affinity for self-constituents exist in the body, and can be activated, for example by lipopolysaccharide. The requirement is to limit the amount, affinity and duration of autoantibody production. A model suggesting how this may be achieved is presented.     

The Florey lecture, 1986. Vaccine prevention of virus-induced human cancers

Carcinogenic viruses have been discovered in numerous animal species over the last 80 years but their role in human cancer has only recently become an important issue. With EB virus involved with endemic Burkitt's lymphoma and undifferentiated nasopharyngeal carcinoma, hepatitis B virus with primary liver cancer, papilloma viruses with carcinoma of the cervix, and T-cell leukaemia virus with adult T leukaemia, 20-25% of all human cancer appears to have a virus component in its causation. By analogy with certain virus-induced animal cancers, vaccine prevention of infection should greatly reduce subsequent tumour development; vaccines against hepatitis B virus are already on trial for this purpose in populations at risk. Experiments are described in which an EB virus subunit vaccine consisting of the virus-determined membrane antigen glycoprotein molecule of molecular mass 340 kDa (MA gp340) has been prepared by two purification methods. Material from one of these has successfully protected cotton-top tamarins against a 100% lymphomagenic dose of challenge virus and investigations are under way to identify an immunogen, based on MA gp340, suitable for use in man. Genetically engineered bacterial, yeast, and mammalian cells expressing the gp340 gene are already available; this gene has also been inserted into vaccinia and varicella virus vectors. Powerful new adjuvants are also considered, together with future strategies for human vaccine studies.     

The Leeuwenhoek lecture, 1986. Environmental carcinogens and papillomaviruses in the pathogenesis of cancer

In many areas of the world there is a geographically localized high incidence of alimentary and bladder cancer in cattle. Studies in western Scotland have demonstrated that this phenomenon is associated with ingestion of bracken fern. However, the affected animals and herds were shown also to have an unusually high infection rate of alimentary papillomas caused by a previously unrecognised bovine papillomavirus (BPV) and that these tumors could undergo malignant transformation. Long-term field and experimental studies were started and indicate that the pathogenesis of the tumours and their relationship to virus infection and food-derived mutagens is complex. Results from these studies, and from cellular and molecular biology experiments, are presented and discussed in the context of recent papillomavirus findings in the human subject.     

The Wellcome Foundation Lecture, 1980: monoclonal antibodies from hybrid myelomas

When the lymphoid cells from immunized animals are fused with myeloma cells adapted to grow permanently in culture, hybrid cells can be isolated that are capable of permanent growth in culture, or as transplantable myeloma tumour in animals, and that at the same time express the antibodies of the immunized donor. Such hybrid cells can be cloned and the antibody produced by each clone is monoclonal. By this procedure therefore it is possible to dissect the heterogeneous immune response of an animal. The monoclonal antibodies can be permanently produced in unlimited quantities and the products are well defined chemical entities, unlike antibodies prepared in animals, which vary from animal to animal and even in different periods within a single animal. These properties have been of great importance in the use of antibodies as biochemical reagents in basic research in a variety of fields. They are also replacing conventional antibodies in standard laboratory practice.     

The calmodulin content of normal and leukaemic lymphocytes

Normal human peripheral blood mononuclear cells (PBMC) and leukaemic cell lines (three of human and one of gibbon origin) were found to contain similar levels of calmodulin (CaM) when expressed relative to the total cell protein. Two of the cell lines examined further were found to contain much higher amounts of CaM per cell (up to 5-fold) than PBMC but this was readily explained by their much greater cell size. Variations in CaM levels were noted during culture of both PBMC and leukaemic cells which were apparently independent of the percentage of cells undergoing active division in these cultures. These results do not support, the contention that transformed cells contain a higher proportion of CaM than normal cells.     

The induction and inhibition of differentiation in normal and leukaemic cells.

For granulocytic-macrophage progenitor populations and their progeny, five glycoproteins have been identified: GM-CSF, G-CSF, multi-CSF, M-CSF and IL-6 that can regulate their proliferative activity, maturation and functional activities. The same glycoproteins also have a capacity to induce irreversible differentiation commitment in normal bipotential granulocyte-macrophage progenitors and in some myeloid leukaemic cell lines, which suggests that common cellular processes exist in both situations. The leukaemia inhibitory factor (LIF) is a glycoprotein, with intriguing properties, which can either induce differentiation in some myeloid leukaemic cell lines or prevent differentiation in normal totipotential embryonic stem cells. The data from the LIF studies suggest a genetic mechanism controlling self-generation that is relatively simple and may be common to all cells. However, the actual cellular response observed appears to depend on the nature of the responding cell.     

Actin and tubulin of normal and leukaemic lymphocytes.

Double labelling and the isolation of actin- and tubulin-derived peptides were used to determine the amounts of these proteins in peripheral lymphocytes from normal donors and from patients with chronic lymphocytic leukaemia. As a precaution against proteolysis, samples were boiled before assay. The actin content of chronic-lymphocytic-leukaemia (CLL) lymphocytes was 8.1 +/- 2.1% of total protein, which was lower (P less than 0.05) than the amount (12.8 +/- 3.0%) of actin found in normal lymphocytes. The tubulin content of CLL lymphocytes was 4.4 +/- 1.5% of total protein, which was also significantly less (P less than 0.05) than that of normal lymphocytes, which was found to be 6.1 +/- 1.1%.     

The Wilhelmine E. Key 1986 invitational lecture. Fifty years of genetic load

The author's involvement with and his successive reactions to the genetic load concept [whose beginning is identified with Haldane's paper. The effect of variation on fitness] is presented in the form of a personal odyssey. The major change in attitude involved the realization that the density- and frequency-independent selection discussed by most population geneticists has little bearing on events transpiring within natural populations; instead, natural selection should be viewed primarily as a density- and frequency-dependent phenomenon. Under this view, the culling of a large number of young zygotes to the considerably smaller number of adults that can be sustained by the environment is an essential process enabling any population's continued existence; to the extent that genetic variation facilitates culling, a genetic load (in direct opposition to the early view) can enhance a population's persistence through time.     

The Leeuwenhoek lecture, 1985. A molecular biologist's view of viral hepatitis

Three forms of viral hepatitis can be distinguished serologically. Hepatitis A virus is a picornavirus, which is being studied increasingly after its propagation in cell cultures. The B virus (HBV) is the prototype of a family now termed hepadna viruses and is by far the best understood. The third, by exclusion, is non-A non-B, about which little else is known. Molecular cloning methods enable copies of viral genes to be propagated and analysed quite readily and provide the means for isolation and expression of individual genes in microbial and animal cells. Determination of the nucleotide sequences of HBV DNA revealed its genetic organization and so guided studies of the mechanism of gene expression both in infected animals and cultures of transformed cells. Replication of the viral genome has also been studied in natural infections, particularly with duck HBV, but also with the human virus. Expression of HBV genes in microbial cells is valuable as a source of antigens for diagnostic reagents and vaccine preparations, but has also been of consequence for the identification of viral gene products not previously recognized and which are of considerable current interest. The methods and materials now available provide additional opportunities for inquiring into the course of viral infection, replication of the virus and, for HBV, the possible role in the development of hepatomas of integration of the viral genome into the host chromosome.     

Cytochemistry of normal and leukaemic lymphocytes: a review.

Findings with six cytochemical reactions demonstrable in normal and leukaemic lymphocytes were reviewed. The two methods which are presently of greater diagnostic value are the acid phosphatase (AP) and alpha-naphthyl acetate esterase (ANAE) reactions. AP has a definitive role in the diagnosis of acute and chronic T-cell leukaemias, where a strong positive reaction helps to distinguish them from most B-cell lymphoproliferative disorders. New findings concerning the ultrastructural localization of this enzyme are presented. ANAE is of value in distinguishing T-lymphocytes (positive localized reaction) from B lymphocytes (negative reaction) and the T micron from the T gamma subpopulation of T-lymphocytes, a positive reaction demonstrable only in the T micron cells. Other reactions reviewed were PAS, beta-glucoronidase, hexosaminidase and alkaline phosphatase.     

The electrophoretic mobility of normal and leukaemic cells of mice

1. The pH-mobility relationships for saline-washed cells from a mouse strain of acute lymphoblastic leukaemia were examined before and after treatment with lower aldehydes, diazomethane and neuraminidase (EC 3.2.1.18). 2. The content of sialic acid released into the supernatant fluid of neuraminidase-treated cells was measured. 3. The stability of the charge-determining structures to temporary changes in environment (pH and ionic strength) was established. 4. Similar measurements were made on lymph-node cells obtained from non-leukaemic mice (a resistant and a leukaemia-susceptible strain were examined). 5. It is deduced that both the malignant and the non-malignant cell possess two dissociable acid functions at the cell surface, a carboxyl group of sialic acid and another acidic group(s), probably carboxyl, of pK 3.0-4.5. The malignant cells, however, have a basic dissociable function not present in the non-malignant types. 6. Suggestions are made as to how the difference in surface chemistry may be related to the problem of malignancy.