Tumour necrosis factors in clinical practice.

The tumour necrosis factors are pleiotropic proteins which have a wide range of biological activities. Tumour necrosis factor-alpha or cachectin is a product of macrophages and is the principal host mediator of septic shock and the cachexia of chronic disease. A related molecule, tumour necrosis factor-beta or lymphotoxin, is produced by T lymphocytes in response to antigen or mitogens. The role of the TNFs in disease processes and in therapy are reviewed. Both agents exert antiproliferative effects on certain tumour cell lines, while normal cells are resistant to these effects. In vivo, they cause haemorrhagic necrosis of certain implantable tumours in mice. Trials of tumour necrosis factor-alpha as an anticancer agent, either singly or in combination with interferon gamma or cytotoxic drugs, are in progress. Understanding the involvement of the tumour necrosis factors in pathological processes may lead to new therapies for endotoxic shock and cancer.

Galloway Memorial Lecture. Protein engineering of tumor necrosis factor-beta and its applications in cancer, septicaemia and cachexia.

The tumour necrosis factors (TNFs) are cytokines, small proteins produced by cells as part of the intercellular signalling network. The exact physiological role of TNFs is unknown, but interest in them focused on three of their capabilities: as potential anti-tumour agents, as humoral mediators of an organism's response to injury and as effector molecules in cachexia. The first TNF to be described, now known as TNF-alpha, has been relatively well studied because the recombinant protein was easily produced since it was cloned in 1984. Studies on TNF-beta or lymphotoxin were hampered by the inability of most groups to express the recombinant protein. This paper describes the expression and purification of recombinant TNF-beta in Escherichia coli, followed by studies to localise the receptor binding site of the molecule through site-directed mutagenesis. Mutants with single amino acid changes at either of two distinct loop regions, at positions aspartic acid-50 or tyrosine-108, were found to have greatly reduced receptor binding and cytotoxic activity. These two regions in TNF-beta correspond to known loop regions where mutations also result in loss of biological activity of TNF-alpha, a related cytokine which shares the same cellular receptors with TNF-beta. This provides evidence for a new hypothesis that both the TNFs bind to their receptors as trimers, each of which is capable of binding simultaneously to three receptors. This leads further to the intriguing possibility of a new mechanism of receptor clustering through simultaneous binding to a single ligand.

Evolution of a hospice home care service in Singapore.

The hospice movement in Singapore was started in 1985 when St Joseph's Home opened its doors to terminally ill patients by setting aside 16 beds for hospice care. A newspaper article about this work brought together a group of volunteers who started a hospice home care service under the auspices of the Singapore Cancer Society in 1987. This service was originally entirely staffed by volunteers until a charitable foundation made possible the employment of a nurse coordinator in 1988. Nearly two years later, in December 1989, the Hospice Care Association, a new charitable organization specifically devoted to the promotion and provision of hospice care, was formed. With charitable funding from the community, the new organization built on the experience of the volunteer-run hospice home care service and developed it into one in which professionals provide most of the care, supported by volunteers. Full-time staff were responsible for the day-to-day running of the service, providing for reliability and setting and maintaining of standards, while the role of volunteers changed to that of supporting, supplementing and enhancing the quality of the care given to patients. This paper chronicles the evolution of this service and describes its present functioning.

Clinico-pathological prognostic factors of large bowel cancer in Singapore: a multivariate analysis.

Clinical and pathological data from 219 cases of large bowel cancer were analysed using the Cox regression model to evaluate their effects on survival. Clinical variables such as sex, change in bowel habit, bleeding per rectum, tumour site, size and duration of symptoms had no significant influence on survival. Individually, age, abdominal distension, Dukes' stage and tumour grade had significant influence on survival. When considered together, Dukes' stage was the only variable selected. Therefore Dukes' stage is the most important prognostic factor in colorectal cancer. The distribution of patients was 25, 18, 26 and 31 percent for Dukes' A, B, C and D respectively. The probabilities of survival at five years were 71, 55, 36 and 16 percent for the respective Duke's stages. These data show that the clinical and pathological variables of patients with colorectal cancer in Singapore and the factors significantly associated with survival are similar to those in western countries.

A survey of symptoms in hospice patients in Singapore.

A descriptive study was done on patients referred to a hospice home care service in Singapore. The demographic profile, type of disease, presenting symptoms and performance status were noted. Particular attention was paid to the incidence of pain and types of analgesics used. The patients referred were representative of the population at large, as was their incidence of cancer. Eastern Cooperative Oncology Group (ECOG) performance status was poor (ECOG 3 or 4) in 93 (93%) of the patients referred, indicative of the lateness of referral in many cases. The number of symptoms per patient averaged 5.8, and pattern of symptoms were similar to those of comparable groups of patients in other parts of the world. Pain was present in 82 (82%) patients and controlled only in 22 (22%) of these at presentation. Strong opioids had been prescribed for 51 (51%) patients. In these, oral morphine mixture was incorrectly prescribed in 17 (47%) out of 36 cases. Education in palliative care is needed so that patients are referred to palliative care services appropriately. Though there is some indication of improvement, medical education in pain management continues to be necessary, particularly in the correct prescription of morphine.

Influence of family history of cryptogenic hypertension, age, sex and race on lymphocyte sodium/potassium pumps.

To determine the effects of age, sex, race, and family history of hypertension on cellular cation transport, we measured specific tritiated-ouabain binding (sodium/potassium pump number), and sodium/potassium pump-mediated rubidium-86 uptake in blood lymphocytes from 105 healthy normotensive adults, comprising 23 Chinese and 19 Indian subjects who had first-degree relatives with cryptogenic hypertension, and 40 Chinese and 23 Indian matched subjects without such history. Both Chinese and Indian subjects with family history had significantly fewer sodium/potassium pumps than control subjects. Sodium/potassium pump-mediated rubidium-86 uptake was similar in these four subject groups. Seven Indian patients with untreated cryptogenic hypertension had fewer sodium/potassium pumps than normotensive Indians without family history. Age did not affect sodium/potassium pump numbers in cells from normotensive subjects. In normotensive subjects without family history. Chinese women had more sodium/potassium pumps than Chinese men, but Indian subjects did not show this pattern. We conclude that a family history of cryptogenic hypertension is associated with fewer lymphocyte sodium/potassium pumps than normal. The reduction in cellular pump number might be only a marker, or an epiphenomenon. However, the reduction in pump number might contribute to the pathogenesis of cryptogenic hypertension if it produces sodium retention in renal tubular or peripheral vascular cells.

c-myc Oncogene expression in colorectal cancer: its use in prognosis and role in colorectal carcinogenesis.

Expression of the c-myc oncogene was studied in 35 cases of colorectal cancer. Elevated expression was found in 46% (16/35) of cases. This elevated c-myc expression was not significantly correlated with tumour stage, tumour size, tumour differentiation or carcinoembryonic antigen gene expression, demonstrating that it is not a useful prognostic indicator in colorectal cancer. The role of c-myc in colorectal carcinogenesis is discussed in the light of these findings and of the recent advances in molecular biology.

Measuring quality of life in different cultures: translation of the Functional Living Index for Cancer (FLIC) into Chinese and Malay in Singapore.

Quality-of-life assessment has become an accepted method of evaluation in clinical medicine. The technique is based on a patient's self-assessment of physical, psychological, and social function, as well as the effects of distressing physical symptoms. The most important aspect of quality-of-life assessment is that it brings into focus a patient-centred view of health outcome, which is broader than the physiologic measures which predominate in Western medicine. Strategies for the development and use of assessment questionnaires have evolved over the past 15 years, and numerous questionnaires have been created. Most originate in Western societies, with English as the most common language of development. Adapting such questionnaires for use in other language and cultural settings is an imprecise practice. Language translation and equivalent cultural meaning must both be addressed. This paper reports on the language translation process and results for the Functional Living Index for Cancer (FLIC) as translated into Chinese and Malay in Singapore. We employed a step-wise process beginning with translation/back translation, followed by structured pilot field trials and population sampling. Taped versions of the questionnaire were devised to meet illiteracy problems in the sample population. Paired comparisons of the Chinese and Malay versions of individual questions with their English counterparts show good correlations and similar means most of the time. Factor analysis on a population sample of 246 (112 Chinese, 35 Malay and 98 English speaking) with cancers of minimal, extensive or palliative extent is convergent with that obtained on a North American population. However, a separate analysis of the Chinese questionnaires showed some differences in factor pattern. Specific language and cultural translation difficulties are discussed. Of note is the predicted significant decrease in total FLIC scores with extent of disease within each of the language preference populations, which provides some evidence for the validity for each language version in the Singapore culture(s). Thus, the FLIC translations into Malay and Chinese in Singapore can be considered for use in local trials, subject to ongoing evaluation.

Structural and functional domains in human tumour necrosis factors.

Human tumour necrosis factors (hTNFs) alpha and beta are related pleiotropic cytokines which share many activities and compete with each other for binding to two receptor components on many cell types. Although structural and biological data indicate that the active form of hTNF-alpha may be a symmetrical trimer, the manner in which hTNFs interact with their receptors to trigger a myriad of cell type-dependent responses is not clear. A combination of chemical modification, epitope mapping and site-directed mutagenesis approaches suggest that at least four distinct peptide sequences are important for the biological activity of hTNF-alpha. In particular, certain peptide sequences between amino acid positions 11 and 35 in hTNF-alpha appear to be critical for receptor binding and triggering biological responses. The recent cloning of the two hTNF-alpha/beta receptors opens the way for precise mapping of the functional domains in hTNFs.

Recombinant human tumor necrosis factor-beta entrapped in liposomes formed by a modification of the dehydration-rehydration method retains potent cytotoxic activity on L929 cells in vitro.

Recombinant human tumor necrosis factor-beta (rhTNF-beta) may be encapsulated with high efficiency in phosphatidylcholine and distearoylphosphatidylcholine liposomes, with entrapment values of 93.4% and 92.3%, respectively, by first entrapping the substance in multilamellar vesicles using a high solute-to-phospholipid ratio followed by freeze-drying and then rehydration. The entrapped cytokine retains potent cytotoxic activity on L929 cells in vitro, causing 100% cytotoxicity, equal to that of free rhTNF-beta at a concentration of about 5 x 10(-8) g/ml.

Aspartic acid 50 and tyrosine 108 are essential for receptor binding and cytotoxic activity of tumour necrosis factor beta (lymphotoxin).

Single amino acid substitutions were generated in predicted hydrophilic loop regions of the human tumour necrosis factor beta (TNF-beta) molecule, and the mutant proteins were expressed in Escherichia coli and purified. Mutants with single amino acid changes at either of two distinct loop regions, at positions aspartic acid 50 or tyrosine 108, were found to have greatly reduced receptor binding and cytotoxic activity. These two regions in TNF-beta correspond to known loop regions where mutations also result in loss of biological activity of TNF-alpha, a related cytokine which shares the same cellular receptors with TNF-beta. The two distinct loops at positions 31-34 and 84-89 in the known three-dimensional structure of TNF-alpha (equivalent to positions 46-50 and 105-110 respectively in TNF-beta), lie on opposite sides of the TNF-alpha monomer. When the TNF-alpha monomer forms a trimer, the two loops, each from a different subunit of the trimer, come together and lie in a cleft between adjacent subunits. Together, these findings suggest that a TNF receptor binds to a cleft between subunits via surface loops at amino acid residues 31-34 and 84-89 in TNF-alpha, and similarly via surface loops including amino acids aspartic acid 50 and tyrosine 108 in TNF-beta.

The lymphotoxin-alpha (LTalpha) subunit is essential for the assembly, but not for the receptor specificity, of the membrane-anchored LTalpha1beta2 heterotrimeric ligand.

The lymphotoxins (LT) alpha and beta, members of the tumor necrosis factor (TNF) cytokine superfamily, are implicated as important regulators and developmental factors for the immune system. LTalpha is secreted as a homotrimer and signals through two TNF receptors of 55-60 kDa (TNFR60) or 75-80 kDa (TNFR80). LTalpha also assembles with LTbeta into a membrane-anchored, heterotrimeric LTalpha1beta2 complex that engages a distinct cognate receptor, the LTbeta receptor (LTbetaR). To investigate the role of the LTalpha subunit in the function of the membrane LTalpha1beta2 complex, gene transfer via baculovirus was used to assemble LTalpha and -beta complexes in insect cells. LTalpha containing mutations at D50N or Y108F are secreted as homotrimers that fail to bind either TNF receptor and are functionally inactive in triggering cell death of the HT29 adenocarcinoma cell line. In contrast, these mutant LTalpha proteins retain the ability to co-assemble with LTbeta into membrane-anchored LTalpha1beta2 complexes that engage the LTbetaR and trigger the death of HT29 cells. Membrane-anchored LTbeta expressed on the cell surface in absence of the LTalpha subunit binds the LTbetaR but is functionally inactive in the cell death assay. These results indicate that the TNF receptor-binding regions of the LTalpha subunit are not necessary for engagement of the LTbetaR, but the LTalpha subunit is required for the assembly of LTbeta into a functional heteromeric ligand.