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G-CSF and GM-CSF have also found application after allogenic or autologous bone marrow transplantation, to accelerate neutrophil recovery (allogenic means that donor and recipient are different individuals, while autologous means that donor and recipient are the same individual).
Bone marrow transplantation, particularly allogenic transplantation, is often a treatment of choice for individuals suffering from acute or chronic leukaemia, aplastic anaemia or various stem cell-related genetic disorders (e.g. thalassaemias).
Allogenic transplantation generally entails removal of approximately 11 of marrow from a matched donor (usually a sibling, preferably an identical twin). The recipient patient is prepared by intensive chemotherapy (or radiotherapy) to kill residual malignant marrow cells. The donated marrow cells are then administered intravenously to the patient, and they re-populate the marrow cavity. The peripheral blood count normally rises within 2-4 weeks.
Autologous bone marrow transplantation involves initial removal of some marrow from the patient, its storage in liquid nitrogen, followed by its re-introduction into the patient subsequent to chemo- or radiotherapy. Leukine is the tradename given to a recombinant human GM-CSF preparation produced in engineered S. cerevisiae (Table 6.4).
LEUKAEMIA INHIBITORY FACTOR (LIF)
LIF is an additional haemopoietic growth factor. It is also known as HILDA (human interleukin for DA cells) and hepatocyte stimulatory factor III. It is produced by a range of cell types, including T cells, liver cells and fibroblasts. LIF is a 180 amino acid, heavily glycosylated, 45 kDa protein. It affects both haemopoietic and non-haemopoietic tissue, often acting in synergy with other cytokines, particularly IL-3. It stimulates the differentiation of macrophages and promotes enhanced platelet formation. It also prompts synthesis of acute phase proteins by the liver and promotes increased bone resorption. The greatest concentrations of LIF receptors are associated with monocytes, embryonic stem cells, liver cells and the placenta. The receptor complex is composed of two transmembrane glycoproteins: the 190 kDa LIFRa-chain which displays affinity for LIF, and a b-chain (gp130), which also forms part of the IL-6 receptor.
Erythropoietin (EPO) is an additional haemopoietic growth factor. It is primarily responsible for stimulating and regulating erythropoiesis (i.e. erythrogenesis, the production of red blood cells) in mammals.
The ‘erythron’ is a collective term given to mature erythrocytes, along with all stem cell-derived progeny that have committed to developing into erythrocytes. It can thus be viewed as a dispersed organ, whose primary function relates to transport of oxygen and carbon dioxide (haemoglobin constitutes up to one-third of the erythrocyte cytoplasm), as well as maintaining blood pH. An average adult contains in the region of 2.3 x1013 erythrocytes (weighing up to 3 kg). They are synthesized at a rate of about 2.3 million cells/s, and have a circulatory life of approximately 120 days, during which they travel almost 200 miles.
EPO is an atypical cytokine in that it acts as a true (endocrine) hormone and is not synthesized by any type of white blood cell. It is encoded by a single copy gene, located on (human) chromosome 7. The gene consists of four introns and five exons. The mature EPO gene product contains 166 amino acids and exhibits a molecular mass in the region of 36 kDa (Figure 6.4) EPO is a glycoprotein, almost 40% of which is carbohydrate. Three N-linked and one O-linked glycosylation site are evident. The O-linked carbohydrate moiety apears to play no
Figure 6.4. 3-D structure of erythropoietin. Photo from Cheetham et al. (1998), by courtesy of the
Protein Data Bank: http://www.pdb.org/
HAEMOPOIETIC GROWTH FACTORS 265
essential role in the (in vitro or in vivo) biological activity of EPO. Interestingly, removal of the N-linked sugars, while having little effect on EPO’s in vitro activity, destroys its in vivo activity. The sugar components of EPO are likely to contribute to the molecule’s:
• cellular processing and secretion;
• in vivo metabolism.
Incomplete (N-linked) glycosylation prompts decreased in vivo activity due to more rapid hepatic clearance of the EPO molecule. Enzymatic removal of terminal sialic acid sugar residues from oligosaccharides exposes otherwise hidden galactose residues. These residues are then free to bind specific hepatic lectins, which promote EPO removal from the plasma. The reported plasma half-life (ti/2) value for native EPO is 4-6 h. The ti/2 for desialated EPO is 2min. Comparison of native human EPO with its recombinant form produced in CHO cells reveal very similar glycosylation patterns.
Circular dichroism studies show that up to 50% of EPO’s secondary structure is a-helical. The predicted tertiary structure is that of four anti-parallel helices formed by variable-sized loops, similar to many other haemopoietic growth factors.