Brian I Lord

Identification of amino acid residues critical for aggregation of human CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES. Characterization of active disaggregated chemokine variants.

Human CC chemokines macrophage inflammatory protein (MIP)-1α, MIP-1β, and RANTES (regulated on activation normal T cell expressed) self-associate to form high-molecular mass aggregates. To explore the biological significance of chemokine aggregation, nonaggregating variants were sought. The phenotypes of 105 hMIP-1α variants generated by systematic mutagenesis and expression in yeast were determined. hMIP-1α residues Asp26and Glu66 were critical to the self-association process. Substitution at either residue resulted in the formation of essentially homogenous tetramers at 0.5 mg/ml. Substitution of identical or analogous residues in homologous positions in both hMIP-1β and RANTES demonstrated that they were also critical to aggregation. Our analysis suggests that a single charged residue at either position 26 or 66 is insufficient to support extensive aggregation and that two charged residues must be present. Solution of the three-dimensional NMR structure of hMIP-1α has enabled comparison of these residues in hMIP-1β and RANTES. Aggregated and disaggregated forms of hMIP-1α, hMIP-1β, and RANTES generally have equivalent G-protein-coupled receptor-mediated biological potencies. We have therefore generated novel reagents to evaluate the role of hMIP-1α, hMIP-1β, and RANTES aggregation in vitro and in vivo. The disaggregated chemokines retained their human immunodeficiency virus (HIV) inhibitory activities. Surprisingly, high concentrations of RANTES, but not disaggregated RANTES variants, enhanced infection of cells by both M- and T-tropic HIV isolates/strains. This observation has important implications for potential therapeutic uses of chemokines implying that disaggregated forms may be necessary for safe clinical investigation.

Tumour induction by methyl-nitroso-urea following preconceptional paternal contamination with plutonium-239

We have investigated the possibility that transgenerational effects from preconceptional paternal irradiation (PPI) may render offspring more vulnerable to secondary exposure to an unrelated carcinogen. 239Pu (0, 128 or 256 Bq g(-1)) was administered by intravenous injection to male mice, 12 weeks before mating with normal females. Two strains of mouse were used -- CBA/H and BDF1. Haemopoietic spleen colony-forming units (CFU-S) and fibroblastoid colony-forming units (CFU-F), a component of their regulatory microenvironment, were assayed independently in individual offspring at 6, 12 and 19 weeks of age. Bone marrow and spleen from each of these mice were grown in suspension culture for 2 or 7 days for assessment of chromosomal aberrations. Female BDF1 were injected with methyl-nitroso-urea (MNU) as a secondary carcinogen at 10 weeks of age and monitored for onset of leukaemia/lymphoma. Mean values of CFU-S and CFU-F were unaffected by preconceptional paternal plutonium-239 (PP-239Pu), although for CFU-F in particular there was an apparent increase in variation between individual animals. There was significant evidence of an increase in chromosomal aberrations with dose in bone marrow but not in spleen. By 250 days, 68% of MNU-treated control animals (no PPI) had developed thymic lymphoma (62%) or leukaemia (38%). The first case arose 89 days after MNU administration. In the groups with PPI, leukaemia/lymphoma developed from 28 days earlier, rising to 90% by 250 days. Leukaemia (65%) now predominated over lymphoma (35%). This second generation excess of leukaemia appears to be the result of PPI and may be related to inherited changes that affect the development of haemopoietic stem cells.

Induction of lympho-haemopoietic malignancy: impact of preconception paternal irradiation

PURPOSE To investigate the effects of preconception paternal irradiation (PPI) from injected 239Pu on the susceptibility to induction of lympho-haemopoietic malignancy by subsequent irradiation or exposure to a chemical carcinogen. MATERIALS AND METHODS The male CBA/H and DBA2 mouse was injected with 0, 128 or 256 Bqg(-1) 239Pu 12 weeks before mating with the normal CBA/H and C57B1 female respectively. CBA/H offspring were exposed to 3.3 Gy gamma-rays total body irradiation: BDF1 offspring were injected with 50 mg kg(-1) methyl nitrosourea (MNU). The offspring were assayed for changes in bone marrow progenitor cell numbers and chromosome aberrations and were followed up for subsequent induction of neoplasia. RESULTS While the untreated mouse showed a normal distribution for cellularity, spleen colony-forming units (CFU-S) and fibroblastoid colony-forming units (CFU-F), significant numbers of PPI offspring presented levels outside the normal range. There was a tendency for them also to show increased, dose-related, levels of chromosomal aberrations. Offspring treated with irradiation or MNU developed an increased incidence of lympho-haemopoietic malignancies. CONCLUSIONS These studies have shown that PPI results in offspring that are more susceptible to the induction of lymphohaemopoietic malignancy on encountering a secondary carcinogenic insult. This may be linked to inherited chromosomal instability and abnormal kinetics of haemopoiesis. The experiments indicate a potential mechanism by which an increased incidence of leukaemia may be linked to PPI.

Continuous infusion of macrophage inflammatory protein MIP-1alpha enhances leucocyte recovery and haemopoietic progenitor cell mobilization after cyclophosphamide

Macrophage inflammatory protein 1alpha (MIP-1alpha) inhibits haemopoietic stem cell proliferation. This property has been exploited in a murine chemotherapy model and has been shown to ameliorate cytotoxic-induced myelosuppression after S-phase-specific cytotoxic therapy. We have now shown that BB-10010, a stable mutant of MIP-1alpha, (a) is more effective when administered as a continuous infusion than when bolus injected and (b), when administered via a 7-day infusion during and after cyclophosphamide treatment, results in an earlier recovery of leucocyte numbers. This effect was accompanied by progenitor cell mobilization into the peripheral blood and included primitive cells with marrow-repopulating ability (MRA). Maximal mobilization and recovery of leucocytes occurred when MIP-1alpha was combined with granulocyte colony-stimulating factor (G-CSF) therapy. The findings suggest that MIP1-alpha used alone or in combination with G-CSF may allow delivery of a greater chemotherapy dose intensity as a consequence of both accelerated leucocyte recovery and maintenance of high-quality mobilized progenitor cells for harvesting and peripheral blood stem cell transplantation.

BB-10010/MIP-1 alpha in vivo maintains haemopoietic recovery following repeated cycles of sublethal irradiation

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is an inhibitor of stem cell proliferation affording protection against damage from agents that express their cytotoxicity specifically in the DNA synthesis phase of the cell cycle. Its ability also to modify the self-renewal capacity of the regenerating cells is now shown to improve and maintain haemopoietic recovery following therapy (sublethal irradiation) whose cytotoxic damage is not limited solely to the DNA-S phase of this cycle. Such non-cell cycle-active cytotoxic agents are used clinically in repeated treatment regimens, which are often limited or terminated because of accumulating haemopoietic damage. BB-10010, a non-aggregating variant of MIP-1 alpha, was administered as a continuous dose (1600 micrograms kg-1 24 h-1) via a subcutaneously implanted pump over a period of 7 days. A dose of 4.5 Gy total-body gamma-rays was given 3-4 h after implantation. Day 8 and 12 spleen colony-forming units (CFU-S) were assayed on days 1, 7 and 14 after irradiation. This cycle of treatment was repeated four times (total 56 days), and on day 14 of the last two cycles the marrow-repopulating ability (MRA) was also measured. In the control bone marrow (no BB-10010) CFU-S fell to < 1% of normal within 1 day of irradiation and recovered to 40% at 14 days. Repeated treatments increased the level of damage, and after four cycles CFU-S recovered to only 10% of normal. BB-10010 afforded little benefit in the first treatment cycle, but by the end of the fourth cycle CFU-S still recovered to 35% of normal. MRA was reduced to 7% of normal by the irradiation protocol-about half that maintained by BB-10010 protection. We conclude that BB-10010 (MIP-1 alpha) reduces the degree of accumulated haemopoietic stem cell damage following repeated non-cell cycle-specific cytotoxic insults-a principle which should be valuable in repeated clinical cytotoxic therapy regimens.

The relative spatial distribution of in vitro‐CFCs in the bone marrow, responding to specific growth factors

Haemopoietic progenitor cells are stimulated by a range of growth factors which promote colony growth in culture. The progenitors are a part of an age‐structured developmental hierarchy in the tissue. The growth factors, although overlapping in their effects, stimulate cells preferentially at different stages in this programme. Femoral bone marrow was fractionated into axial (close to the central venous sinus) and marginal (close to the bone surface) cells. Progenitors which responded to IL‐3, GM‐CSF, G‐CSF, M‐CSF and SCF were then assayed in soft agar cultures. Consequent plots of their spatial distributions showed that the more primitive cells in vitro (responding to IL‐3) were concentrated close to the bone surface. The peak concentrations of cells responding primarily to growth factors with progressively more affinity to more mature progenitor cells correspondingly appeared progressively further from the bone surface and closer to the point of release at the central venous sinus. This suggests that the developmental/maturational process in haemopoiesis is accompanied by a progressive movement of cells from the bone surface towards the central axial regions of the bone cavities. The most primitive cells are however exposed, close to the centre of the cavity, by a combination of SCF and G‐CSF (or by a 50‐fold increase in G‐CSF concentration alone). These results corroborate earlier data which indicate a developmental movement of cells from the centre of the marrow tissue towards the bone surface and back again, sequentially encountering a series of growth factors which promote their differentiation into mature cells, for release at the central venous sinus.

Microdistribution and localized dosimetry of the alpha-emitting radionuclides 239Pu, 241Am and 233U in mouse femoral shaft.

Purpose: To analyse the temporal change in microdistribution of 239Pu, 241Am and 233U in mouse femur and to compare the calculated radiation doses with regions of the bone marrow thought to contain target cells for osteosarcoma and leukaemia with relative risk for those diseases. Materials and methods: Neutron-induced and alpha-track autoradiographs were prepared from femora of the CBA/H mouse that had been injected with 40kBqkg 1 radionuclide between 1 and 448 days previously. Computer-based image analysis of the autoradiographs was performed and dosimetric methods applied to obtain radiation dose-rates to different regions of the marrow cavity. Results: Initially each radionuclide deposited on endosteal and periosteal bone surfaces; 241Am was additionally deposited on vascular canal surfaces. Redistribution resulted in 233U being incorporated into bone, while 239Pu and 241Am showed transfer into both bone volume and marrow. Accumulation in the central marrow peaked at 112-224 days post-injection, but subsequently was cleared by 448 days. Cumulative doses to both osteosarcomagenic and myeloid leukaemogenic target cell regions showed the trend 239 Pu> 241 Am> 233 U. Conclusions: Calculation of cumulative doses to a 10-mum layer of marrow adjacent to bone surfaces appears to be a suitable predictor for risk of osteosarcoma. Risks of myeloid leukaemia in the mouse are better predicted by considering the central marrow as the target region rather than average dose to all marrow.PURPOSE To analyse the temporal change in microdistribution of 239Pu, 241Am and 233U in mouse femur and to compare the calculated radiation doses with regions of the bone marrow thought to contain target cells for osteosarcoma and leukaemia with relative risk for those diseases. MATERIALS AND METHODS Neutron-induced and alpha-track autoradiographs were prepared from femora of the CBA/H mouse that had been injected with 40 kBq kg(-1) radionuclide between 1 and 448 days previously. Computer-based image analysis of the autoradiographs was performed and dosimetric methods applied to obtain radiation dose-rates to different regions of the marrow cavity. RESULTS Initially each radionuclide deposited on endosteal and periosteal bone surfaces; 241Am was additionally deposited on vascular canal surfaces. Redistribution resulted in 233U being incorporated into bone, while 239Pu and 241Am showed transfer into both bone volume and marrow. Accumulation in the central marrow peaked at 112-224 days post-injection, but subsequently was cleared by 448 days. Cumulative doses to both osteosarcomagenic and myeloid leukaemogenic target cell regions showed the trend 239Pu > 241Am > 233U. CONCLUSIONS Calculation of cumulative doses to a 10-microm layer of marrow adjacent to bone surfaces appears to be a suitable predictor for risk of osteosarcoma. Risks of myeloid leukaemia in the mouse are better predicted by considering the central marrow as the target region rather than average dose to all marrow.

Tumorigenic target cell regions in bone marrow studied by localized dosimetry of 239Pu, 241Am and 233U in the mouse femur

Purpose : To study the temporal change in microdistribution of plutonium-239, americium-241 and uranium-233 in the mouse distal femur and to compare and combine calculated radiation doses with those obtained previously for the femoral shaft. Also, to relate doses to relative risks of osteosarcoma and acute myeloid leukaemia. Materials and methods : Computer-based image analysis of neutron-induced and α -track autoradiographs of sections of mouse femora was used to quantify the microdistribution of 239 Pu, 241 Am and 233 U from 1 to 448 days after intraperitoneal injection. Localized dose-rates and cumulative doses over this period were calculated for different regions of the marrow spaces in trabecular bone. The results were then combined with previous data for doses to the cortical marrow of the femoral shaft. A morphometric analysis of the distal femur was carried out. Results : Initial deposition on endosteal surfaces and dose-rates near to the trabecular surfaces at 1 day were two to four times greater than corresponding results for cortical bone. Burial was most rapid for 233 U, about twice the rate in cortical bone. As in cortical bone, subsequent uptake into the marrow was seen for 239 Pu and 241 Am but not 233 U. Cumulative doses to 448 days for different regions of trabecular marrow were greater than corresponding values for cortical marrow for each radionuclide. Combined doses reflected the greater overall volume of cortical marrow. Conclusions : Cumulative radiation doses to the 10 μ m thick band of marrow adjacent to all endosteal surfaces were in the ratio of ~7:3:1 for 239 Pu: 241 Am: 233 U. This ratio is not inconsistent with observed incidences of osteosarcoma induction by the three nuclides. Analysis of doses to different depths of marrow, however, showed that although ratios were probably not significantly different to that for a 10 μ m depth, better correlations with osteosarcomagenic risk were obtained with 20-40 μ m depths. For acute myeloid leukaemia, the closest relationship between relative risk and doses was obtained by considering only the central 5-10% of marrow, which gave a dose ratio of ~12:11:1 for 239 Pu: 241 Am: 233 U respectively.

Engineering, Biology, and Clinical Development of hMIP-1α

The side effects of anticancer chemotherapy on normal cells often limit the severity of treatment that can be tolerated. If side effects could be reduced or managed effectively, then escalation of chemotherapy may lead to better clinical responses. Typical side effects of chemotherapy include depletion of bone marrow causing neutropenia, gastrointestinal mucositis, and hair loss. Of these, perhaps the most limiting is the effect of chemotherapy on bone marrow. Bone-marrow recovery after treatment with cytotoxic drugs depends on the survival of hematopoietic stem cells with the ability to replenish the hematopoietic progenitor pool indefinitely. Such cells are normally quiescent, rendering them insensitive to cycle-specific cytotoxic therapy. However, prolonged doses or repeat cycles of chemotherapy stimulate the division (self-renewal) of these multipotent stem cells and their differentiation into more committed progenitors (1). Therefore, once dividing, their survival is in jeopardy during cytotoxic drug therapy and a specific inhibitor of stem cell division might be expected to find use as an adjunct to chemotherapy. In clinical oncology, the expectation would be that stem-cell protection could provide a means of facilitating the recovery of circulating blood cells that have been depleted by multiple cycles of chemotherapy. Because the depletion of circulating neutrophils and platelets during cytotoxic drug therapy is the major dose-limiting toxicity, the use of a recombinant stem-cell inhibitor during such treatment should allow dose intensification and, therefore, more effective therapy. Here we describe the evaluation of the clinical potential of the stem-cell inhibitor macrophage inflammatory protein-1a (MIP-1α), a multifunctional CC chemokine.