Marrow function reconstitution by fraction 3 of Percoll-density-gradient-separated cells.

Eight children with advanced tumors or acute myeloid leukemia were treated either with very-high-dose multi-agent chemotherapy (4) or marrow ablative high-dose melphalan (4) followed by autologous marrow rescue, using only fraction 3 from a Percoll-discontinuous density gradient separation of bone marrow buffy coats. Each patient received less than 20% of the number of cells usually reinfused from the buffy coat. The yield of CFU-c in Percoll gradient 3 was similar to the yield obtained from whole buffy coats of bone marrow. Reconstitution of marrow function with a neutrophil count greater than 0.5 X 10(9)/L and platelet count greater than 50 X 10(9)/L occurred in 7 patients in a medium time of 15 and 16 days, respectively--a time comparable to that following infusion of whole buffy coat in 20 other patients. In one patient, hemopoietic recovery was considerably delayed, suggesting that fraction 3 from the Percoll gradient had been relatively ineffective in marrow reconstitution. We conclude that fraction 3 from marrow separated on Percoll gradients has the advantages of small volume and good recovery of marrow stem cells and can promptly reconstitute marrow function in the majority of children treated with very-high-dose chemotherapy or marrow ablative doses of melphalan.

Establishment and partial characterisation of a human fibrosarcoma cell line MR-83.

We describe a mycoplasma-free human fibrosarcoma cell line, MR-83, which grows readily in liquid culture and as clones in semi-solid agar with a plating efficiency of about 0.5%. It has a stable karyotype consisting of a modal number of 49-51 chromosomes, with two translocations and a deletion. The cell line shows resistance to adriamycin in semi-solid agar assay, and responds to Epidermal Growth Factor (EGF) by increased DNA synthesis, as measured by thymidine uptake.

Experience with high dose multiagent chemotherapy and autologous bone marrow rescue in the treatment of twenty-two children with advanced tumours.

Intensive chemotherapy followed by infusion of cryopreserved autologous bone marrow (ABMR) was used in the treatment of 22 children with advanced tumours. In nine this was their initial therapy; in eight it was used after partial or complete remission had been achieved with standard therapy; and in five, after relapse had occurred. Recovery of marrow function occurred in 20 patients with a mean time of 13.2 and 18.2 days to recovery of neutrophils (greater than 0.5 X 10(9)/l) in newly diagnosed and previously treated patients respectively. Platelet count recovery to greater than 50 X 10(9)/l occurred in a mean time of 13.4 days in newly diagnosed and 20.4 days in previously treated patients. Control of extensive local tumour was obtained in three of three evaluable patients with abdominal non-Hodgkin's lymphoma (NHL). Metastatic bony and marrow disease was controlled in two of two patients with retinoblastoma. In Ewing's sarcoma, temporary control of widespread metastatic disease occurred in one patient. In the other, eradication of extensive local mass disease at the primary site had been achieved. Poor response to treatment has been seen in seven of eight patients with Stage III or IV rhabdomyosarcoma, three patients with neuroblastoma and four of five patients with recurrent disease. Apart from the anticipated bone marrow toxicity, the major complications were severe mucositis, anaphylaxis following bone marrow infusion and haemorrhagic cystitis. The presence of herpes simplex infection appeared to aggravate mucosal complication.(ABSTRACT TRUNCATED AT 250 WORDS)

The development of a microassay for human committed progenitor cells.

A microassay for human committed progenitor cells (CFU-c) has been developed using 24-well, 16 mm diameter culture dishes. Comparisons were made of simultaneous cultures of 21 samples in both 35 mm and 16 mm culture dishes employing two sources of colony-stimulating factor (CSF). The microassay does not differ significantly from the standard 1 ml 35 mm assay, apart from some enhancement of colony numbers in the 16 mm dish. Other advantages of the microassay are that it is economical with respect to cells, media, and space; and it is possible to increase the number of experiments fivefold which can be performed with the same number of cells.

Murine anti-lymphocyte monoclonal antibodies fail to act as opsonins for allogeneic human peripheral blood polymorphonuclear phagocytes.

Seven murine monoclonal antibodies (MoAbs) to antigenic determinants present on human lymphocytes did not promote phagocytosis of allogeneic human lymphocytes by human peripheral blood polymorphonuclear leucocytes (PML). Similarly there was no significant metabolic activation of PML as determined by incorporation of radiolabelled iodide. The opsonic activity of MoAbs was not enhanced by addition of human or rabbit complement nor by the use of sheep anti-mouse IgG, rabbit anti-mouse immunoglobulin or goat anti-mouse immunoglobulin as second antibody. Whole serum from mice immunized with human lymphocytes did not act as an opsonin when tested with human PML. Hyperimmune mouse antiserum to Bordetella pertussis antigen acted only weakly as an opsonin. Human PML may not have an in vivo role in the removal of allogeneic lymphocytes treated with murine anti-lymphocyte MoAbs.

The use of discontinuous Percoll gradients to separate populations of cells from human bone marrow and peripheral blood.

Cells from human bone marrow samples were separated on discontinuous Percoll gradients and the effects of variations in osmolality were studied. Yield of committed progenitor cells as determined by CFU-c and BFU-E assays was improved when a gradient of discontinuous osmolality (360-320 mosM) was compared with gradients of uniform osmolality (320 mosM and 290 mosM). The CFU-c and BFU-E containing fraction recovered from this gradient also contained fewer T cells. When used for separation of peripheral blood samples, the same gradient yielded populations of mononuclear cells, neutrophils and platelets of more than 90% purity.

CFU-c enrichment from human bone marrow using a discontinuous Percoll gradient and soybean agglutinin in comparison with Ficoll-paque.

This study compares the efficiency of different methods of separation of human bone marrow in vitro prior to transplantation. Separation on Ficoll-paque resulted in a median recovery of 11.3 X 10(4) CFU-c/10(9) nucleated cells harvested. The recovery from the major CFU-c containing fraction following separation on a discontinuous Percoll gradient was 10.8 X 10(4) CFU-c/10(9) nucleated cells. The CFU-c recovered from Percoll were contained in a smaller number of cells, resulting in enrichment for CFU-c of 1.6 times that of Ficoll-paque. Further CFU-c enrichment to approximately three times that of Ficoll-paque separation was possible by treatment of Percoll fractionated cells with soybean agglutinin (SBA). However this caused an overall loss of 67% of CFU-c. Cells remaining after SBA treatment of the CFU-c containing Percoll fraction had increased spontaneous DNA synthesis and decreased PHA responses. There was no significant change in the mixed lymphocyte reaction in comparison with Ficoll-paque.

Attempted reconstitution of a foal with primary severe combined immunodeficiency.

A foal with primary severe combined immunodeficiency, diagnosed within the first two weeks of life, was maintained with its dam in semi-isolation. The foal received continuous prophylactic antibiotic therapy, plasma from a sibling hyperimmunised with equine adenovirus vaccine, and intensive general nursing care. A full sibling female was selected as a bone marrow donor on the basis of red blood cell cross-matching and mixed lymphocyte reactions. Cyclophosphamide was given before two bone marrow transfusions at 35 and 73 days of age. To prevent graft versus host disease graft versus host disease the foal was maintained on methotrexate therapy. Reconstitution was not achieved nor were there signs of graft versus host disease. The foal died suddenly four days after the second bone marrow transfer when 77 days old. It had remained clinically free of any life threatening infectious disease and at necropsy a remarkable degree of freedom from infectious disease was confirmed. The most notable necropsy findings were bilateral nephrosis and myocardial degeneration and fibrosis. The likely cause of death was an electrolyte imbalance, particularly hypokalaemia, which secondarily affected the myocardium. Renal toxicity caused by the cytotoxic drugs, especially cyclophosphamide, may have contributed to the electrolyte imbalance.

Autologous bone marrow rescue in the treatment of advanced tumors of childhood.

High-dose multiagent chemotherapy followed by autologous marrow rescue was used in the treatment of 13 patients with Stage III or IV childhood tumors. Encouraging results are being obtained in abdominal lymphoma (1/3 complete remissions (CR); rhabdomyosarcoma (2/4 CR); and retinoblastoma (1/2 CR). In neuroblastoma, the results are disappointing, with only one of four patients in CR; this patient developed a lymphoma associated with Epstein-Barr virus infection. Marrow reconstitution was obtained in 11 patients, with recovery of neutrophils to greater than 0.5 x 10(9)/liter between six and 30 days and platelet recovery to greater than 50 x 10(9)/liter between seven and 38 days. Investigations on the numbers of cells or committed granulocyte precursors ()CFU-c's) infused and parameters of hematologic recovery show poor correlation and suggest that a more accurate and reliable assay for the predictability of cryopreserved marrow to reconstitute marrow function within a reasonable time is necessary. Nonhematologic toxicities of high-dose multiagent chemotherapy are the principal dose-limiting factors.

A reliable technique for making permanent preparations of bone marrow cultures.

By modifying several techniques for the permanent preparation of bone marrow cultures in semi-solid agar, we have developed a system which give reliable results in terms of a high success rate, morphological quality and cytochemical flexibility.

Poor outlook for childhood acute lymphoblastic leukaemia with relapse.

Results of treatment in 72 children with a relapse of acute lymphoblastic leukaemia were analysed. A second remission was induced in 57 children (79%). Remission rates were significantly higher in males, and in patients with white cell counts less than 30 X 10(9)/L. The median duration of the second remission was only four months, and the median duration of survival from the time of relapse was nine months. The median duration of survival from the time of diagnosis was 27 months. Among children who suffered a relapse was a small group who had prolonged survival and prolonged second remissions. It is uncertain whether some of these may have a chance of "cure".

Treatment of childhood lymphocytic leukaemia with high white-cell counts.

Combination chemotherapy with cytosine arabinoside, cyclophosphamide and L-asparaginase (Asnase) was given to 22 children with acute lymphocytic leukaemia (ALL) with a white-cell count greater than 30 X 10(9)/1, and other features suggestive of poor prognosis. Complete remission was induced in all patients--in 19 after 2 courses of chemotherapy and in the remainder after a third course. During induction, neutropenia occurred in 18 and severe infection in 3. Anaphylaxis to Asnase occurred in 8 patients after the second course and one other had transient Asnase-induced diabetes. All patients received central-nervous-system prophylaxis after achieving remission, during which they were also treated with weekly vincristine and a 2-week course of prednisolone. Continuation therapy consisted of short cycles of intermittent chemotherapy and BCG inoculation or long cycles of intermittent chemotherapy +/- BCG. Life-table analysis shows 46% complete remission rate at 28 months, with 6 patients all in complete remission followed up between 28 and 41 months. There were minimal complications of continuation therapy, and BCG inoculation was well tolerated.

Cytokinetic studies in children with untreated acute lymphocytic leukaemia (ALL): relationship to "T" cell markers.

"The labelling index (LI), mitotic index (MI) of marrow lymphoblasts and the percent of peripheral blood lymphocytes and lymphoblasts that form rosettes with sheep red cells at 4 degrees C ("T4"), were measured at presentation in 33 children with ALL. There was a significant correlation between LI and MI, but no significant correlations between them and the total white cell count at diagnosis or the "T4" percent. Three patients had greater than 55% "T4" rosettes and showed increased LI and MI, and 2 have relapsed; 12 had less than 20% "T4" rosettes, showed an increased LI (LI greater than 8% in 8 of 12) but not MI, and 5 have relapsed; 18 had 20--55% "T4" rosettes and generally had the lowest LI (LI less than 8% in 11 of 18), and only 3 have relapsed. Our findings suggest that patients with high and low percentage of "T4" rosettes in the peripheral blood have an increased fraction of leukaemic cells in DNA synthesis and have a diminished chance of prolonged remission.

The defensive function of cyanogenesis in natural populations.

Detailed ecological studies of some maritime populations of Lotus corniculatus L. have shown that the distribution of the cyanogenic form of this plant is directly related to the distribution and density of molluscs which graze selectively the acyanogenic form. This work, on a genetic polymorphism, is interpreted as giving direct evidence of chemical defence in natural populations.