Enhanced capacity of cytosine-arabinoside-treated murine bone marrow to maintain hematopoiesis in long-term culture.

A study of bone marrow of C57B1 mice administered cytosine-arabinoside (Ara-C) was carried out in long-term bone marrow culture (LTBMC). Two days after administration of two consecutive i.p. Ara-C injections (200 mg/kg each) at 6-h intervals, the bone marrow becomes hypocellular, yet in the process of regeneration, with an enriched and/or concentrated content of progenitors and stem cells. Ara-C-treated marrow was observed to sustain hematopoiesis in vitro better than physiological marrow; it produced a higher cell yield, a higher proportion of young-type myeloid cells, and higher levels of granulocyte-macrophage colony-forming cells and colony-forming units in diffusion chamber than control marrow. In addition, stromal cell cultures (SCC), devoid of hematopoiesis and engrafted with hematopoietic cells from LTBMC of Ara-C-treated marrow, were observed to produce hematopoietic cells for longer periods of time than SCC engrafted with control cells. In view of its increased capacity for regeneration, it is suggested that regenerative marrow should be used in autologous bone marrow transplantation in humans.

Improved prognosis in mice with advanced myeloid leukemia following administration of GM-CSF and cytosine arabinoside.

Acute myeloid leukemia (AML) was induced in C57Bl mice through the i.v. innoculation of C-1498 cell line. One week later, i.e. at mid-term disease, the leukemic mice received an i.p. injection of 200 ng rmGM-CSF and 24 h later, two consecutive i.p. cytosine arabinoside (ara-C) injections at 6 h intervals (2 x 200 mg/kg). The leukemic mice received 3-4 weekly courses of combined therapy and survived 4-5 weeks following leukemia induction. Control mice received ara-C only and survived 2-3 weeks. Moreover, leukemic mice administered both GM-CSF and ara-C had a lower marrow leukemic load than mice treated with ara-C only. From these findings, we conclude that therapy of murine AML with combined rmGM-CSF and ara-C is more effective than ara-C only. Leukemic mice treated with GM-CSF and ara-C had a longer life expectancy and a smaller leukemic load than mice administered ara-C only.

Bone marrow stromal deficiency in acute myeloid leukemia in mice.

A study of bone marrow stroma in acute myeloid leukemia (AML) in mice was carried out. AML was induced in C57B1 mice by i.v. inoculation of the C1498 cell line. There was a direct correlation between the marrow leukemic load and the degree of marrow stromal deficiency. This was expressed by reduced in vitro fibroblastoid colony formation. In co-cultures of normal mouse bone marrow and leukemic cells, and also in cultures of normal marrow with added conditioned medium (CM) of leukemic cells, marked inhibition of fibroblast-colony-forming units (CFU-F) from normal marrow was observed. In additional experiments, leukemic mice were treated with two consecutive injections of cytosine arabinoside (Ara-C) (2 X 200mg/kg) and sacrificed 48 h later. Bone marrow samples of treated animals formed in vitro an increased number of fibroblastoid colonies despite relatively high levels of marrow leukemia. It is concluded that: (a) there is a direct correlation between the incidence of marrow leukemic cells and the degree of stromal deficiency; (b) leukemic cells produce in vitro--and probably in vivo CFU-F inhibitory factors and (c) administration of restricted doses of cytosine arabinoside to leukemic mice reduces the marrow leukemic cell content to some extent and increases the capacity of CFU-F to form fibroblastoid colonies in vitro.

Effects of chemotherapy on bone marrow stroma in mice with acute myelogenous leukemia. Correlation with CFU-C and CFU-D.

This study describes changes in bone marrow stroma in murine acute myelogenous leukemia (AML). The AML was induced in C57B1 mice by intravenous (i.v.) transfusion of C4198 myelogenous leukemic cells. In untreated leukemic mice, the colony-forming unit fibroblasts (CFU-F) were severely inhibited. In leukemic mice treated by three chemotherapy protocols of cytosine-arabinoside (Ara-C) and adriamycin there was a 200% increase in the life span as compared to untreated leukemic animals and marked reduction of marrow leukemic load. In these mice the stromal inhibition was temporarily relieved, expressed by peaks of CFU-F2-3 days following each protocol. In between the peaks, CFU-F decreased to subnormal levels, remaining low to the end of the disease. In normal mice administered a similar chemotherapy regimen, there were peaks of CFU-F activation after each protocol and normal levels in between the peaks. Granulocyte/macrophage progenitors (CFU-C) of leukemic-treated and normal-treated mice showed increased levels following each chemotherapy protocol. Whereas CFU-C decreased below normal levels in leukemic mice towards the end of the disease, the level of these progenitors remained high in normal mice receiving Ara-C and adriamycin. Colony-forming units in diffusion chamber (CFU-D) showed mild fluctuations in both leukemic and normal mice receiving three protocols of Ara-C and adriamycin. It is possible that despite treatment, the bone marrow stroma in leukemia becomes irreversibly deficient towards the end of the disease and cannot support the residual normal hematopoiesis.

Minimal residual disease in vitro of a pre-B-lymphoma.

We have studied the survival of clonogenic neoplastic cells of a murine pre-B-lymphoma (BCl-1) of the spleen in culture. We have found quantitative deficiencies such as reduced surface adherence of stromal cells and impaired CFU-F (colony forming units-fibroblast) and pre-CFU-F colony and layer formation in stromal cultures of lymphoma bearing spleen, as compared to cultures from normal spleen. There are two populations of clonogenic BCl-1 lymphoma cells surviving in culture: one population is surface adherent, and the other is non-adherent. Both populations transmit the lymphoma to healthy indicator mice. We hope that this model will be helpful in studying minimal residual leukemic disease [1] in culture.

Radiotherapy of non-metastatic ewing sarcoma.

Comparing the radiotherapy data of two groups of patients with non-metastatic Ewing sarcoma, we came to the following conclusions: intensive chemotherapy does not substitute for effective radiotherapy. Irradiation fields should be large enough to include the whole affected bone and the entire extra-osseous compartments, exempting only uninvolved epiphyses. Marginal recurrences are a major risk of the shrinking field technique. Single doses per fraction should be high and the total dose raised to the point of maximal irradiation tolerance of normal tissues.

Adjuvant chemotherapy in osteosarcoma - effects of cisplatinum, BCD, and fibroblast interferon in sequential combination with HD-MTX and adriamycin. Preliminary results of the COSS 80 study.

In a cooperative adjuvant chemotherapy study of osteosarcoma (COSS-80), 192 patients were registered from December 1979 to March 1982. Forty-one patients have been excluded from study because of their nonadjuvant situation, therapy-limiting clinical conditions, or inadequate diagnosis. One hundred and fifty-one patients have been randomized to receive either the drug combination bleomycin + cyclophosphamide + dactinomycin (BCD) or cisplatinum (CPL) within a course of sequential multidrug chemotherapy including adriamycin (ADR) and high dose methotrexate (HDMTX). After exclusion of 51 patients with some deviation in history and/or management 100 selected patients were randomized once more to receive in addition or not fibroblast interferon after preoperative chemotherapy and surgical removal of the primary tumor. Patients were stratified for age and sex and for site and extension of tumor as well in both randomizations. Median follow up is now 12 (1-16) months. The expected 2-year disease free survival (DFS) rate of the total doubly randomized group is 78% and of the single randomized group 76%. No difference could be discerned between recombined groups receiving BCD vs CPL or interferon vs no interferon. The effect of preoperative chemotherapy on the tumor was evaluated clinically and by histopathologic grading; 66/85 (78%) patients were judged clinically as responders with pathohistologic verification of this finding in 71% of these cases. No adverse effect arose from delaying definite surgery for preoperative chemotherapy, but initial application of chemotherapy as well as planning, preparing, and performing of the surgical procedure have been facilitated. The majority of patients received some kind of limb-salvage treatment without local recurrences so far. A statistically insignificant but intriguing tendency for a slightly higher incidence of pulmonary metastases after resection as opposed to amputation could be detected. Similar to observations in the previous study COSS-77.

Changes of bone marrow stromal cells (MSC) in mice after drug induced eradication of hematopoietic cells: in vitro effects on normal bone marrow.

This study deals with murine marrow stromal cells (MSC) during hematopoietic regeneration. Regenerative marrow was induced in infant mice by two consecutive i. p. injections of hydroxyurea (HU) (2 X 1,000 mg/kg) which kill most cells in DNA synthesis. Two days later the marrow becomes enriched in pluripotent stem cells (CFU-S) and committed progenitors. It was found that fibroblastoid colony-forming-units (CFU-F) become concentrated approximately four fold above controls and give rise to significantly larger fibroblastoid colonies than CFU-F in control marrow. In split-phase semisolid agar cultures, i. e. adherent cells of HU-treated marrow (in under layers) and normal bone marrow cells (in upper layers), inhibition of G/M colonies in the upper layer in observed. In control split-phase cultures of both under layers and upper layers of normal bone marrow, CFU-C inhibition is by far less pronounced. In liquid cultures, pre-established adherent layers of HU-treated marrow have a stimulatory effect on normal marrow CFU-C. It is concluded that during increased demand for pluripotent stem cells and progenitors, such as after HU administration, marrow stromal cells and possibly other adherent cells produce inhibitory factor(s) of CFU-C differentiation. The inhibitory activity possibly is an effective mechanism for stem cell pool preservation. The differing effect of adherent cells of HU-treated marrow on CFU-C in liquid and in semi-solid medium suggests production of two different factors with opposing influences on CFU-C, depending upon the culture conditions.

[Multidrug chemotherapy of osteogenic sarcoma (author's transl)]. / Die Behandlung des Osteosarkoms--Modell einer chemotherapeutischen Strategie.

Osteogenic sarcoma may be treated effectively by radical surgical removal of the primary tumor and combined chemotherapy, including Adriamycin and high dose Methotrexate. In order to render any protocol a safe procedure, strict precautions are required to avoid drug toxicity. We present a protocol, "COSS 77", presently employed in several university hospitals of West Germany and Austria. Final results concerning long term prognosis and long term side effects are not yet available.

Paediatric oncology in developing countries.

Paediatric oncology in developing countries is a specialty in its own right that has so far been largely neglected by the western medical profession. It has specific features of genetic cancer predisposition and of external factors influencing phenotypic cancer manifestations. We point out here some of the specific features of cancer presentation in children of developing countries.

Fetal blood-borne myelo- and thrombopoietic stem cells in diffusion chambers.

The potential of hematopoietic stem cells from cord blood for proliferation and differentiation have been studied by the diffusion chamber technique. In a pilot study, it was shown that blast cell production and myelopoiesis from the original lymphoid cell suspension starts 6 days after implantation. Mature granulocytes and megakaryocytes respectively appear in the chambers from the 16th and 10th days onwards. Myelopoiesis is ten times more active in fetal than in adult blood. There were thrombopoietic stem cells in 7 of 10 cord blood samples but in only one of 10 adult blood specimens. It is concluded that the high hematopoietic activity of the bone marrow at term is reflected by an increase in the number of stem cells circulating in the blood. Myelo- and thrombopoietic stem cells must be sought among the lymphoid cells of the original cord blood cell suspension.

The ontogeny of the gut mucosal immune system and the susceptibility to infections in infants of developing countries.

In this review we summarize data on the human gut mucosa associated lymphatic tissues as part of the common mucosal immune system. Its embryonal-fetal and post-natal ontogeny becomes severely distorted and compromised by mal-/undernutrition which is so prevalent in developing countries. Pathogenetic interdependencies exist between maternal-fetal undernutrition, the ontogeny of the immune system, constant antigenic stimulation of the mucosal immune system post-natally, and the 14 million deaths annually from infections in children below the age of 5 years in developing countries. A detailed knowledge of these interdependencies is required for effective prevention and treatment in an attempt to reduce the high morbidity and mortality rates of children in developing countries.

Fanconi's anemia. II. Are multiple endocrine insufficiencies a substantial part of the disease?

Three children with Fanconi's anemia belonging to a family where 6 children had the disease were investigated. One child had growth hormone deficiency, a second child showed subnormal response of testosterone to gonadotropin stimulation and the third child had a missing insulin release following arginine. This report shows that growth hormone deficiency is not necessarily liniked with Fanconi's anemia when it occurs in a family. Multiple endocrine insufficiencies do no appear to be part of the disease.

Long-term survival of murine erythroid progenitors in long-term bone marrow culture and stromal cell culture: differentiation in peritoneal diffusion chamber culture.

Bone marrow cells of normal and cytosine-arabinoside (Ara-C) treated C57B1 mice were cultured in primary long-term culture (LTBMC) for a period of eight weeks. Non-adherent cells collected at weekly culture feedings consisted of neutrophils, macrophages and megakaryocytes. These were transferred into a) secondary peritoneal diffusion chamber cultures (DC) and b) secondary stromal cell cultures (SCC) first, and then into tertiary DC cultures. While in LTBMC and SCC there was no evidence of erythropoiesis, many erythroid colonies developed in DC cultures. It appears that undifferentiated erythroid progenitors may have a long survival in LTBMC and SCC devoid of erythropoietin and then differentiate in vivo in DC cultures in host mice without specific erythropoietic stimuli. Terminal differentiation and maturation of erythroid progenitors occurs to a limited extent in conventional DC cultures. The large number of erythroid colonies in DC observed in the present study could be due to increased sensitivity of undifferentiated erythroid progenitors from LTBMC to physiological levels of Epo in host mice of DC.

Haematopoietic stem cells (CFUc) in human cord blood.

Colony forming units (CFUc) giving rise to myelocytic colonies in methylcellulose cultures were found among non-adherent mononuclear cells of human cord blood with a frequency of one in 1678. The number decreased makredly during the first 8 to 10 days of life. They were rarely detected in adult blood by this technique.

Differential characterization of colony-forming units in diffusion chambers from pluripotent stem cells and progenitors of granulocytes/macrophages.

Colony-forming units in diffusion chambers (CFU-D), pluripotent stem cells (CFU-S) and colony-forming units in agar cultures (CFU-C) were studied in the bone marrow of weanling mice injected twice with hydroxyurea (HU). CFU-D, CFU-S and CFU-C were assayed 2 h, 2 days and 4 days after HU administration. After 2 h the total number of CFU-D/femur marrow cells did not change, while CFU-S and CFU-C decreased to 16.6 and 13.5% of control levels, respectively. On Day 2, CFU-D and CFU-S increased to 25 and 16.6%, respectively, above control levels and CFU-C was only 52% of the normal level. Four days after HU administration, CFU-S decreased steeply to 7.6%, CFU-D 80% and CFU-C to 32% of their pretreatment levels. Bone marrow of HU-treated weanling mice was also incubated in liquid medium where CFU-S decreased more rapidly than did CFU-D. It is concluded that CFU-D in weanling mice represent a noncycling and more primitive stem cell pool than CFU-S and probably constitute a reserve pool, the mobilization of which is secondary to that of CFU-S.

Circulating myelopoietic stem cells (CFUc): high levels in healthy pre-term infants and reduced levels in sick pre-term infants.

In healthy and diseased pre-term infants at birth, levels of circulating CFUc are 2,586 +/- 3,063 (s. d.) and 269 +/- 292 (s.d.) per 5 X 10(5) lymphoid cells, as compared to data from the literature of 5 CFUc per 5 X 10(5) mononuclear cells from the blood of adults [8]. Hypotrophic infants have the lowest concentration of CFUc in the blood, namely 78 +/- 105 (s.d.). In formerly sick infants recovering from their perinatal illness, blood CFUc levels increase to approach those from healthy infants of the same age. Our results provide evidence that blood CFUc levels in pre-term infants reflect the strong myelopoietic activity of fetal bone marrow and suggest a correlation to the clinical status of the infant.