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Objectives: This review is to explore whether potential gene interactions in the cell cycles of gametes, zygotes, and embryonic stem (ES) cells are associated with the development of cancer., Methods: MEDPILOT at the Central Library of the University of Cologne, Germany (Zentralbibliothek Köln) that covers 5,800 international medical journals and 4,300 E-journals was used to collect data. The initial searches were done in December 2012 and additional searches in October 2013-May 2015. The search terms included "cancer development," "gene interaction," and "ES cells," and the time period was between 1998 and 2015. A total of 147 articles in English language only were included in this review., Results: Transgenerational gene translation is implemented in the zygote through interactions of epigenetic isoforms of transcription factors (TFs) from parental gametes, predominantly during the first two zygote cleavages. Pluripotent transcription factors may provide interacting links with mutated genes during zygote-to-ES cell switches. Translation of post-transcriptional carcinogenic genes is implemented by abnormally spliced, tumor-specific isoforms of gene-encoded mRNA/non-coding RNA variants of TFs employing de novo gene synthesis and neofunctionalization. Post-translationally, mutated genes are preserved in pre-neoplastic ES cell subpopulations that can give rise to overt cancer stem cells. Thus, TFs operate as cell/disease-specific epigenetic messengers triggering clinical expression of neoplasms., Conclusion: Potential gene interactions in the cell cycle of gametes, zygotes, and ES cells may play some roles in the development of cancer.

Unlabelled: This review focuses on gene transcription patterns of leukemogenic S-phases in mitotic cell cycles for identification of enzymatic reactions as potential targets for epigenetics-based drug therapy. Transcription of leukemic genes is triggered by reprogrammed transcription factors (TFs) mediated by chromatin histones. Reprogrammed TFs originate from transcriptional alterations of CpG methylation patterns of mutated epigenetic genes. They preserve memory information of earlier leukemogenic exposures, even transgenerationally via the zygote, through small (e.g. pi)RNA transmitted between cells by exosomes. Normally, reprogrammed TFs are enzymatically silenced and stored as markers in heterochromatic domains. Failure of intra S-phase surveillance (IS) permits the formation and continual operation of DNA replication forks in spite of persisting genotoxic stress. Silenced TFs are re-activated by euchromatin, most likely through leakages of insulator barriers of cis-regulating chromatin modulators (CRM) that normally separate hetero- from euchromatin domains. During transport by sliding nucleosomes, reprogrammed leukemogenic TFs are misplaced at transcription factor binding-/starting-sites (TFBS /TSS) allowing them to interact with and trigger replication of mutated leukemic genes., In Conclusion: Interactions of enzymatically reprogrammed TFs, transcribed from mutated epigenetic genes, with replicating leukemic genes at TFBS/TSSs are key driving forces in leukemogenesis. Probably, epigenetic genes, although mutated, still retain their control of replication of leukemic genes. Epigenetics-based enzyme inhibitors must target reprogrammed TFs. Prudently, therapeutic corrections should be introduced within the frame of conventional, cytoreductive treatment protocols. Alternatively, reprogrammed TFs could be replaced by cell populations with regular TF production. Clinically, classification of leukemias should be based on their epigenetic presentation.

Objectives: Processing of epigenomic transcriptional information by cell cycle phase G(1) and decision-making at checkpoint G(1)/S are the final organizational steps preceding gene replication in transcriptional reorientation programs (i.e., switches from proliferation to cycle arrest and neoplastic transformation). Further analyses of cycle progression will open up new approaches in antineoplastic therapy., Materials and Methods: The following bibliographic databases were consulted: Central Medical Library Cologne, PubMed (English), the last search was done on April 23,2008 and key words searched were: cell cycle, cell memory, DNA methylation, embryonal/neoplastic stem cells, enzyme-modulated chromatin, G(1)-G(1)/S checkpoint, genomic/epigenomics, genomic viral DNA, histones, telomere/telomerases, transcription factors, neoplastic transformation, senescence., Results: Gene transcription and epigenomic surveillance form a functional entity. In proliferation programs, transcriptional information is mediated by chromatin and DNA methylation, analyzed and processed in G(1) phase, and converged on the parental checkpoint G(1)/S for final decision-making on DNA replication. Genomic reorientation appears to be associated with transcriptional instability, which normally is corrected, possibly during the G(2)/M phase, to new levels of epigenomic equilibria. We speculate that daughter stem cells inherit persistent neoplasm-specific transcriptional instabilities through failure of the parental G(1)/S checkpoint. Foreign, silenced, potentially oncogenic DNA sequences, i.e. regular components of the human genome such as endogenous retroviruses, could conceivably be activated for expression in neoplastic transformation by epigenomic histone deacetylase/acetyl transferase/histone methyltransferase-mixed lineage leukemia deregulations., Conclusions: Failure of cell cycle G(1)/S decision-making for DNA replication is the final and possibly a major cause in neoplastic transformation. Therefore, further analysis of the dynamics of G(1)-G(1)/Sphases could provide new opportunities for therapeutic strategies.

Reducing the blood supply of tumors is one modality to combat cancer. The objective of this study was to evaluate such an approach in the treatment of localized murine AML (acute myelogenous leukemia). For this purpose we designed an experimental model in which leukemic cells were embedded in 1% agar discs before subcutaneous implantation in C57Bl female mice. The C-1498 AML cell line (Frederick Inst., NCI, MD, USA) was used. Thirty experimental mice received on alternate days injections of 5 x 2.5 microg anti-VEGF (vascular endothelial growth factor) and 5 x 2.5 microg anti-Flk-1 (VEGFR2) antibodies to the site of cell implantation over a period of 10 d. Fifteen control mice received daily PBS injections. All mice were sacrificed 16 d after AML implantation. Of the 30 experimental animals, macroscopic examination showed in 21 animals (70%) small sized, pale tumors (0.5 g); in six mice (20%) the tumors were replaced completely by necrotic tissue, while in three mice (10%), there were large (2.5 g), highly vascularized tumors. In all 15 control mice large highly vascularized tumors were seen. A separate group of mice was studied for total survival following AML implantation. While 12 mice in the control group not treated with antibodies survived for 16 d post-implantation, survival was prolonged in 15 antibody treated mice by approximate 30 d to a total survival time of 48 d. Tumor specimens were processed for histology, immunohistochemistry (IHC) for CD31 endothelial cell antigen, and tube-like formation assay. The small, pale tumors of antibody treated animals consisted of degenerate hyaline material with remnant nests of leukemic cells, whereas large tumors showed sheets of leukemic cells and numerous blood vessels. Specimens processed for CD31 antigen showed scarce or absence of blood vessels in the small, pale tumors in contrast to intensive staining from a rich network of blood vessels in the large, highly vascularized tumors. Tube-like formation assays disclosed rudimentary Grade 1 endothelial cell tubes in the small, pale tumors as opposed to polygonal Grade 4 tube formation in control animals. In conclusion, this murine model of localized AML allows assessment of anti-angiogenic tumor regression. Anti-angiogenic antibodies against VEGF and Flk-1 have therapeutic effects in murine AML., ((c) Blackwell Munksgaard 2005.)

Embryonal stem (ES) cells are the earliest ontogenetically identifiable stem cells of the embryo proper for all subsequent mesenchymal stem cells and for highly specialized differentiated cells. This review characterizes, in a working hypothesis, the role of reversible EMT/MET (epithelialmesenchymal transition) as a manifestation of cell plasticity 1) in the development of ES cells to adult stem cells (hematopoietic stem cells) and 2) in metastasizing cancer cells. Animal studies support the concept that EMT/MET is a key manifestation of cell plasticity in the development of ES cells to adult stem cells, and in conversion of localized to metastasizing cancer cells. In fact, ES cells may persist to postnatal life, in cytologically verifiable form and/or within the frame of EMT/MET, as ultimate reservoir for adult stem cells. Furthermore, EMT could possibly serve as a conceptional link between physiologic and pathologic signaling pathways. Clonal confirmation in humans is necessary.

The life-long interdependencies/interactions between hemato- and endotheliopoiesis suggest that they form a supplementary functional entity. This view is compatible with the concept of stem cell plasticity as a reversible continuum and is substantiated by the common hematopoietic-endothelial stem cell, i.e., hemangioblasts, with bidirectional, reversible gene transcription and persistence in postnatal life. Indeed, embryonal stem cells/hemangioblasts appear to form a reservior in the adult with the possibility of dedifferentiation of more differentiated progenitor cells back to hemangioblasts. The recent detection of BCR/ABL fusion proteins in endothelial cells during vascular neoangiogenesis in CML suggests that endothelial cells are part of the neoplastic clone, and extends the concept of a functional entity to include CML angiogenesis. Thus, hemangioblasts rather than committed hematopoietic stem cells appear to be target cells for the first oncogenic hit in CML, which could occur as early as during the first steps of embryonal stem cell differentiation towards hemato-endotheliopoiesis and/or in hemangioblasts persisting in adults. The relation of the other leukemias to hemangioblasts is not known.

Epithelial mesenchymal transitions are a remarkable example of cellular plasticity. These transitions are the hallmark of embryo development, are pivotal in cancer progression, and seem to occur infrequently in adult organisms. The reduced incidence of transitions in the adult could result from restrictive functions of the microenvironment that stabilizes adult cell phenotypes and prevents plastic behavior. Multipotential progenitor cells exhibiting a mesenchymal phenotype have been derived from various adult tissues. The ability of these cells to differentiate into all germ layer cell types, raises the question as to whether mesenchymal epithelial transitions occur in the adult organism more frequently than presently appreciated. A series of cytokines are known to promote the transitions between epithelium and mesenchyme. Moreover, several transcription factors and other intracellular regulator molecules have been conclusively shown to mediate these transitions. However, the exact molecular basis of these transitions is yet to be resolved. The identification of the restrictive mechanisms that prevent cellular transitions in adult organisms, which seem to be unleashed in cancerous tissues, may lead to the development of tools for therapeutic tissue repair and effective tumor suppression.

Unlabelled: Scientists, non-governmental experts (NGOs) and governmental officials from the Central Asian Republics and an international group of invited scientists and NGO representatives participated in a workshop on the disastrous health problems in the Aral Sea Region. Various serious problems were reported in more than 20 presentations. Particular emphasis was put on the way in which adverse environmental factors such as contaminated water and food have contributed to the deterioration of human health, particularly that of mothers and children., Conclusion: There is an urgent request that the international community assists local scientists to develop programmes to improve the health of the population in the Aral Sea Region.

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.

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.

The morbidity rates from neoplastic disease have been studied in children below the age of 15 years, residing at the time of their diagnosis within a diameter of 25 km of the nuclear power plant of Würgassen (FRG). A total of 42 patients were registered between the years of 1980 and 1988: 15 patients with neoplasms of the hematolymphopoietic system, 14 neoplasms of the central nervous system (CNS), and 13 with solid tumors outside of the CNS. The data show that children living in the vicinity of this nuclear power plant do not have an increased risk of developing neoplastic disease, as compared to the entire pediatric population of the FRG. However, a tendency for an elevated risk, not reaching the level of statistical significance, cannot be fully excluded.

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.

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.

A study of bone marrow stromal elements in murine acute myeloid leukemia (AML) was carried out. Our previous studies had indicated marrow stromal deficiency in murine AML. In the current investigation, separate stromal cells were cultured and the results obtained have shown that, while marrow stromal macrophages are normal in leukemia and express adequate amounts of IL-1, the fibroblasts are markedly reduced. However, if sufficient fibroblasts are pooled in vitro, they produce adequate amounts of CSF. Test of TNF alpha in leukemic cells CM, as possible cause of marrow stromal inhibition in leukemia, had not disclosed this cytokine. Further, it was observed that total body lethal irradiation of leukemic mice aggravates the stromal deficiency, confirming results of our previous investigations. It is concluded that bone marrow stromal deficiency in murine AML is due to decreased fibroblasts and, implicitly, reduced CSF production.

A study of treated murine acute myeloid leukemia (AML) with an emphasis on the bone marrow stromal function is reported. Leukemia was induced in C57Bl mice through intraperitoneal (i.p.) inoculation of C-1498 myelogenous leukemic cells. The leukemic mice were administered: (1) total body lethal X-irradiation (t.b.i.); (2) two i.p. cytosine-arabinoside (Ara-C) injections followed by X-irradiation. Control mice received similar regimens. Bone marrow of experimental and control mice was processed for stromal cell cultures (SCC) and in vitro engraftment of hematopoietic cells onto the cultures. The results of this study indicate that the bone marrow stromal deficiency which occurs in leukemia is aggravated by Ara-C and irradiation treatments. Moreover, SCC of treated leukemic mice sustain in vitro hematopoiesis only to a limited degree. Stromal deficiency, as possible cause for graft failure in bone marrow transplanted leukemic patients, is discussed.

Hypothyroid goiter is a rare but well recognized complication following long term administration of iodine containing expectorants and disinfectants in children. Only few reports exist on iodine-induced hypothyroidism after a single injection of the iodized radiopaque dye Lipiodol. A 15-year-old boy with previously normal thyroid function is described who developed hypothyroid goiter within six weeks following bipedal lymphography. Urinary iodine excretion was extremely elevated up to 18 mg/day while serum concentrations of total thyroxine were below the euthyroid range and thyrotropin levels were elevated. After oral L-thyroxine treatment the goiter disappeared. Thyroid function remained normal when treatment was discontinued after five months although iodine excretion was still 50 times higher (2.5 mg/day) than in normal age matched children. The observed alterations of the thyroid gland were caused by a long lasting Wolff-Chaikoff effect with a delayed adaptation to high iodide concentrations.

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.

Regenerating bone marrow of newborn random bred Sabra mice (9-13 days old) was obtained by the administration of two consecutive i.p. injections of hydroxyurea (HU) (2 x 100 mg/kg body wt), three days prior to collection of the marrow cells. The bone marrow of HU-treated newborn mice was assayed for CFU-S, CFU-C and plasma-clot-diffusion-chamber (PCDC) progenitor cells. A fourfold content of CFU-S was found in the regenerating bone marrow compared with that of the control marrow, while the level of CFU-C and PCDC progenitor cells was the same in treated and untreated newborn mice. In lethally irradiated adult, random bred Sabra recipient mice, transfused with regenerating bone marrow from newborn mice, the initial survival rate was greater than in irradiated animals receiving normal newborn marrow (75% as against 50%); marrow repopulation, 10-14 days after transfusion, was also greater in the former than in the latter group of animals (1.5-2x10(6) nucleated cells per femur as compared with 08.-2x10(5)). The bone marrow of these groups of mice was assayed for CFU-S, CFU-C and PCDC progenitor cells; with a cell inoculum of 5 x 10(4) i.v., 10(5) in vitro and 5 x 10(4) per DC, respectively, pluripotent and committed stem cells were detected in the experimental group and were lacking in control recipients. Regenerating bone marrow of newborn mice was also transfused into lethally irradiated splenectomized recipients. In this experimental group there was high mortality, low marrow repopulation and lack of CFU-S (5-10 x 10(4) cell inoculum). The results of this study indicate that, despite genetic differences among random bred Sabra mice, regenerating bone marrow of newborn mice "takes better" than normal marrow in lethally irradiated recipients. Improved marrow acceptance is possibly due to the increased content of activated CFU-S and/or pre-CFU-S in the regenerating bone marrow

A suspension of fine carbon particles was added to cord blood of healthy premature and full-term infants, and the mixture was incubated for 3 h, after which the uptake of carbon particles by blood leukocytes was examined. The results were compared with those from the blood of adults. A gradient of phagocytic activity was observed. The most active uptake of carbon was by the leukocytes of premature infants, the least active by leukocytes of adults. In cord blood and blood of adults, phagocytic activity was evident in both monocytes and neutrophils. In addition, two types of what have been termed 'lymphocytoid' phagocytes were seen. These resemble lymphocytes in their morphology. One type possesses basophilic cytoplasm, and has been found only in premature cord blood. The presence of lymphocytoid phagocytes affords a further indication of the differences between the circulating lymphocyte population in the prenatal and perinatal period as compared with the adult.

Glucosephosphate isomerase deficiency as the cause of macrocytic congenital nonspherocytic hemolytic anemia is described in three unrelated families. The biochemical properties of the variant glucosephosphate isomerases indicate that the patients have new variants, designated as GPI Kiel, GPI Hamburg, and GPI Homburg. The severity of the clinical symptoms depended on the amount of residual GPI activity and the biochemical properties of the variant enzyme. Thus the patient with GPI Kiel (34% residual activity) whose variant GPI was slightly unstable showed a mild chronic hemolytic anemia. The patient with GPI Homburg (7% residual activity) whose variant enzyme was stable and had a reduced specific activity, suffered from severe congenital hemolytic anemia and neuromuscular symptoms. Due to the special properties of GPI Homburg, we assume that both the hematological and neuromuscular symptoms of the patient with GPI Homburg are caused by his GPI deficiency. The twins with GPI Hamburg (27% residual activity) had a distinctly unstable variant enzyme and had suffered from hemolytic crises since birth. Only GPI Homburg showed an altered electrophoretic mobility and an increased affinity for fructose-6-phosphate. The other two variants had normal values.

L-asparaginase is an effective antileukaemic drug and a potent inhibitor of hepatic protein synthesis. Its effect on lipid metabolism was studied in two cohorts of children with ALL, one of whom received L-asparaginase concomitantly with three other drugs (protocol BFM 79). In the second protocol (BFM 83) administration of L-asparaginase was arranged to follow the other three drugs in time sequence. The two major findings of this study were elevated serum levels of total cholesterol and a strong increase in serum triglycerides. The former change was due to an increase in alpha-cholesterol and could not be attributed to L-asparaginase because it was also found following protocol BFM 83 before the administration of the drug. Elevations of total triglycerides were due to high levels of exogenous chylomicron bound triglycerides and were limited in occurrence almost exclusively to the period of L-asparaginase monotherapy. Hypothyroidism was excluded as a possible pathogenetic mechanism. These changes in lipid metabolism induced by L-asparaginase during intensive remission induction chemotherapy are fully reversible.

The existence of pluripotential haemopoietic stem cells raises the possibility of stem cell competition when two or more differentiating stimuli are given either simultaneously or in close succession. The experiments now reported deal with the effects on guinea pig bone marrow of successive stimulation and they fall into two groups. In one group, erythropoiesis was first stimulated by means of hypoxia, after which granulopoiesis was stimulated by the intraperitoneal injection of typhoid vaccine. In another group, these two stimuli were given in the reverse order. Bone marrow changes were evaluated both quantitatively and by differential counts. The experimental animals were compared with controls given only one stimulus, either hypoxia or vaccine, and also with normal untreated animals. As judged by the output of granulocytes or erythrocytes, no stem cell shortage developed in the experimental animals. A marked fall in transitional cells in the bone marrow of the experimental animals is consistent with the view, though not affording actual proof, that the pluripotential stem cells are to be found in the transitional cell compartment.

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.

One hundred and twenty-one febrile episodes (FE) were studied in 58 aplastic children with acute leukemia. All patients received a prophylactic regimen of trimethoprim-sulfamethoxazole (TS) and colistin sulfate orally, and amphotericin B locally from the beginning of their antineoplastic therapy. Eighty episodes were analysed retrospectively. Forty-one episodes in 24 patients were treated prospectively with a standard regimen of gentamicin + cefotaxime. The results show that the prophylactic regimen does not eradicate potentially pathogenic organisms from the throat, urine, or stools. Sixty-three per cent of FE were of unknown origin (FUU). 40.5% of all isolated organisms were TS resistant. In the prospective group, 9 of 41 FE (22%) responded to gentamicin + cefotaxime with lysis of fever within 48 hours, and 63% required no further antibiotics. Defeverescence occurred in the whole group within 5 +/- 4 days. No patients were lost to infectious complications. The combination of gentamicin + cefotaxime as initial therapy of a febrile episode must be supplemented within 48 to 72 hours by additional antibiotics in the absence of clinical improvement.

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.

During fetal life, haematopoiesis is in a physiologic state of rapid expansion. The activity of the pluripotent haematopoietic stem cell compartment of the bone marrow probably is reflected by the number and proliferative potentiality of CFUc in the blood circulation. We have studied, by the methylcellulose culture technique and without addition of exogenous colony stimulating factor (CSF), blood CFUc in pre-term and full-term newborn infants, in full-term infants 10 to 17 d of age, and in adults. Two groups of pre-term infants were studied: otherwise healthy infants and sick infants, including infants small-for-date and in perinatal distress. In healthy pre-term infants and in full-term infants 147 +/- 25 (SD) and 105 +/- 41 (SD) colonies per 100 000 lymphoid cells were formed, respectively. This is clear evidence for the presence in the blood of both. CFUc and CSF producing cells. By contrast, there was no or only slight spontaneous colony formation by sick pre-term infants, by older infants, and by adult.

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.

26 children with acute lymphoblastic leukemia (ALL) and 7 additional patients with lymphosarcoma in leukaemic transformation (LSA) have been studied with respect to the content of myelopoietic stem cell (CFUc) in blood and bone marrow. The methylcellulose culture technique (Iscove et al 1974) was employed in the absence of an exogenous source of colony stimulating factor (CSF). During active disease, CFUc colony formation was absent from patients with ALL, but was present in 2 patients with LSA. 2 therapeutic regimens were employed. Colony formation from bone marrow CFUc was highly variable during remission maintained by either regimen, with no clear relation to clinical stage, number of monocytes or circulating neutrophils. Patients with LSA consistently had high numbers of bone marrow CFUc. CFUc were low or absent from the blood. In conclusion, CFUc are absent from the bone marrow in active ALL, but may be present in active LSA. For the purpose of monitoring children with ALL during therapy, determination of blood or bone marrow CFUc was not found in this study to be helpful.

Stromal precursor cells from bone marrow aspirates of children have been studied in culture. In 7 day liquid cultures normal individuals and patients with acute leukemia in remission grew 110 +/- 50 CFU-F and 100 +/- 40 CFU-F (colony forming unit--fibroblasts) respectively, per 6 X 10(5) buffy coat mononuclear cells. Staining with monoclonal antibodies suggests that stromal cells from CFU-F colonies are fibroblasts. CFU-F colony growth from the bone marrow of patients with active leukemia was low. After cultivation periods of more than 21 days, we observed, in addition, still more immature, clonogenic fibroblast precursor cells, "pre CFU-F", and round cells attached to stromal cells from pre CFU-F colonies. From the round cells, we have passaged pre CFU-F and CFU-GM (colony forming unit--granulocytic, monocytic) in secondary cultures. Our observations are in agreement with the concept that the bone marrow stromal cell matrix serves as a sanctuary for reversibly attached clonogenic cells of both the hematopoietic and fibroblast lineages.

Cord blood from 16 premature infants and 10 full-term infants, and blood from 10 healthy adults, was incubated for 30 minutes with tritiated thymidine, after which an autoradiographic study was made of spontaneously labeling cells. Apart from a varying number of erythroblasts, myelocytes, and an occasional blast cell, two main types of spontaneously labeling cells were observed: transitional cells and large lymphoid cells, previously shown to be phagocytic. Spontaneously labeling cells were 12 times more frequent in cord blood of premature and full-term infants than in the blood of adults. No transitional cells were seen in adult blood.

71 patients with resectable osterosarcoma received chemotherapy for one year including high-dose methotrexate (18 x 200 mg/kg), adriamycin (5 x [2 x 45] mg/m2) and cyclophosphamide (6 x 1200 mg/m2). During the initial 15 weeks adriamycin was used preferentially and cytostatic agents were applied in a higher frequency than later on. 41/71 patients are continuously free of disease with a median follow up of 39 (24-54) months. The latest appearance of pulmonary metastases was observed at 28 months so far. 18/27 (67%) patients with extension of tumor lesion beyond 1/3 long bones length by x-ray examination relapsed in contrast to 12/43 (28%) patients with smaller lesions. 1 patient died from adriamycin induced cardiomyopathy. Generally therapy was well tolerated. An average of 70-80% of planned drug dosages could be realized without measurable influence of individual differences on outcome.

A study of murine adherent marrow cells (AMC) under conditions of high and low concentrations of hematopoietic stem cells and progenitors was carried out. In one group of weanling mice, decreased marrow cellularity and increased concentrations of CFU-S, CFU-D, and CFU-C were observed two days after administration of two consecutive intraperitoneal (i.p.) cytosine arabinoside (Ara-C) injections (2 x 200 ng/kg, at 6 h interval). Fibroblast colony-forming units (CFU-F) of this marrow were studied. In a second group of mice, which were given three i.p. phenylhydrazine (PHZ) injections (3 x 60 mg/kg on days 0, 1, and 3), CFU-S and CFU-C levels were unchanged or lowered 6 days after the start of PHZ administration. In these animals, however, the number of CFU-D was four times higher than in controls. The study of CFU-F in experiments of groups 1 and 2 indicated a high concentration of these progenitors in group 1 and lower concentration in group 2. Furthermore, fibroblastoid colonies produced in vitro by CFU-F of Ara-C-treated marrow were significantly larger than colonies from control marrow, and they markedly inhibited G/M colonies in split-phase agar cultures. By contrast, fibroblastoid colonies produced by PHZ-treated marrow were of regular size and did not inhibit G/M colonies from test bone marrow.

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.

Cord blood cells from 8 full-term and 12 premature infants as well as blood leukocytes from 10 adults were incubated with 3H-thymidine and were subsequently exposed to autoradiographic emulsion for prolonged periods of 45 and 90 days. Cytoplasmic label -- which were interpreted as evidence of mitochondrial DNA synthesis -- was found in 1.7 per cent of lymphoid cells from adult blood, in 2.9 per cent and 4.5 per cent of lymphoid cells from full-term and premature infants, respectively. Statistical analysis of the data shows that the number of labelled cells is significantly larger in the blood of premature infants than in adults (p less than 0.01).

Myelopoietic progenitor cells (CFU-MG) have been studied from peripheral blood of healthy, sick, and hypotrophic pre-term infants. Methylcellulose cultures were prepared simultaneously with and without exogenous colony stimulating factor. It was found that large numbers of circulating CFU-M are present at birth in healthy infants, smaller numbers in sick infants, and very few in hypotrophic infants. Exogenous CSF increases the number of colonies in cultures of healthy infants at birth. A limiting factor in spontaneous colony formation is the production of CSF by the cells in culture. This is particularly evident in sick infants. During the postnatal course similar levels of circulating CFUc, higher than in adult blood, are found in all three groups of pre-term infants.

In three young patients with erythroleukemia in whom a partial reversion to the fetal pattern of erythropoiesis occurred there was found additionally an imbalance of globin chain synthesis. The synthesis of beta- plus gamma-chains exceeded that of the alpha-chains. In contrast, physiologic hemoglobin F production occurring in newborn infants and increased hemoglobin F production due to rapidly regenerating erythropoiesis in hereditary spherocytosis and after acute erythroblastopenia are characterized by a well balanced globin chain synthesis. These studies indicate that in distinct cases of juvenile erythroleukemia the genuine reversion to fetal erythropoiesis may be associated not only with a depression of hemoglobin synthesis but also with an imbalanced globin chain synthesis. Unlike adult cases of erythroleukemia without reversion to fetal erythropoiesis the imbalance of globin chain synthesis seems to be a more generalized phenomenon in these cases of juvenile erythroleukemia which is not confined to a particular red cell population.

19 children with AML were treated using the combination of high dose cytosine-arabinoside and mitoxantrone. All children were initially treated according to protocol AML-BFM-83. 6 children with refractory AML, 9 children with bone-marrow relapse during or after maintenance therapy and 4 children with residual blasts (5-25%) after remission induction and consolidation therapy AML-BFM 83 were treated with the relapse protocol. 6 of 15 children with refractory AML and all 4 children with residual blasts achieved a complete remission. 2 children died in bone-marrow aplasia and 1 child did not respond. One child died after further mitoxantrone treatment due to toxic cardiomyopathy. All children went into severe bone marrow aplasia, which lasted in median 27 days. These data indicate a high antileukemic activity of HD-ARA C/mitoxantrone in childhood AML.

CFU-F (colony forming units-fibroblast) were studied from cord blood and, as controls, from normal bone marrow of older children and adults. Numbers of CFU-F in cord blood buffy coat cells are lower by a factor of 10 in comparison to bone marrow CFU-F. Cytomorphology and staining with monoclonal antibody identify the progeny cells of CFU-F as fibroblasts. Cord blood CFU-F derived fibroblasts have properties supporting hematopoiesis: They produce CSF (colony stimulating factor) to which fresh cord blood CFU-GM (colony forming units-granulocytic, monocytic) react by colony formation in a dose-response manner. In addition, fibroblast colonies discharge clonogenic round cells into the medium forming CFU-GM and CFU-F colonies in secondary methyl cellulose cultures. We conclude that fetal blood contains clonogenic stromal cells (CFU-F) that give rise to fibroblasts with properties of hematopoietic support.