Zina Ben-Ishay

CFU-F circulating in cord blood

SummaryCFU-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.

Bone marrow stromal dysfunction in mice administered cytosine arabinoside

Abstract: Objective: The aim of the study was to investigate ex‐vivo the bone marrow (BM) stroma of mice under conditions of low‐ and high‐dose cytosine arabinoside (Ara‐C), a cycle‐specific drug (S‐phase) and to assess possible stromal damage, apart from the killing of hematopoietic cells. Stroma consists of mesenchymal elements generally not in the cell cycle; therefore it could not be a target for the killing effect of Ara‐ C. Materials and Methods: The stromal function was studied by the following: the incidence of stromal stem cells, i.e. CFU‐F; formation of stromal layers under growth conditions of long‐term culture (LTC) followed by irradiation and overlayering of test cells in contact and non‐contact co‐cultures; subsequent culture of the test cells in a semi‐solid medium to assay the incidence of hyperproliferative potential cells (HPPC); production of GM‐CSF, IL‐3, IL‐4, IL‐6 and IFNγ in the conditioned medium (CM) of confluent stromal layers. All tests and assays were carried out on BM specimens, 1–4 d after Ara‐C administration and on controls. Results: Low‐dose Ara‐C induces a marked decrease of CFU‐F, compensated by cycle induction of pre‐CFU‐F, young‐type stromal stem cells. High‐dose Ara‐C causes a CFU‐F decrease to almost zero level. The time length to layer confluency is normal after low‐dose Ara‐C (∼10 d) and prolonged after a high dose (∼30 d). The confluent layers from mice receiving low‐ or high‐dose Ara‐C support hematopoiesis adequately. Among the growth factors and cytokines assayed, only IL‐6 is detected in CM layers. IL‐6 decreases after a low dose of Ara‐C and increases after a high dose. The cause of IL‐6 fluctuations is yet to be investigated. It is, however, evident that IL‐6 is not an essential factor in support of hematopoiesis. Conclusions: Taken together, the current study in mice indicates that Ara‐C administration, in particular a high dose, induces bone marrow stromal damage and/or disfunction. The long period of time to reach layer confluency after a high Ara‐C dose might reflect the in‐vivo situation of slow stromal regeneration.

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.

Anti-angiogenic effects and regression of localized murine AML produced by anti-VEGF and anti-Flk-1 antibodies

Abstract:  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 × 2.5 μg anti‐VEGF (vascular endothelial growth factor) and 5 × 2.5 μg 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.

Interaction between normal or irradiated macrophages and lymphocytes in mice

Abstract Electron microscopic observations showed that normal peritoneal macrophages and lymphocytes are found in close apposition with one another and that extensive areas of close flattened surfaces exist between the two cell types. By contrast, peritoneal macrophages, irradiated in vivo , did not display similar areas of interaction with added normal lymph node lymphocytes. The failure of irradiated macrophages to interact closely with lymphocytes may be responsible for their inability to induce antibody production against several antigens.

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.

Effect of hydroxyurea on two different types of hematopoietic stem cells (CFU-S and DCPC) of newborn mice.

ZusammenfassungPluripotente hämatopoetische Stammzellen neugeborener Mäuse (Alter: 9–13 Tage) wurden nach Gabe von Hydroxy-Harnstoff (1000 mg/ kg) untersucht. Nach einmaliger und nach acht Injektionen war das Knochenmark innerhalb von 7 Tagen völlig wiederhergestellt. Während der Regenerationsphase war die Proliferation von Stammzellen in den Diffusionskammern (DCPC) aus dem Knochenmark behandelter Mäuse stärker mit einer grö\eren Zellausbeute als bei den Kontrolltieren. Dagegen waren die kolonienbildenden Einheiten (Milz) (CFU-S) Spiegel während der gleichen Zeit auf 15 bis 40% der Kontrollwerte abgesunken, wahrscheinlich als Ausdruck der geringen Reserven von ruhenden CFU-S bei neugeborenen Mäusen. Die Ergebnisse werden so interpretiert, da\ es sich bei den DCPC um nichtproliferierende Stammzellen handelt, die weniger differenziert als CFU-S sind. Danach würden bei neugeborenen Mäusen unter dem Stre\ zur hämatopoetischen Rekonstitution DCPC schnell das Stadium der CFU-S passieren, um direkt in die Myelopoese überzugehen.SummaryPluripotent hematopoietic stem cells from the bone marrow of newborn mice (9–13 days of age) have been studied following the administration of hydroxyurea (HU, 1,000 mg/kg). Following a single injection as well as a regimen of eight injections complete bone marrow reconstitution was achieved after 7 days. During the recovery phase diffusion chamber progenitor cells (DCPC) from pretreated newborn mouse bone marrow proliferated strongly to give significantly higher cell yields than DCPC from control animals. By contrast (colonyforming units-spleen [CFU-S]) levels were reduced at the same time to between 15 and 40% of controls suggesting a small-sized reserve pool of resting CFU-S in newborn mice. Assuming that DCPC are less differentiated than CFU-S and are non-cycling stem cells they may possibly pass through the CFU-S stage rapidly in newborn mice under the stress of hematopoietic reconstitution and proceed directly to myelopoiesis.

Crystalline-like surface charge array of murine macrophages and lymphocytes: Visualization with cationized ferritin

The distribution of negative charges on the surface of murine macrophages and lymphocytes was assessed by labeling fresh unfixed cells with cationized ferritin at a physiological pH. Ultrastructural examination of the ferritin labeled cells demonstrated in places a crystalline pattern of surface charges on both marcophages and lymphocytes. The possible significance of this crystalline-like arrangement is discussed.

Fibrogenesis in the human myocardium: An electron microscopic study

Summary Biopsies of auricular appendages of patients with congenital and chronic rheumatic heart disease have been studied by electron microscopy. The fine structure of the microfibrils, elastic and collagen fibers in areas of fibrosis of the myocardium is described. Morphological evidence is presented which indicates that both fibroblast and cardiac muscle cell participate in the production of the different extracellular fibers. The stimulus for the production of these extracellular elements by cardiac muscle cells is unknown.

Growth of Diffusion Chamber Hematopoietic Colonies Derived from Spleen Cells of Rats Administered Hydroxyurea

Donor rats of the Hebrew University strain were administered a single intraperitoneal injection of hydroxyurea (400 mg/kg body weight). 1--3 h following the administration of the drug, a suspension of spleen cells, the majority of which consisted of lymphocytes, was prepared. Spleen cells were placed in diffusion chambers and these were implanted in the peritoneal cavity of preirradiated mice. 5--8 days following implantation, erythroid and granulocytic colonies developed in 30.3% of the diffusion chambers studied. However, in most chambers, macrophages were observed. In control experiments with implantation of spleen cells of normal rats, granulocytic colonies did not grow and in only 3.1% of the chambers erythroid colonies were noted. Macrophage colonies, however, developed in all 32 control cultures. Our previous studies showed that administration of a single dose of hydroxyurea strips the rat bone marrow of approximately 50% of replicating cells within 9--10 h. The results of the present study indicate that such a severe depletion of rat marrow cells results in early committment of spleen stem cells to various blood cell lines.